BIO-engineered mitochondria for targeted delivery to cells

Abstract

The present disclosure provides engineered mitochondria and antibody-mitochondrion conjugates for targeted delivery to cells. The present disclosure also provides methods for using the engineered mitochondria and antibody-mitochondrion conjugates for treating a cardiovascular disease (CVD) or mitochondrial dysfunction (mt-dys) in a subject.

Description

PATENT Attorney Docket No.: 079445-014410PC-1488490 Client Reference No.: S24-026 BIO-ENGINEERED MITOCHONDRIA FOR TARGETED DELIVERY TO CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 560,421, filed March 1, 2024, the disclosure of which is herein incorporated by reference in its entirety for all purposes. BACKGROUND

[0002] Cardiovascular disease (CVD) is a major cause of mortality worldwide and in the US. Roughly 45% of the US adult population in 2016 (approximately 112 million people)1, 2had some form of CVD, with a higher prevalence of CVD development and related mortality in the elderly population2-5. Additionally, the elderly population in the US is projected to reach around 74 million by 2030. The elderly cost of care in the US increased by $100 billion from 2001 to 20116and will continue to rise, further adding urgency toward understanding the complex pathology that underlies CVD.

[0003] In the context of aging, mitochondrial dysfunction (mito-dysfunction, or mt- dys) inspecific immune cells and vascular cells, such as macrophages (M s), T cells, endothelial cells(ECs), and vascular smooth muscle cells (VSMCs), underpin multiple forms of CVD. Mt-dys includes reduced mitochondrial DNA (mtDNA) quality, inefficient electron transport chain(ETC) activity, abnormal mitochondrial membrane potential ( m), and abnormal generationof reactive oxygen species (mt-ROS). While current strategies to combat mt-dys such as antioxidant supplementation or modulation of critical pathways related to mitochondrial biogenesis temporarily augment mitochondrial health, they fail to repair the underlying dysfunction and restore mtDNA quality.

[0004] Mitochondrial transplantation (mito-transplantation, or mt-tfr) is as a novel approach that replaces damaged / aged mitochondria with healthier, more functional counterparts, which may restore mitochondrial health and may rescue cellular dysregulations7. However, the underlying mechanisms of how mt-tfr drives cellular reprogramming and subsequent physiological benefits still need to be explored.SUMMARY

[0005] In one aspect, the present disclosure provides an engineered mitochondrion comprising one or more modified phospholipids on the mitochondrial membrane (MM) of the mitochondrion. In some embodiments, the mitochondrial membrane (MM) is the outer mitochondrial membrane (OMM). In some embodiments, the mitochondrial membrane (MM) is the inner mitochondrial membrane (IMM). In some embodiments, the mitochondrial membrane (MM) is a combination of the OMM and the IMM. In some embodiments, the modified phospholipids comprise azide-modified phospholipids and / or alkyne-modified phospholipids.

[0006] In some embodiments, the modified phospholipids are derived from phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines (PSs), phosphatidylinositol (PLs), phosphatidic acids (Pas), cardiolipins (CLs), or sphingomyelins (SMs). In some embodiments, the azide-modified phospholipids comprise azido-ethyl- phosphocholine (Az-Cho), azide-modified phosphatidylethanolamine (Az-PE), azide-modified phosphatidylserine (Az-PS), azide-modified phosphatidylinositol (Az-PI), azide-modified phosphatidic acid (Az-PA), azide-modified cardiolipin (Az-CL), azide-modified sphingomyelin (Az-SM), and / or any variant thereof. In some embodiments, the alkyne- modified phospholipids comprise propargyl choline (P-Cho), alkyne-modified phosphatidylethanolamine (Ak-PE), alkyne-modified phosphatidylserine (Ak-PS), alkyne- modified phosphatidylinositol (Ak-PI), alkyne-modified phosphatidic acid (Ak-PA), alkyne- modified cardiolipin (Ak-CL), alkyne-modified sphingomyelin (Ak-SM), and / or any variant thereof.

[0007] In some embodiments, the engineered mitochondrion is linked to an antibody and / or a therapeutic payload through the modified phospholipids. In some embodiments, the engineered mitochondrion is linked to the antibody and / or the therapeutic payload via an azide- alkyne cycloaddition reaction. In some embodiments, the antibody binds to a transmembrane protein of a cell. In some embodiments, the transmembrane protein is intercellular adhesion molecule type 1 (ICAM-1, also known as CD54). In some embodiments, the transmembrane protein is lymphocyte function-associated antigen 1 (LFA-1, also known as Integrin, alpha L, ITGAL, or CD11a). In some embodiments, the transmembrane protein is platelet endothelial cell adhesion molecule type 1 (PECAM-1, also known as CD31).

[0008] In some embodiments, the cell is a vascular cell or an immune cell. In some embodiments, the vascular cell is selected from the group consisting of an endothelial cell (EC), a vascular smooth muscle cell (VSMC), a smooth muscle cell, a cardiomyocyte, a pericyte, a fibroblast, a cardiac fibroblast, an endothelial progenitor cell, a pacemaker cell, and an adventitial cell. In some embodiments, the immune cell is selected from the group consistingof a macrophage (M ), a T cell, a B cell, a dendritic cell (DC), a NK cell, a neutrophils, aneosinophil, a basophil, a mast cell, a helper t cell (th1, th2, th17), a regulatory t cell (treg), a memory B cell, a plasma cell, a follicular dendritic cell, a monocyte, an innate lymphoid cell(ilc), and a gamma delta t cell ( t cell).

[0009] In some embodiments, the engineered mitochondrion comprises one or more modified phospholipids on the OMM linked to the antibody and one or more modified phospholipids on the IMM linked to the therapeutic payload. In some embodiments, the therapeutic payload is Coenzyme Q10 (CoQ10), Mito-TEMPO, or a gene therapy agent.

[0010] In another aspect, the present disclosure provides an antibody-mitochondrion conjugate, comprising an antibody and a mitochondrion. In some embodiments, the antibody and the mitochondrion are linked via a covalent, non-covalent, or hybrid linker, including but not limited to click chemistry, thiol-based conjugation, or affinity-based binding.

[0011] In some embodiments, the antibody and the mitochondrion are covalently linked through a linker. In some embodiments, the linker comprises an azide-modified phospholipid and / or an alkyne-modified phospholipid. In some embodiments, the azide-modified phospholipid comprises azido-ethyl-phosphocholine (Az-Cho), azide-modified phosphatidylethanolamine (Az-PE), azide-modified phosphatidylserine (Az-PS), azide- modified phosphatidylinositol (Az-PI), azide-modified phosphatidic acid (Az-PA), azide- modified cardiolipin (Az-CL), azide-modified sphingomyelin (Az-SM), and the alkyne- modified phospholipid comprises propargyl choline (P-Cho), alkyne-modified phosphatidylethanolamine (Ak-PE), alkyne-modified phosphatidylserine (Ak-PS), alkyne- modified phosphatidylinositol (Ak-PI), alkyne-modified phosphatidic acid (Ak-PA), alkyne- modified cardiolipin (Ak-CL), alkyne-modified sphingomyelin (Ak-SM), and / or any variant thereof. In some embodiments, the antibody and the mitochondrion are covalently linked via an azide-alkyne cycloaddition reaction.

[0012] In some embodiments, the antibody binds to a transmembrane protein of a cell. In some embodiments, the transmembrane protein is intercellular adhesion molecule type 1(ICAM-1). In some embodiments, the transmembrane protein is lymphocyte function- associated antigen 1 (LFA-1). In some embodiments, the transmembrane glycoprotein is platelet endothelial cell adhesion molecule type 1 (PECAM-1).

[0013] In some embodiments, the cell is a vascular cell or an immune cell. In some embodiments, the vascular cell is selected from the group consisting of an endothelial cell (EC), a vascular smooth muscle cell (VSMC), a smooth muscle cell, a cardiomyocyte, a pericyte, a fibroblast, a cardiac fibroblast, an endothelial progenitor cell, a pacemaker cell, and an adventitial cell. In some embodiments, the immune cell is selected from the group consistingof a macrophage (M ), a T cell, a B cell, a dendritic cell (DC), a NK cell, a neutrophils, aeosinophil, a basophil, a mast cell, a helper t cell (th1, th2, th17), a regulatory t cell (treg), a memory B cell, a plasma cell, a follicular dendritic cell, a monocyte, an innate lymphoid cell(ilc), and a gamma delta t cell ( t cell).

[0014] In another aspect, the present disclosure further provides a composition comprising the engineered mitochondrion or the antibody-mitochondrion conjugate described herein. Further disclosed is a pharmaceutical composition comprising the composition and a pharmaceutically acceptable carrier.

[0015] In another aspect, the present disclosure further provides a kit for treating a subject comprising the pharmaceutical composition, wherein the subject has a cardiovascular disease (CVD) or mitochondrial dysfunction.

[0016] In another aspect, the present disclosure further provides a method of treating a cardiovascular disease (CVD) or mitochondrial dysfunction in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. In some embodiments, the subject is an aged individual. For purposes of the present invention, an "aged individual" is defined as a subject who is 60 years or older, preferably 65 years or older, in alignment with clinical and regulatory standards for aging-related conditions such as cardiovascular disease and mitochondrial dysfunction.

[0017] In another aspect, the present disclosure further provides a method of delivering a mitochondrion to a cell, comprising: a. generating an antibody-mitochondrion conjugate by covalently linking the mitochondrion to an antibody, wherein the antibody recognizes and binds to a transmembrane protein of the cell; and b. contacting the cell with the mitochondrion- targeting conjugate, wherein the targeting moiety facilitates binding to the transmembrane protein, leading to endocytosis-mediated internalization of the mitochondrion into the cell.

[0018] In some embodiments, the antibody and the mitochondrion are covalently linked through a linker. As disclosed herein, any linker that undergoes a click chemistry reaction can be applied to covalently link the antibody and the mitochondrion. In some embodiments, the linker comprises a bioorthogonal functional group, including but not limited to azide-modified phospholipids, alkyne-modified phospholipids, tetrazine-functionalized lipids, thiol-reactive linkers, or oxime-based ligation systems. In some embodiments, the linker is derived from phospholipids, polymers, peptides, or other biocompatible linker materials. In some embodiments, the linker comprises an azide-modified phospholipid and / or an alkyne-modified phospholipid. In some embodiments, the azide-modified phospholipid comprises azido-ethyl- phosphocholine (Az-Cho), azide-modified phosphatidylethanolamine (Az-PE), azide-modified phosphatidylserine (Az-PS), azide-modified phosphatidylinositol (Az-PI), azide-modified phosphatidic acid (Az-PA), azide-modified cardiolipin (Az-CL), azide-modified sphingomyelin (Az-SM), and the alkyne-modified phospholipid comprises propargyl choline (P-Cho), alkyne-modified phosphatidylethanolamine (Ak-PE), alkyne-modified phosphatidylserine (Ak-PS), alkyne-modified phosphatidylinositol (Ak-PI), alkyne-modified phosphatidic acid (Ak-PA), alkyne-modified cardiolipin (Ak-CL), alkyne-modified sphingomyelin (Ak-SM), and / or any variant thereof.

[0019] In some embodiments, the antibody and the mitochondrion are covalently linked via a click chemistry reaction, including but not limited to azide-alkyne cycloaddition, tetrazine ligation, thiol-maleimide coupling, oxime ligation, or sulfur fluoride exchange. In some embodiments, the transmembrane protein is intercellular adhesion molecule type 1 (ICAM-1, also known as CD54). In some embodiments, the transmembrane protein is lymphocyte function-associated antigen 1 (LFA-1, also known as Integrin, alpha L, ITGAL, or CD11a). In some embodiments, the transmembrane glycoprotein is platelet endothelial cell adhesion molecule type 1 (PECAM-1, also known as CD31).

[0020] In some embodiments, the cell is a vascular cell or an immune cell. In some embodiments, the vascular cell is selected from the group consisting of an endothelial cell (EC), a vascular smooth muscle cell (VSMC), a smooth muscle cell, a cardiomyocyte, a pericyte, a fibroblast, a cardiac fibroblast, an endothelial progenitor cell, a pacemaker cell, and an adventitial cell. In some embodiments, the immune cell is selected from the group consistingof a macrophage (M ), a T cell, a B cell, a dendritic cell (DC), a NK cell, a neutrophils, aeosinophil, a basophil, a mast cell, a helper t cell (th1, th2, th17), a regulatory t cell (treg), a memory B cell, a plasma cell, a follicular dendritic cell, a monocyte, an innate lymphoid cell(ilc), and a gamma delta t cell ( t cell). In some embodiments, the antibody-mitochondrionconjugate comprises an anti-PECAM-1 ( -PECAM-1) antibody bound to a mitochondrion, andthe cell is a vascular cell. In some embodiments, the vascular cell is an endothelial cell (EC).In some embodiments, the antibody-mitochondrion conjugate comprises an anti-LFA-1 ( -LFA-1) antibody bound to a mitochondrion, and the cell is an immune cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 depicts both mitochondria with 1-Azidoethyl-choline (Az-Cho-mito) andmitochondria with propargyl choline (P-Cho-mito) are taggable to macrophages (M s). A)Frequency (%) of M s labeled with fluorescent alkyne (DBCO-Cy5+ M s), B) the medianfluorescent intensity (MFI) of DBCO-Cy5+ M s, and C) Fold change in MFI of DBCO-Cy5+M s after mitochondrial transplantation with Az-Cho-mito. D) Frequency (%) of M s labeledwith Alexa Fluor 647 (Az-647 + M s), E) the MFI of Az-647+ M s, and F) Fold change inMFI of Az-647+ M s after mitochondrial transplantation with P-Cho-mito. p< 0.05 = * via 1-way ANOVA, n = 4.

[0022] FIG. 2 depicts mitochondrial transplantation (mt-tfr) modulates macrophage (M )and endothelial cell (EC) function. A) Fold change in Arg1+ M ’s after IL-4 stimulation andtreatment with 25 or 75 ug of mitochondria. In plots B-C), M s were cultured in LPS / IFNy for48h, subjected to mt-ftr and then cultured in IL-4 for up to 72 h (M1 M2 polarization). B)The Ratio of Arg1+ / iNOS+ M s and C) the MFI of Arg1+ M s at 24, 48 and 72 h post IL-4stimulation. D) Extracellular acidification rate (ECAR) of early (p6) and late (p13) passaged microvascular ECs at 24 h post mt-tfr. E) mitochondrial volume and F) proliferation of early & late passage ECs. p< 0.05 = significant (*) using paired Student’s T-test, n=3 per group.

[0023] FIG. 3 depicts confocal images of donor mitochondria entering recipient cells with Mito-tracker Deep Red, a mitochondrial membrane potential sensitive probe. The mitochondria of recipient cells were stained with Mito-tracker Deep Red. The Donor mitochondria were tagged with 2 different ligands; Azido-pycolly-488 and Azido modified -PECAM-1 ( -CD31), that was already fluorescently conjugated to PE. This allows for dualdetection of mitochondria after delivery. The cells (endothelial cells) were incubated with the tagged mitochondria for 24h, after which the cells were fixed and visualized. The images demonstrate that donor mitochondria have colocalize and seemingly integrate into the recipient cell’s native mitochondrial network. This is proof of principle that antibody-tagged mitochondria can be effectively delivered to cells and are likely functional (i.e. have amembrane potential). A) depicts punctates identified as the internalized -CD31-mito-tags mito-tags. The -CD31-mito-tags contained a R-phycoerythrin (PE) fluorophore attached to the -CD31 antibody. B) depicts punctates identified as the internalized -CD31-mito-tags. The fluorescence from 488-Picolyl-Azide-tagged mitochondria verifies dual tagging. C) depicts fluorescence of Mito-tracker Deep red, which stains mitochondria, in a membrane potential dependent manner. D) depicts fluorescence from Hoechst 33342 staining the nuclei. E) shows the Merged / overlay of the confocal channels (panels A – D).

[0024] FIG. 4 depicts confocal images of donor mitochondria entering recipient cells with Mito-tracker Deep Red, a mitochondrial membrane potential sensitive probe. The mitochondria of recipient cells were stained with Mito-tracker Deep Red. The Donor mitochondria were tagged with 2 different ligands; Azido-pycolly-488 and Azido modified - PECAM-1 (CD31), that was already fluorescently conjugated to PE. A) depicts punctates identified as the internalized -CD31-mito-tags mito-tags. The -CD31-mito-tags contained a R-phycoerythrin (PE) fluorophore attached to the -CD31 antibody. B) depicts punctates identified as the internalized -CD31-mito-tags. The fluorescence from 488-Picolyl-Azide- tagged mitochondria verifies dual tagging. C) depicts fluorescence of VE-Cadherin staining (with -VE-Cadherin antibody) at the surface of Endothelial cells. VE-Cadherin is an endothelial cell-cell junction marker. D) depicts fluorescence from Hoechst 33342 staining the nuclei. E) shows the Merged / overlay of the confocal channels (panels A – D).

[0025] FIG. 5 depicts mitochondrial dysfunction (mt-dys) in cells and tissue isolated from mice with surgically induced abdominal aortic aneurysms (AAA). A) Heatmap of electron transport chain (ETC) associated genes in infrarenal aortic tissue isolated from AAA mice at 3-, 7-, 14-, and 28-days post-surgery, expressed as Z-Score (log2Fold normalized to rows). B) Umap of AAA and control (SHAM) mice at 7 days post-surgery. Differential expression ofgenes (DEGs) related to mitochondrial processes, in C) M and D) ECs at 7 days post AAAsurgery.

[0026] FIG. 6 depicts impact of mito-transfer with P-cho mito on mitochondrial ROSproduction in macrophages (M s). P-cho-Az 647 mito were transplanted into macrophages,after which the levels of mitochondrial superoxide production, another indicator of mitochondrial function / dysfunction was examined using the fluorescent probe MitoSOX (5 uM) and quantified by flow cytometry using the PE channel. These data show thatmitochondrial ROS production significantly decreased in M s treated with difference doses ofP-cho-Az 647 mito.

[0027] FIG. 7 depicts internalization of unmodified mitochondria (nkd-mito) vs -CD31 mito-tags in ECs. A) depicts ECs exposed to ndk-mito (punctates), with nuclear counterstain Hoechst 33342. B) depicts ECs exposed to ndk-mito (punctates), the recipient cell’s mitochondrial network, and with nuclear counterstain Hoechst 33342, C) depicts ECs exposed to -CD31 mito-tags (punctates), with nuclear counterstain Hoechst 33342. D) depicts ECs exposed to -CD31 mito-tags, the recipient cell’s mitochondrial network, and with nuclear counterstain Hoechst 33342.

[0028] FIG.8 depicts quantification of the internalization of unmodified mitochondria (nkd- mito) vs -CD31 mito-tags in ECs. A) Cells treated with -CD31 mito-tags exhibited a significantly higher number of particles per nucleus, averaging 310.05 punctate, compared to 27.67 punctate in cells treated with nkd-mito. B) The -CD31 mito-tags occupied significantly more cellular area (3.4%) compared to nkd-mito (0.3%).

[0029] FIG. 9 presents the results of colocalization analysis performed using Just Another Colocalization Plugin (JACoP) in ImageJ to evaluate the specificity of mito-tags integrating into the recipient cells’ mitochondrial network. The Thresholded Overlap Coefficient quantifies the proportion of overlapping pixels between the two fluorescence signals after applying an intensity threshold. The Costes 2D correlation analysis is a statistical approach commonly used in colocalization studies to determine whether two fluorescence signals are spatially related within an image. The Pearson Costes 2D correlation method was applied toassess the spatial relationship between delivered mitochondria ( -CD31-PE mito-tag) and therecipient cell’s endogenous mitochondrial network (stained with Mito Tracker Deep Red). A) presents the Thresholded Overlap Coefficient values. B) displays the Random Pearson Costes 2D values.

[0030] FIG. 10 depicts the specificity of the -CD31-PE mito-tags evaluated through co- culture experiments involving endothelial cells (ECs, CD31+) and vascular smooth muscle cells (VSMCs, CD31-), and visualized using confocal microscopy (A-E). A) depicts -VE- Cadherin-stained endothelial cells. B) depicts -CD31-PE mito-tags (punctates / dots). C) depicts Hoechst 33342 stained nuclei. D) depicts vascular smooth muscle cells stained with anti-alpha-smooth actin. E) depicts a merged confocal image of Panels A-D.

[0031] FIG. 11 depicts the specificity of the -CD31-PE mito-tags evaluated through co- culture experiments involving endothelial cells (ECs, CD31+) and vascular smooth muscle cells (VSMCs, CD31-), and quantified by flow cytometry. Nearly all ECs exhibit significant uptake of -CD31-PE mito-tags, while VSMCs demonstrate minimal internalization.

[0032] FIG. 12 depicts the selective uptake of -CD31-PE mito-tags by bone marrow- derived macrophages (BMDMs) relative to vascular smooth muscle cells (VSMCs). A) depicts BMDMs stained with -CD68 antibod. BMDM express the CD31 receptor. B) depicts - CD31-PE mito-tags (punctates / dots). C) depicts vascular smooth muscle cells stained with anti-alpha-smooth actin. D) depicts a merged confocal image of Panels A-C.

[0033] FIG.13 depicts impact of -CD31 mito-tags on mitochondrial abundance (baseline). A) depicts that endothelial cells (ECs) treated with -CD31 mito-tags exhibit a significant increase in mitochondrial abundance, as measured by MFI-FITC-TOMM20, compared to untreated control cells (*p<0.05). B) depicts that treatment with -CD31 mito-tags (Y-mt-tag and O-mt-tag) significantly enhances mitochondrial content in both young (Y) and old (O) endothelial cells (ECs), as measured by MFI of Mitoview Green (MTV-G), compared to untreated controls (Y-ctrl and O-ctrl) and cells treated with non-targeted mitochondria (Y-ndk and O-ndk).

[0034] FIG. 14 illustrates the relative mean fluorescence intensity (MFI) of DCF-DA, a fluorescent probe used to detect reactive oxygen species (ROS) under different experimental conditions, including Control, -CD31 (antibody only), naked mitochondria (nk-mito), andantibody-conjugated mitochondria ( -CD31 mito-tag).

[0035] FIG. 15 depicts a Seahorse XF Real-Time ATP Rate Assay report highlighting the bioenergetic improvements achieved through -CD31 mito-tag treatment in aged aortic endothelial cells (O-AoECs). A) depicts an ATP production graph. B) depicts an energetic map. C) an ATP Rate Index measurements.

[0036] FIG. 16 depicts the functional impact of mito-tags on endothelial cells (ECs). A) depicts the relative mean fluorescence intensity (MFI) of DCF-DA, a reactive oxygen species(ROS) indicator, under different experimental conditions involving IFN treatment, -CD31antibody (control), naked mitochondria (nkd-mt), and -CD31 mito-tags. B) depicts the mean fluorescence intensity (MFI) of MitoSOX, an indicator of mitochondrial superoxide production, across various experimental groups involving young (Y) and old (O) endothelialcells (ECs) treated with or without interferon gamma (IFN ), naked mitochondria (nkd), or -CD31 mito-tags (mito-tags).

[0037] FIG. 17 depicts the functional impact of mito-tags on endothelial cells (ECs) by assessing bioenergetics approximately 24 hours after exposure to mito-tags. The analysis compared aged ECs treated with mito-tags to untreated controls, as well as to ECs exposed to nkd-mitochondria or mito-tags loaded with damaged mitochondria in specific instances. A) Basal mito respiratory, B) maximum mito respiratory, C) Spare respiratory capacity, D) Mito- ATP, and E) Non-mito oxygen consumption rates are analyzed.

[0038] FIG. 18 depicts the impact of -CD31 mito-tags treatment on cell’s basal and compensatory glycolysis. A) depicts basal glycolysis assessment. B) depicts compensatory glycolysis assessment.

[0039] FIG. 19 depicts the impact of mitochondrial transfer on endothelial cell (EC) proliferation in A) young (2–3 months) and B) old (18–22 months) mouse aortic endothelial cells (AoEC) in single time point (48 hours post-mitochondrial transfer).

[0040] FIG.20 depicts depicts the impact of mitochondrial transfer on endothelial cell (EC) proliferation in A) young (2–3 months) and B) old (18–22 months) mouse aortic endothelial cells (AoEC) in two time points (48 and 96 hours post-mitochondrial transfer).

[0041] FIG.21 depicts the effects of mitochondrial transfer on the proliferation of mtDNA- deficient rho-null endothelial cells (Rho ECs), which rely on exogenous uridine for survival and proliferation. A) shows single time point (48 hours post-mitochondrial transfer). B) shows two time points (48 and 96 hours post-mitochondrial transfer).

[0042] FIG.22 depicts the biodistribution of MitoTracker deep-red-stained mitochondria 24 hours after injection into mice. A) depicts a mouse without an injection (control). B) depicts a mouse injected with naked mitochondria. C) depicts a mouse injected with -CD31 mt-tag (Antibody-Tagged Mitochondria).

[0043] FIG. 23 depicts confocal images of isolated aortic tissue show donor mitochondria (MitoTracker Deep Red-stained) in endothelial cells (ECs) stained for PECAM1 and nuclei. A) depicts naked mitochondria. B) depicts -CD31 mt-tag (Antibody-Tagged Mitochondria).

[0044] FIG.24 depicts the uptake of -CD11a mito-tagged mitochondria compared to naked mitochondria (nkd-mito) by splenocytes isolated from aged mice in an ex vivo system. A)shows Flow Cytometry Scatter Plots of naked mitochondria (nkd-mito). B) shows Flow Cytometry Scatter Plots of -CD11a Mito-Tag. C) shows percentage of mitochondria-positive cells quantified from Q2-UR and Q2-LR of A) and B). D) shows mean fluorescence intensity (MFI) quantified from Q2-UR and Q2-LR of A) and B).

[0045] FIG.25 depicts the uptake of -CD11a mito-tagged mitochondria compared to naked mitochondria (nkd-mito) by splenocytes isolated from aged mice in an ex vivo system. A) shows Flow Cytometry Scatter Plots of naked mitochondria (nkd-mito). B) shows Flow Cytometry Scatter Plots of -CD11a Mito-Tag. C) shows mean fluorescence intensity (MFI) quantified from Q2-UR of A) and B).

[0046] FIG.26 depicts confocal (cross sectional) images of splenocytes illustrated a merge of CD45+ and -CD11a mito-tags (A), CD45+ only (B), and -CD11a mito-tags only (C). DETAILED DESCRIPTION I. Introduction

[0047] Aging-associated mitochondrial dysfunction (mito-dysfunction, or mt- dys) affects every cell system in our body. Mito-dysfunction includes reduced quality of mitochondrial DNA (mtDNA), irregular generation of reactive oxygen species, and membrane potential. In the context of the cardiovascular system, mito-dysfunction exacerbates inflammatory immune cell activity, vascular inflammation, and oxidative stress. These factors are primary drivers of cardiovascular disease (CVD). Consequently, the elderly face higher risks of developing CVD. An innovative approach, mitochondrial transplantation (mito-transplantation), replaces damaged mitochondria in cells and holds significant potential for fixing a root cause of aging- associated dysfunctions. The inventor has discovered that mito-transplantation improves the activation and proliferation of aged T-cells and the anti-inflammatory polarization of macrophages in ex vivo assays. Additionally, mito-transplantation reestablishes proper metabolism in aged endothelial cells and reduces oxidative stress in vascular smooth muscle cells. However, while immune cells can be reintroduced through the periphery, delivering vascular cells is more challenging, posing a barrier to the broader application of mito-transfer for CVD.

[0048] The present disclosure provides a pioneering strategy: bioengineering mitochondria for targeted delivery using Click Chemistry (CC). CC, a highly selective biochemical reaction involving azide and alkyne functional groups, can be harnessed to tag cellular components likephospholipids abundant in the mitochondrial membrane (MM) including both the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). This innovative approach posits that taggable phospholipids of the MM can act as hooks for receptor-mediated internalization by cells.

[0049] The present disclosure is centered around bioengineering mitochondria for targeted delivery with broad therapeutic application to conditions involving mitochondrial dysfunction. Mitochondrial transplantation has shown promise in rescuing dysfunctional cells and this technology aims to further develop this method by using mitochondria as their delivery package via Click Chemistry. Considering that the outer OMM is made up of about 40-50% Phosphatidylcholine and 25-30% Phosphatidylethanolamine, it is feasible for click chemistry reactions to occur on the OMM following the introduction of azide or alkyne-modified phospholipids. This allows antibodies or other homing molecules to be attached directly to mitochondria, which can facilitate their uptake without the need for encapsulation. The shared phospholipid composition between the OMM and IMM suggests that if an engineered mitochondrion is modified at the OMM, some of these modifications could persist in the IMM, allowing for targeted drug delivery.

[0050] Described herein are compositions of engineered mitochondria or antibody- mitochondrion conjugates for targeted delivery to cells, tissue, and organs. Additionally, kits containing the engineered mitochondria or antibody-mitochondrion conjugates and methods for preventing or treating cardiovascular disease (CVD) or mitochondrial dysfunction in a subject in need thereof are provided. In some embodiments, the subject is an aged individual. II. Definitions

[0051] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0052] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0053] The terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.

[0054] Also, the words “comprise,” “comprising,” “contains,” “containing,” “include,” “including,” and “includes,” when used in this specification and in the following claims, are intended to specify the presence of stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, acts, or groups.

[0055] As used herein, the term “antibody” means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of an antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and specifically covers a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antigen-binding fragments including but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity.

[0056] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0057] As used herein, the term “administering” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0058] The term “treating” refers to an approach for obtaining beneficial or desired results including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. Therapeutic benefit can also mean to effect a cure of one or more diseases, conditions, or symptoms under treatment.

[0059] The term “effective amount” or “sufficient amount” refers to the amount of the engineered mitochondria or the antibody-mitochondrion conjugates or other composition that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific amount may vary depending on one or more of: the particular agent chosen, the target cell type, the location of the target cell in the subject, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, and the physical delivery system in which it is carried.

[0060] For the purposes herein, an effective amount is determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect in a subject suffering from target disease(s). The desired therapeutic effect may include, for example, amelioration of undesired symptoms associated with the disease(s), prevention of the manifestation of such symptoms before they occur, slowing down the progression of symptoms associated with the disease(s), slowing down or limiting any irreversible damage caused by the disease(s), lessening the severity of or curing the disease(s), or improving the survival rate or providing more rapid recovery from the disease(s).

[0061] The effective amount depends, inter alia, on the type and severity of the disease to be treated and the treatment regime. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the distribution profile of atherapeutic agent (e.g., engineered mitochondria or antibody-mitochondrion conjugates) or composition within the body, the relationship between a variety of pharmacological parameters (e.g., half-life in the body) and undesired side effects, and other factors such as age and gender, etc.

[0062] The term “pharmaceutically acceptable carrier” refers to a substance that aids the administration of an active agent to a cell, an organism, or a subject. “Pharmaceutically acceptable carrier” refers to a carrier or excipient that can be included in the compositions of the disclosure and that causes no significant adverse toxicological effect on the subject. Non- limiting examples of pharmaceutically acceptable carriers include water, sodium chloride, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonic and absorption delaying agents, and the like. The carrier may also be substances for providing the formulation with stability, sterility and isotonicity (e.g. antimicrobial preservatives, antioxidants, chelating agents and buffers), for preventing the action of microorganisms (e.g. antimicrobial and antifungal agents, such as parabens, chlorobutanol, sorbic acid and the like) or for providing the formulation with an edible flavor etc. In some instances, the carrier is an agent that facilitates the delivery of the engineered mitochondria or the antibody-mitochondrion conjugates to a target cell or tissue. One of skill in the art will recognize that other pharmaceutical carriers are useful in the present disclosure. III. Detailed Description of the Embodiments

[0063] The present disclosure is based, in part, on the inventor’s discovery that centrifugation is a highly efficient mt-tfr method for the delivery of naked mitochondria to CD4+ T cells isolated from aged mice (18-24 months). In addition, the inventor has found that Mt-tfr to recipient-aged CD4+ T cells resulted in reduced mt-ROS production, and increased ETC activity & oxidative phosphorylation (Ox-Phos). Mt-tfr also significantly improved activation, cytokine production, and proliferation of aged CD4+ T cells. In experiments with immune- deficient RAG-KO mice, the aged CD4+ T cells were enhanced with mt-tfr, provided better protection against influenza and tuberculosis infections, compared to mice receiving untreated old CD4+ T cells8. This work strongly supports the use of mt-tfr as a strategy for extending health span.

[0064] Extracellular delivery of mitochondria (mitochondrial transplantation) to recipient cells is known to occur by two methods; direct uptake of naked mitochondria and by the uptake of mitochondria within microvesicles (mEVs). While the mechanisms behind naked mitochondrial uptake are unclear, mEV-mediated transfer is believed to involve specific ligand-receptor interactions. Independent of mitochondrial transplantation, receptor-mediated uptake has also influenced the development of antibody-mediated delivery methods, where antibodies or their fragments target specific receptors on cell surfaces for cargo delivery. For example, adhesion molecules on ECs, like ICAM-1 and PECAM-1, have each been previously exploited for intracellular delivery of biological materials. This new modification to mitochondria bypasses the need for encapsulation and removes the bottleneck placed on mitochondrial transplantation due to supply shortcomings. A. Engineered Mitochondria

[0065] A mitochondrion is an organelle in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is used throughout the cell as a source of chemical energy. The most prominent roles of mitochondria are to produce the energy currency of the cell, ATP (i.e., phosphorylation of ADP), through respiration and to regulate cellular metabolism. The central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs cycle, and oxidative phosphorylation. In addition to the production of ATP, the mitochondrion also has many other functions, such as mitochondrial fatty acid synthesis (mtFASII), uptake, storage and release of calcium ions, cellular proliferation regulation, and other metabolic tasks.

[0066] In one aspect, the present disclosure provides an engineered mitochondrion comprising one or more modified phospholipids on the mitochondrial membrane (MM) of the mitochondria. In some embodiments, the one or more modified phospholipids are on the outer mitochondrial membrane (OMM). In some embodiments, the one or more modified phospholipids are on the inner mitochondrial membrane (IMM). In some embodiments, the one or more modified phospholipids are on both the OMM and IMM of the engineered mitochondrion. Since some of the phospholipids found on the OMM are also present in the IMM, the antibodies or other homing molecules can also target the IMM for drug or molecule delivery via transient modifications of shared phospholipids. As disclosed herein, targeting the IMM for drug or molecule delivery requires specific conditions that allow for IMMaccessibility without complete mitochondrial disruption. Since the IMM is highly impermeable to most molecules, modifications or drug delivery into the IMM require specialized techniques. Transient permeabilization methods, such as detergents (e.g., saponin, digitonin) or mitochondrial stress inducers, could provide temporary access to the IMM without destroying mitochondrial function. The nonlimiting mitochondrial permeabilization strategies include a. Detergent Permeabilization: Saponin or digitonin selectively permeabilizes the OMM, enabling molecules to enter the intermembrane space and potentially reach the IMM; b. Mitochondrial Stress-Induced Porosity: Certain conditions (e.g., mitochondrial fission, mitophagy signaling) can transiently expose the IMM; c. Use of Rho-Mitochondria as Vesicles. As disclosed herein, (rho-zero) mitochondria lack mtDNA and functional OxPhos complexes. These mitochondria could be permeabilized and loaded with drugs, essentially functioning as a novel mitochondrial-derived nanoparticle delivery system. In addition, synthetic mtDNA can be loaded in these rho-zero mitochondria for delivery to different cells.

[0067] In some embodiments, the engineered mitochondrion comprises one or more modified phospholipids on both the OMM and IMM. For example, the engineered mitochondrion comprises one or more modified phospholipids on the IMM tagged with a therapeutic payload and one or more modified phospholipids on the OMM tagged with an antibody.

[0068] Tagging the IMM with therapeutic payloads allows for the precise conjugation of drugs to mitochondria. In some embodiments, the therapeutic payload tagged to the modified phospholipids on the IMM can be a bio-orthogonal drug or gene therapy agent. Non-limiting examples of therapeutic payloads include, coenzyme Q10 (CoQ10), Mito-TEMPO, and CRISPR / Cas9-mRNA. Coenzyme Q10 (CoQ10), an antioxidant agent, can be utilized to support mitochondrial bioenergetics when loaded into mitochondria. Mito-TEMPO, a cell- permeable antioxidant specifically targeted to mitochondria, is effective in eliminating mitochondrial superoxide and can be used to protect against oxidative stress when loaded into mitochondria. Additionally, CRISPR / Cas9-mRNA can be conjugated to the modified phospholipids on the IMM via click chemistry, enabling mitochondrial genome editing.

[0069] Tagging the OMM with antibodies enables the specific delivery of the drug-loaded mitochondria to target cells, thereby facilitating internalization of the drug-loaded mitochondria by the target cells. In some embodiments, the antibody attached to the modified phospholipids on the OMM selectively binds to a transmembrane protein on the recipient cell.Once these drug-loaded mitochondria are introduced into a cell or system, they integrate with the recipient cell’s endogenous mitochondrial network through fusion. This approach enables highly specific mitochondrial drug delivery while maintaining cellular specificity, as targeting moieties on the OMM ensure selective uptake by the intended cell types.

[0070] In some embodiments, the modified phospholipids are azide-modified phospholipids. In some embodiments, the modified phospholipids are alkyne-modified phospholipids. In some embodiments, the modified phospholipids are a combination of azide- and alkyne- modified phospholipids. In some embodiments, the modified phospholipids are derived from phosphatidylcholines (PCs). In some embodiments, the modified phospholipids are derived from phosphatidylethanolamines (PEs). In some embodiments, the modified phospholipids are derived from phosphatidylserines (PSs). In some embodiments, the modified phospholipids are derived from phosphatidylinositol (PLs). In some embodiments, the modified phospholipids are derived from phosphatidic acids (PAs). In some embodiments, the modified phospholipids are derived from cardiolipins (CLs). In some embodiments, the modified phospholipids are derived from sphingomyelins (SMs). In some embodiments, the modified phospholipids are derived from any combination of PCs, Pes, PSs, PLs, PAs, CLs, and SMs. Unlimited examples of the modified phospholipids include azido-ethyl-phosphocholine (Az-Cho), propargyl choline (P-Cho), and any variant thereof. As disclosed herein, the engineered mitochondrion is a functional mitochondrion. In some embodiments, the engineered mitochondrion comprises a mitochondrial membrane potential. The term “mitochondrial membrane potential” or “membrane potential” refers to an electrical potential difference across the inner membrane of mitochondria generated by the electron transport chain (ETC). A healthy / functional mitochondrion (e.g., the engineered mitochondrion) has a detectable membrane potential, often visualized or quantified using mitochondrial membrane potential sensitive dyes, such as mito- tracker deep red.

[0071] In some embodiments, the engineered mitochondrion disclosed herein links to an antibody either covalently or non-covalently. In some embodiments, the engineered mitochondrion links to an antibody and / or a therapeutic payload through the modified phospholipids disclosed herein. In some embodiments, the engineered mitochondrion links to the antibody and / or the therapeutic payload via an azide-alkyne cycloaddition reaction. In some embodiments, the antibody specifically binds to a transmembrane protein of a cell, thereby delivering the linked mitochondrion close to the cell membrane and intake themitochondrion inside of the cell. More description of anti-transmembrane antibodies is disclosed below in next section. B. Antibody-mitochondrion conjugates (AMCs)

[0072] In another aspect, the present disclosure provides an antibody-mitochondrion conjugate (AMC, or Ab-MC) comprising an antibody and a mitochondrion. As disclosed herein, the AMC comprises a functional mitochondrion. In some embodiments, the AMC comprises a mitochondrial membrane potential. In some embodiments, the antibody and the mitochondrion of the AMC are covalently or non-covalently linked. In some embodiments, the antibody is non-covalently linked to the mitochondrion. For example, in some embodiments, the antibody is attached to a biotin moiety, and the mitochondrion is attached to a streptavidin, thereby the antibody and the mitochondrion is non-covalently linked. In some embodiments, the antibody is covalently linked to the mitochondrion. In particular embodiments, the antibody is linked to the mitochondrion through a linker. Antibodies

[0073] As disclosed herein, any antibodies that binds to a surface protein / epitope / moiety / molecule of target cells / tissue / organ can be used to generate antibody- mitochondrion conjugates. Potential antibodies for antibody-mitochondrion conjugates can recognize and bind to a target listed, but are not limited to, in Table 1. Table 1. List of Antibody Targets

[0074] In some embodiments, the antibody specifically binds to a transmembrane protein of a cell, thereby delivering the linked mitochondrion close to the cell membrane and intake the mitochondrion inside of the cell. The term “transmembrane protein” refers to a type of integral membrane protein that spans the entirety of the cell membrane. Many transmembrane proteins function as gateways to permit the transport of specific substances across the membrane. Transmembrane proteins can be classified by structure as alpha-helical or beta barrels transmembrane proteins, or by the position of the protein N- and C-termini on the different sides of the lipid bilayer as types I, II, III or IV transmembrane proteins. As disclosed herein, any transmembrane protein facilitates intracellular delivery of biological materials can be chosen as the target antigen of the antibody.

[0075] In some embodiments, the transmembrane protein is intercellular adhesion molecule type 1 (ICAM-1). ICAM-1 (Intercellular Adhesion Molecule 1) also known as CD54 (Cluster of Differentiation 54) is a transmembrane protein possessing an amino-terminus extracellular domain, a single transmembrane domain, and a carboxy-terminus cytoplasmic domain. ICAM- 1 is a member of the immunoglobulin superfamily, the superfamily of proteins including antibodies and T-cell receptors. ICAM-1 plays an important role in stabilizing cell-cell interactions and facilitating leukocyte endothelial transmigration. In addition, ICAM-1 binds to macrophage adhesion ligand-1 (Mac-1; ITGB2 / ITGAM), leukocyte function associated antigen-1 (LFA-1), and fibrinogen. These three proteins are generally expressed on endothelial cells and / or leukocytes, and they bind to ICAM-1 to facilitate transmigration of leukocytes across vascular endothelia in processes such as extravasation and the inflammatory response. Therefore, ICAM-1 plays a key role in intercellular adhesion. As disclosed herein, an engineered mitochondrion or an antibody-mitochondrion comprises a binding fragment or an antibody which recognizes and binds to ICAM-I on a cell (e.g., an endothelial cell), the engineered mitochondrion or the antibody-mitochondrion conjugate can be easily delivered to the cell (e.g., an endothelial cell).

[0076] In some embodiments, the transmembrane protein is lymphocyte function-associated antigen 1 (LFA-1, also known as ITGAL or CD11a). LFA-1 belongs to the integrin superfamily of adhesion molecules, involved in cellular adhesion and costimulatory signaling. LFA-1 plays a key role in leukocyte emigration from the bloodstream into the tissues and mediating firm arrest of leukocytes. Additionally, LFA-1 is involved in the process of cytotoxic T cell mediated killing and antibody-mediated killing by granulocytes and monocytes. LFA-1 can be found on all immune cells and expressed on immune cell precursors,such as hematopoietic stem cells. LFA-1 has five well-established ligands: ICAM-1, ICAM- 2, ICAM-3, ICAM-4, and ICAM-5. LFA-1 and ICAM-1 interactions have been shown to stimulate signaling pathways that influence T cell differentiation. As disclosed herein, an engineered mitochondrion or an antibody-mitochondrion comprises a binding fragment or an antibody which recognizes and binds to LFA-1 on a cell (e.g., an endothelial cell), the engineered mitochondrion or the antibody-mitochondrion conjugate can be easily delivered to the cell (e.g., an endothelial cell).

[0077] In some embodiments, the transmembrane protein is platelet endothelial cell adhesion molecule-1 (PECAM-1). PECAM-1, also known as cluster of differentiation 31 (CD31), is a transmembrane glycoprotein composed of an N-terminal extracellular domain (574 amino acids), a transmembrane domain (19 amino acids), and a C-terminal cytoplasmic domain (118 amino acids). The extracellular domain contains six Ig-like domains, which facilitate homophilic interactions with other PECAM-1 molecules and heterophilic interactions with non-PECAM-1 molecules. PECAM-1 is normally found on endothelial cells, platelets, macrophages and Kupffer cells, granulocytes, lymphocytes (T cells, B cells, and NK cells), megakaryocytes, and osteoclasts, and plays an important role on endothelial cell intercellular junctions. As disclosed herein, an engineered mitochondrion or an antibody-mitochondrion comprises a binding fragment or an antibody which recognizes and binds to PECAM-I on a cell (e.g., an endothelial cell), the engineered mitochondrion or the antibody-mitochondrion conjugate can be easily delivered to the cell (e.g., an endothelial cell). Linkers

[0078] As disclosed herein, the mitochondrion may be linked to the anti-transmembrane protein antibody via a linker. Any linkers known in the art good for antibody-drug conjugates (ADCs) can be also useful in the present disclosure. Non-limiting exemplary linkers are listed in Table 2. Table 2. List of linkers

[0079] In some embodiments, the linker is a click chemistry linker. As disclosed herein, any linker that undergoes a click chemistry reaction can be applied to link the antibody and the mitochondrion. In some embodiments, the linker comprises a bioorthogonal functional group, including but not limited to azide-modified phospholipids, alkyne-modified phospholipids, tetrazine-functionalized lipids, thiol-reactive linkers, or oxime-based ligation systems. In some embodiments, the linker is derived from phospholipids, polymers, peptides, or other biocompatible linker materials. In some embodiments, the linker comprises an azide-modified phospholipid. In some embodiments, the linker comprises an alkyne-modified phospholipid.In some embodiments, the linker comprises both an azide-modified phospholipid and an alkyne-modified phospholipid. In certain embodiments, the mitochondrion comprises one or more azide-modified phospholipids and the anti-transmembrane protein antibody comprises one or more alkyne-modified phospholipids, thereby the mitochondrion and antibody are conjugated through an azide-alkyne cycloaddition reaction. In other embodiments, the mitochondrion comprises one or more alkyne-modified phospholipids and the anti- transmembrane protein antibody comprises one or more azide-modified phospholipids, thereby the mitochondrion and antibody are conjugated through an azide-alkyne cycloaddition reaction. In yet other embodiments, both the mitochondrion and antibody comprise a mix of azide-and alkyne-modified phospholipids, thereby the mitochondrion and antibody are conjugated through an azide-alkyne cycloaddition reaction. In such embodiments, the linker comprises both azide-and alkyne-modified phospholipids (e.g., Az-Cho and P-Cho).

[0080] Other linkers known in the art good for antibody-drug conjugates (ADCs) can be also useful in the present disclosure. Such linkers can be up to 30 carbon atoms in length. For example, the linkers can each independently be from 5 to 20 carbon atoms in length. The types of bonds used to link the linker to the cytotoxic agent and antibody of the present disclosure include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonate and thioureas.

[0081] Attachment of a linker to an antibody can be accomplished in a variety of ways, such as through surface lysines on the antibody, reductive coupling to oxidized carbohydrates on the antibody, or through cysteine residues on the antibody liberated by reducing interchain disulfide linkages. Alternatively, attachment of a linker to an antibody may be achieved by modification of the antibody to include additional cysteine residues (see, for example, U.S. Patent Nos. 7,521,541; 8,455,622 and 9,000,130) or non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine or p- azidomethyl-L-phenylalanine (see, for example, Hofer et al., Biochemistry, 48:12047-12057 (2009); Axup et al., PNAS, 109:16101-16106 (2012); Wu et al., PNAS, 106:3000-3005 (2009); Zimmerman et al., Bioconj. Chem., 25:351-361 (2014)), to allow for site-specific conjugation.

[0082] A linker may comprise one or more linker components. Typically, a linker will comprise two or more linker components. Exemplary linker components include functional groups for reaction with the antibody, functional groups for reaction with the drug, stretchers, peptide components, self-immolative groups, self-elimination groups, hydrophilic moieties,and the like. Various linker components are known in the art, some of which are described below.

[0083] Certain useful linker components can be obtained from various commercial sources, such as Pierce Biotechnology, Inc. (now Thermo Fisher Scientific Corporation, Waltham, MA) and Molecular Biosciences Inc. (Boulder, Colo.), or may be synthesized in accordance with procedures described in the art (see, for example, Toki et al., J. Org. Chem., 67:1866-1872 (2002); Dubowchik et al., Tetrahedron Letters, 38:5257-60 (1997); Walker, M. A., J. Org. Chem., 60:5352-5355 (1995); Frisch et al., Bioconjugate Chem., 7:180-186 (1996); U.S. Patent Nos. 6,214,345 and 7,553,816, and International Patent Application Publication No. WO 02 / 088172).

[0084] In certain embodiments, the linker is a cleavable linker. Suitable cleavable linkers include, for example, linkers comprising a peptide component that includes two or more amino acids and is cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. A peptide component may comprise amino acid residues that occur naturally and / or minor amino acids and / or non-naturally occurring amino acid analogues, such as citrulline. Peptide components may be designed and optimized for enzymatic cleavage by an enzyme, for example, a tumor-associated protease, cathepsin B, C or D, or a plasmin protease.

[0085] In certain embodiments, the linker comprised by the AMCs may be a peptide- containing linker. In some embodiments, the linker comprised by the AMCs may be a dipeptide-containing linker, such as a linker containing valine-citrulline (Val-Cit) or phenylalanine-lysine (Phe-Lys). Other examples of suitable dipeptides for inclusion in the linker include Val-Lys, Ala-Lys, Me-Val-Cit, Phe-homoLys, Phe-Cit, Leu-Cit, Ile-Cit, Trp- Cit, Phe-Arg, Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met- (D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me3Lys-Pro, PhenylGly- (D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys and Met-(D)Lys. Cleavable linkers may also include longer peptide components such as tripeptides, tetrapeptides or pentapeptides. Examples include, but are not limited to, the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe- Phe-Lys and (D)Ala-Phe-Lys, and the tetrapeptides Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Ala-Leu-Ala-Leu. In some embodiments, the linker comprised by the AMCs may be a peptide- containing linker, where the peptide is between two and five amino acids in length, for example, between two and four amino acids in length.

[0086] Additional examples of cleavable linkers include disulfide-containing linkers, such as, N-succinimydyl-4-(2-pyridyldithio) butanoate (SPBD) and N-succinimydyl-4-(2- pyridyldithio)-2-sulfo butanoate (sulfo-SPBD). Disulfide-containing linkers may optionally include additional groups to provide steric hindrance adjacent to the disulfide bond to improve the extracellular stability of the linker, for example, inclusion of a geminal dimethyl group. Other suitable linkers include linkers hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Linkers comprising combinations of these functionalities may also be useful, for example, linkers comprising both a hydrazone and a disulfide are known in the art.

[0087] A further example of a cleavable linker is a linker comprising a -glucuronide, which is cleavable by -glucuronidase, an enzyme presents in lysosomes and tumor interstitium (see, for example, De Graaf et al., Curr. Pharm. Des., 8:1391–1403 (2002)).

[0088] Cleavable linkers may optionally further comprise one or more additional components such as self-immolative and self-elimination groups, stretchers or hydrophilic moieties.

[0089] Self-immolative and self-elimination groups that find use in linkers include, for example, p-aminobenzyloxycarbonyl (PABC) and p-aminobenzyl ether (PABE) groups, and methylated ethylene diamine (MED). Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PABC or PABE group such as heterocyclic derivatives, for example 2-aminoimidazol-5-methanol derivatives as described in U.S. Patent No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 2:223-227 (1995)) and 2- aminophenylpropionic acid amides (Amsberry et al., J. Org. Chem., 55:5867-5877 (1990)).

[0090] Stretchers that find use in linkers for AMCs include, for example, alkylene groups and stretchers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and caproamide. Other stretchers include, for example, glycine-based stretchers, polyethylene glycol (PEG) stretchers and monomethoxy polyethylene glycol (mPEG) stretchers. PEG and mPEG stretchers also function as hydrophilic moieties.

[0091] Examples of components commonly found in cleavable linkers include, but are not limited to, SPBD, sulfo-SPBD, hydrazone, Val-Cit, maleidocaproyl (MC), MC-Val-Cit, MC- Val-Cit-PABC, Phe-Lys, MC-Phe-Lys, MC-Phe-Lys-PABC, maleimido triethylene glycolate (MT), MT-Val-Cit, MT-Phe-Lys, TFP and adipate (AD).C. Click Chemistry (CC)

[0092] As disclosed herein, the engineered mitochondria can be linked to an antibody via Click Chemistry (CC). The term “Click Chemistry” or “CC” refers to well-known, selective methods of conjugation, wherein two components comprising a click reactive functional group are reacted to link the two components. For example, for the antibody-mitochondrion conjugates (AMCs) as described herein, the antibody comprises a first click reactive functional group (such as an azide-modified phospholipid), and the mitochondrion is suitably modified to comprise a second click reactive functional group (such as an alkyne-modified phospholipid), which is reactive with the first click reactive functional group (such as an azide-modified phospholipid). In some embodiments, the antibody and the mitochondrion are covalently linked via a click chemistry reaction, including but not limited to azide-alkyne cycloaddition, tetrazine ligation, thiol-maleimide coupling, oxime ligation, or sulfur fluoride exchange.

[0093] The click reactive functional group includes, without limitation, an azide group, a nitrone group or an alkyne group. In some embodiments click chemistry comprises reaction of an azide group with an alkyne group to form a triazole group linking the two components, or the reaction of a nitrone group with an alkyne group to form an isoxazoline group linking the two components. In some embodiments, the alkyne group is a dibenzocyclooctyne (DBCO) group or a difluorooctyne (DIFO) group. In some embodiments, the click chemistry is Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC) or strain-promoted alkyne-nitrone cycloaddition (SPANC). See also Jewett, John C. and Bertozzi, Carolyn, R., Cu-free click cycloaddition reactions in chemical biology. Chem Soc Rev. 39(4), 1272-1279 (2010); Agard et al., A Comparative Study of Bioorthogonal Reactions with Azides. ACS Chem. Biol., 1(10), 644-648 (2006); MacKenzie et al., Strain-promoted cycloadditions involving nitrones and alkynes—rapid tunable reactions for biorthogonal labeling. Current Opinion in Chemical Biology 21, 81-88 (2014), the disclosures of which are hereby incorporated by reference in their entirety. D. Target Cells

[0094] As disclosed herein, the engineered mitochondria and / or the antibody-mitochondrion conjugates can be delivered to a target cell. These taggable mitochondria can be delivered to any cell of the subject in need (such as a patient undergoing mitochondrial dysfunction). Non- limiting exemplary target cells are listed in Table 3.Table 3. Target Cells for engineered mitochondria and / or the antibody-mitochondrion conjugates

[0095] In some embodiments, the engineered mitochondrion or the antibody-mitochondrion conjugate is delivered to a vascular cell. In some embodiments, the vascular cell is selected from the group consisting of an endothelial cell (EC), a vascular smooth muscle cell (VSMC), a smooth muscle cell, a cardiomyocyte, a pericyte, a fibroblast, a cardiac fibroblast, an endothelial progenitor cell, a pacemaker cell, and an adventitial cell. In some embodiments, the engineered mitochondrion or the antibody-mitochondrion conjugate is delivered to an immune cell. In some embodiments, the immune cell is selected from the group consisting ofa macrophage (M ), a T cell, a B cell, a dendritic cell (DC), a NK cell, a neutrophils, aeosinophil, a basophil, a mast cell, a helper t cell (th1, th2, th17), a regulatory t cell (treg), a memory B cell, a plasma cell, a follicular dendritic cell, a monocyte, an innate lymphoid cell(ilc), and a gamma delta t cell ( t cell).E. Target Diseases

[0096] As disclosed herein, the engineered mitochondria and / or the antibody-mitochondrion conjugates can be delivered to a subject in need for disease treatment. For examples, a potential primary application could be in creating specialized therapeutic agents that replace dysfunctional mitochondria in patients with cardiovascular diseases (atherosclerosis, endothelial dysfunction, vascular degeneration), immune dysfunctions (T cell exhaustion associated with cancer or chronic infections, immune cell senescence), and aging-associatedabnormalities. The engineered mitochondria and / or the antibody-mitochondrion conjugates disclosed herein can be used to prevent and treat any pathology or dysfunction that impacts mitochondrial health / function. This includes, but not limited to, aging associated conditions and conditions associated with radiation damage (cancer therapy, space travel), conditions that induce persistent and chronic inflammation including viral and non-viral infections. Additionally, this may be useful for treating combat-related injuries or as a means to enhance recovery. Non-limiting exemplary target diseases are listed below, as breakdown by systems of the body.

[0097] Cardiovascular: coronary artery disease (CAD), cardiomyopathies such as dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), heart failure (HF) including both systolic and diastolic heart failure, ischemic heart disease, myocardial infarction (MI), arrhythmias, atherosclerosis, peripheral artery disease (PAD), stroke due to vascular dysfunction, hypertension, pulmonary arterial hypertension (PAH), diabetic cardiomyopathy, congenital heart defects, myocarditis, endocarditis, pericarditis, congenital heart defects that affect mitochondrial integrity, and metabolic syndrome, which is closely linked to cardiovascular health through mitochondrial pathways.

[0098] Nervous: Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), epilepsy, stroke, migraine, neuropathies (such as diabetic neuropathy), spinal muscular atrophy, Friedreich's ataxia, Charcot-Marie-Tooth disease, traumatic brain injury (TBI), Leber's hereditary optic neuropathy (LHON), Kearns-Sayre syndrome, autism spectrum disorders (ASD), bipolar disorder, schizophrenia, major depressive disorder, dystonia, ataxias beyond Friedreich's ataxia, Rett syndrome, Wilson's disease, and Creutzfeldt-Jakob disease, neurofibromatosis, tuberous sclerosis, and Lewy body dementia, optic neuropathies beyond Leber's hereditary optic neuropathy, such as dominant optic atrophy.

[0099] Immune: Rheumatoid arthritis, Systemic lupus erythematosus (SLE), Multiple sclerosis (MS), Type 1 diabetes mellitus, Inflammatory bowel disease (IBD) encompassing Crohn's disease and ulcerative colitis, Psoriasis, Scleroderma (systemic sclerosis), Sjögren's syndrome, Myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Addison's disease, Celiac disease, Guillain-Barré syndrome, Autoimmune hepatitis, Vitiligo, Pernicious anemia, Ankylosing spondylitis, Alopecia areata, Antiphospholipid syndrome, Primary biliarycholangitis, Dermatomyositis, Polymyositis, Wegener's granulomatosis (Granulomatosis with polyangiitis), and Chronic fatigue syndrome (CFS), COVID-19.

[0100] Respiratory: Chronic Obstructive Pulmonary Disease (COPD), Asthma, Pulmonary Fibrosis, Pulmonary Hypertension, Cystic Fibrosis, Acute Respiratory Distress Syndrome (ARDS), Lung Cancer, Bronchiectasis, Respiratory Syncytial Virus (RSV) infection, and Tuberculosis.

[0101] Digestive: Crohn's Disease, Ulcerative Colitis, Hepatic Steatosis (Non-Alcoholic Fatty Liver Disease), Cirrhosis, Pancreatitis, Colorectal Cancer, Gastroesophageal Reflux Disease (GERD), Celiac Disease, Gastric Cancer, and irritable bowel syndrome (IBS).

[0102] Endocrine: Type 1 and Type 2 Diabetes Mellitus, Thyroid Disorders (such as Hashimoto's Thyroiditis and Graves' Disease), Adrenal Insufficiency, Polycystic Ovary Syndrome (PCOS), Obesity, Metabolic Syndrome, Osteoporosis, Addison's Disease, and Pituitary Adenomas, affecting hormone production, regulation, and energy metabolism.

[0103] Musculoskeletal: Osteoarthritis, Rheumatoid Arthritis, Muscular Dystrophies, Fibromyalgia, Osteoporosis, Sarcopenia (age-related muscle loss), Lupus Erythematosus, Ankylosing Spondylitis, Paget's Disease of Bone, and Tendinopathy.

[0104] Skin: Psoriasis, Atopic Dermatitis, Vitiligo, Skin Cancer (including Melanoma and Non-Melanoma Skin Cancers), Rosacea, Photoaging, Scleroderma, Lupus Erythematosus, and Acne, affecting skin cell renewal, pigmentation.

[0105] Reproductive: Endometriosis, Polycystic Ovary Syndrome (PCOS), Erectile Dysfunction, Ovarian Cancer, Premature Ovarian Failure, Preeclampsia, Miscarriage, and Testicular Cancer.

[0106] Urinary: Chronic Kidney Disease (CKD), Acute Kidney Injury (AKI), Polycystic Kidney Disease, Bladder Dysfunction, Renal Cell Carcinoma, Nephrolithiasis (kidney stones), and Interstitial Cystitis.

[0107] Sensory: Age-related Macular Degeneration (AMD), Glaucoma, Diabetic Retinopathy, Cataracts, Hearing Loss (including age-related hearing loss), Usher Syndrome, Retinitis Pigmentosa, and Olfactory Dysfunction.F. Compositions

[0108] In one aspect, the present disclosure provides a composition comprising the engineered mitochondrion or the antibody-mitochondrion conjugate described herein. In some embodiments, the engineered mitochondrion comprising one or more modified phospholipids on the uter mitochondrial membrane (MM) of the mitochondrion, wherein the modified phospholipids comprise azide-modified phospholipids and / or alkyne-modified phospholipids. In some embodiments, the mitochondrial membrane (MM) is the outer mitochondrial membrane (OMM) or the inner mitochondrial membrane (IMM). In some embodiments, the mitochondrial membrane (MM) is a combination of the OMM and the IMM. In some embodiments, the antibody-mitochondrion conjugate, comprising an antibody and a mitochondrion, wherein the antibody and the mitochondrion are covalently linked through a linker. In some embodiments, the antibody and the mitochondrion are covalently linked via an azide-alkyne cycloaddition reaction..

[0109] In another aspect, the present disclosure provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the compositions described herein and a pharmaceutically acceptable carrier. For example, the pharmaceutical composition may comprise an engineered mitochondrion comprising one or more modified phospholipids on the outer mitochondrial membrane (OMM) and / or inner mitochondrial membrane (IMM) of the mitochondrion, wherein the modified phospholipids comprise azide- modified phospholipids and / or alkyne-modified phospholipids. Further, the engineered mitochondrion is linked to an antibody through the modified phospholipids.

[0110] The pharmaceutical compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the disease or symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” A therapeutically effective dose is determined by monitoring a patient’s response to therapy. Typical benchmarks indicative of a therapeutically effective dose includes the amelioration of symptoms of the disease in the patient. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient’s health, including other factors such as age, weight, gender, administration route, and the like single or multiple administrations of the engineered mitochondrion or the antibody-mitochondrion conjugate may be administered depending on the dosage and frequency as required and tolerated by thepatient. In any event, the methods provide a sufficient quantity of the engineered mitochondrion or the antibody-mitochondrion conjugate to effectively treat the patient.

[0111] The pharmaceutical composition can be administered by any suitable means, including, for example, parenteral, intrapulmonary, and intranasal, administration, as well as local administration, such as intratumor administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the composition may be administered by insufflation. In an illustrative embodiment, the composition may be stored at 10 mg / ml in sterile isotonic aqueous saline solution for injection at 4°C and is diluted in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient. In some embodiments, the composition is administered by intravenous infusion over the course of 1 hour at a dose of between 0.01 and 25 mg / kg. In other embodiments, the composition is administered by intravenous infusion over a period of between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via sub-cutaneous bolus injection.

[0112] The dose of composition is chosen to provide effective therapy for the patient and is in the range of less than 0.01 mg / kg body weight to about 25 mg / kg body weight or in the range 1 mg – 2 g per patient. Preferably the dose is in the range 0.1 – 10 mg / kg or approximately 50 mg – 1000 mg / patient. The dose may be repeated at an appropriate frequency which may be in the range once per day to once every three months, or every six months, depending on the pharmacokinetics of the composition (e.g., half-life of the composition in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect of the composition). In some embodiments, the in vivo half-life of between about 7 and about 25 days and composition dosing is repeated between once per week and once every 3 months or once every 6 months. In other embodiments, the composition is administered approximately once per month.

[0113] In an illustrative embodiment, the composition may be stored at 10 mg / ml or 20 mg / ml in a sterile isotonic aqueous solution. The solution can comprise agents such as buffering agents and stabilizing agents. For example, in some embodiments, a buffering agent such as histidine is included to maintain a formulation pH of about 5.5. Additional reagents such as sucrose or alternatives can be added to prevent aggregation and fragmentation in solution and during freezing and thawing. Agents such as polysorbate 80 or an alternative can be included to lower surface tension and stabilizes the composition against agitation-induced denaturationand air-liquid and ice-liquid surface denaturation. In some embodiments, the solution for injection is stored at 4°C and is diluted in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient.

[0114] In another aspect, the present disclosure provides a kit for treating a subject comprising the pharmaceutical composition described herein. In some embodiments, the subject has a cardiovascular disease (CVD) or mitochondrial dysfunction. In some embodiments, the kit comprises an engineered mitochondrion, an antibody-mitochondrion conjugate, a composition, and / or a pharmaceutical composition of the present disclosure described herein. The kits are useful for treating any diseases that impacts mitochondrial health / dysfunction. This includes, but not limited to, aging associated conditions and conditions associated with radiation damage (cancer therapy, space travel), conditions that induce persistent and chronic inflammation including viral and non-viral infections. Additionally, this may be useful for injuries obtained during combat / etc or used as a recovery boost.

[0115] Materials and reagents to carry out the various methods of the present disclosure can be provided in kits to facilitate execution of the methods. As used herein, the term “kit” includes a combination of articles that facilitates a process, assay, analysis, or manipulation. In particular, kits of the present disclosure find utility in a wide range of applications including, for example, diagnostics, prognostics, therapy, and the like.

[0116] Kits can contain chemical reagents as well as other components. In addition, the kits of the present disclosure can include, without limitation, instructions to the kit user, apparatus and reagents for sample collection and / or purification, apparatus and reagents for product collection and / or purification, apparatus and reagents for administering the engineered mitochondria or the antibody-mitochondrion conjugates or other composition(s) of the present disclosure, apparatus and reagents for determining the level(s) of biomarker(s) and / or the activity and / or number of immune cells, apparatus and reagents for detecting diseases, sample tubes, holders, trays, racks, dishes, plates, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and / or control samples. Kits of the present disclosure can also be packaged for convenient storage and safe shipping, for example, in a box having a lid. For instance, the kits may be stored and shipped at room temperature, on wet ice or with cold packs, or frozen in the vapor phase of liquid nitrogen or in dry ice.G. Methods for Treatment

[0117] In one aspect, the present disclosure provides a method of treating a cardiovascular disease (CVD) in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. In some embodiment, the pharmaceutical composition comprises an engineered mitochondrion comprising one or more modified phospholipids on the outer mitochondrial membrane (OMM) and / or the inner mitochondrial membrane (IMM) of the mitochondrion, wherein the modified phospholipids comprise azide-modified phospholipids and / or alkyne-modified phospholipids. In some embodiments, the pharmaceutical composition comprises an antibody-mitochondrion conjugate, comprising an antibody and a mitochondrion. In some embodiments, the antibody and the mitochondrion are covalently linked through a linker. In some embodiments, the antibody and the mitochondrion are covalently linked via an azide-alkyne cycloaddition reaction.

[0118] In another aspect, the present disclosure provides a method of treating mitochondrial dysfunction in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. In some embodiment, the pharmaceutical composition comprises an engineered mitochondrion comprising one or more modified phospholipids on the outer mitochondrial membrane (OMM) and / or the inner mitochondrial membrane (IMM) of the mitochondrion, wherein the modified phospholipids comprise azide-modified phospholipids and / or alkyne-modified phospholipids. In some embodiments, the pharmaceutical composition comprises an antibody-mitochondrion conjugate, comprising an antibody and a mitochondrion. In some embodiments, the antibody and the mitochondrion are covalently linked through a linker. In some embodiments, the antibody and the mitochondrion are covalently linked via an azide-alkyne cycloaddition reaction.

[0119] As disclosed herein, administering a pharmaceutical composition comprising an engineered mitochondrion or antibody-mitochondrion conjugate can reduce reactive oxygenspecies (ROS) levels and alleviate oxidative stress (e.g., reduce IFN -induced mitochondrialsuperoxide production). In some embodiments, the administration of the pharmaceutical composition disclosed herein can further enhance glycolysis and promote endothelial regeneration. In some embodiments, the administration of the pharmaceutical composition disclosed herein modulates cellular energy metabolism, reprogram metabolism to meet energydemands, and / or restore mitochondrial function in cells with compromised bioenergetics. This treatment is particularly beneficial for subjects suffering from inflammation-driven pathologies and / or age-related mitochondrial disfunctions. In some embodiments, the engineered mitochondrion or antibody-mitochondrion conjugate disclosed herein can be used to prevent or treat inflammatory conditions. In some embodiments, the engineered mitochondrion or antibody-mitochondrion conjugate disclosed herein can be used to promote endothelial regeneration or restore cellular mitochondrial function with compromised bioenergetics in aged subjects. In some embodiments, the engineered mitochondrion or antibody-mitochondrion conjugate is an anti-PECAM-1 antibody conjugated with a mitochondrion, useful for treating inflammation-driven pathologies and / or mitochondrial disfunctions in a subject. In some embodiments, the subject is an aged individual.

[0120] In some embodiments, the engineered mitochondrion or antibody-mitochondrion conjugate can be used to deliver mitochondria specifically to immune cells, offering potential applications in immunomodulation by enhancing immune cell function, regulating inflammatory responses, and / or restoring cellular energy balance. In some embodiments, the antibody-mitochondrion conjugate is an anti-LFA-1 antibody conjugated with a mitochondrion, useful for treating immune dysfunctions in a subject. In some embodiments, the subject is an aged individual. H. Methods for Delivery Mitochondria to Cells

[0121] In one aspect, the present disclosure provides a method of delivering a mitochondrion to a cell, comprising: a) generating an antibody-mitochondrion conjugate by covalently linking the mitochondrion to an antibody, wherein the antibody recognizes and binds to a cell surface protein of the cell; and b) contacting the cell with the antibody-mitochondrion conjugate, wherein the antibody binds to the cell surface protein of the cell, thereby delivering the mitochondrion to the cell.

[0122] In some embodiment, the antibody and the mitochondrion are covalently linked through a linker. In some embodiment, the linker comprises an azide-modified phospholipid and / or an alkyne-modified phospholipid. In some embodiment, the azide-modified phospholipid comprises azido-ethyl-phosphocholine (Az-Cho), azide-modified phosphatidylethanolamine (Az-PE), azide-modified phosphatidylserine (Az-PS), azide- modified phosphatidylinositol (Az-PI), azide-modified phosphatidic acid (Az-PA), azide- modified cardiolipin (Az-CL), azide-modified sphingomyelin (Az-SM), and / or any variantthereof. In some embodiment, the alkyne-modified phospholipid comprises propargyl choline (P-Cho), alkyne-modified phosphatidylethanolamine (Ak-PE), alkyne-modified phosphatidylserine (Ak-PS), alkyne-modified phosphatidylinositol (Ak-PI), alkyne-modified phosphatidic acid (Ak-PA), alkyne-modified cardiolipin (Ak-CL), alkyne-modified sphingomyelin (Ak-SM), and / or any variant thereof. In some embodiment, the antibody and the mitochondrion are covalently linked via an azide-alkyne cycloaddition reaction.

[0123] In some embodiments, the cell surface protein is selected from the antibody targets listed in Table 1. In some embodiments, the cell surface protein is a transmembrane protein. In some embodiment, the transmembrane protein is intercellular adhesion molecule type 1 (ICAM-1). In some embodiments, the transmembrane protein is lymphocyte function- associated antigen 1 (LFA-1). In some embodiment, the transmembrane glycoprotein is platelet endothelial cell adhesion molecule type 1 (PECAM-1).

[0124] In some embodiments, the cell is selected from the target cells listed in Table 3. In some embodiment, the cell is a vascular cell. In some embodiments, the vascular cell is selected from the group consisting of an endothelial cell (EC), a vascular smooth muscle cell (VSMC), a smooth muscle cell, a cardiomyocyte, a pericyte, a fibroblast, a cardiac fibroblast, an endothelial progenitor cell, a pacemaker cell, and an adventitial cell. In some embodiment, the vascular cell is an endothelial cell (EC) or a vascular smooth muscle cell (VSMC). In some embodiment, the cell is an immune cell. In some embodiments, the immune cell is selectedfrom the group consisting of a macrophage (M ), a T cell, a B cell, a dendritic cell (DC), a NKcell, a neutrophils, a eosinophil, a basophil, a mast cell, a helper t cell (th1, th2, th17), a regulatory t cell (treg), a memory B cell, a plasma cell, a follicular dendritic cell, a monocyte,an innate lymphoid cell (ilc), and a gamma delta t cell ( t cell). In some embodiment, theimmune cell is a macrophage (M ), a T cell, a B cell, or a dendritic cell (DC).

[0125] As disclosed herein, the targeted delivery of the engineered mitochondrion or antibody-mitochondrion conjugate disclosed herein can improve systemic circulation and ensure precise mitochondrial targeting. In some embodiments, the engineered mitochondrion or antibody-mitochondrion conjugate can be used to deliver mitochondria specifically to immune cells, offering potential applications in immunomodulation by enhancing immune cell function, regulating inflammatory responses, and restoring cellular energy balance. In some embodiments, the engineered mitochondrion or antibody-mitochondrion conjugate can be used to deliver drugs / toxins / small molecules to induce apoptosis and / or cell death of unwanted cells,such as cancer cells or senescence cells. This targeted approach may also be effective in treating mitochondrial dysfunction associated with a wide range of pathologies, including autoimmune diseases, chronic inflammatory conditions, neurodegenerative disorders, and metabolic syndromes, thereby addressing both localized and systemic dysfunctions.

[0126] In some embodiments, an antibody-mitochondrion conjugate comprising an anti- PECAM-1 antibody bound to a mitochondrion, enables the targeted delivery of mitochondria into endothelial cells (e.g., CD31+ cells). In some embodiments, an antibody-mitochondrion conjugate comprising an anti-LFA-1 antibody bound to a mitochondrion, enables the targeted delivery of mitochondria into immune cells (e.g., CD45+ cells). IV. Examples

[0127] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Example 1: Extracellular Tagging Capacity of Bioengineered Mitochondria

[0128] This example illustrates a safe level of azide and alkyne modifications on the outer mitochondrial membrane (OMM) does not impair mitochondrial function.

[0129] Background: Click Chemistry (CC) refers to highly efficient and selective chemical reactions, notably between azide and alkyne groups, through two methods: copper-catalyzed (CuAAC) and strain-promoted (SPAAC)9. In lipid research, the introduction of alkyne and azide-modified phospholipids such as Phosphatidylcholine (PC) and Phosphatidylethanolamine (PE), facilitates the study of lipid biosynthesis kinetics, subcellular localization, and lipid-protein interactions.

[0130] Considering that the outer OMM is made up of about 40-50% PC and 25-30% PE10, it is feasible for CC reactions to occur on the OMM following the introduction of azide or alkyne-modified phospholipids. In the preliminary experiments, the inventor explored whether mitochondria with azide modified OMM could be successfully tagged with fluorescent markers, and additionally determined whether these tagged mitochondria are detectable in recipient cells after mitochondrial transplantation (mt-tfr).

[0131] In the experiments, mouse fibroblasts (MEF) were grown overnight in DMEM enriched with an azide variant of phosphocholine, specifically azido-ethyl-phosphocholine (Az-Cho; 50 M), or in standard DMEM (Wt-Cho). Mitochondria from both groups (Az-Cho- mito and Wt-Cho-mito) were then isolated and incubated in mitochondrial buffer (SHE) containing the fluorescent alkyne, DBCO-Cy5. After a 15 min incubation with DBCO-Cy5,both types of mitochondria were transplanted into RAW 264.7 M s via centrifugation. thefluorescence was then measured in the recipient M s using flow cytometry and confocalmicroscopy.

[0132] M s that received Az-Cho-mito exhibited significantly higher frequencies of DBCO-Cy5+ mitochondria (~60% vs 15% for Wt-Cho-mito), as well as a 4-fold increase in medianfluorescent intensity (MFI) of DBCO-Cy5+ M s (FIGs. 1A-F). Confocal orthogonalprojections (Z-stacks) of M s with Az-Cho-mito also confirmed the presence of the DBCO-Cy5 signal. These data collectively indicate that Az-Cho-mito can be effectively tagged extracellular, and the tags are maintained after mt-tfr. Methods:

[0133] Azide / Alkyne OMM modifications: primary mouse embryonic fibroblasts (MEF) can be cultured in DMEM supplemented with either Az-Cho or propargyl choline (P-Cho) at concentrations of 10, 50, 250, 500, and 1000 μM, or using an unsupplemented DMEM (Wt- Cho) as a control.

[0134] Impact of OMM modifications on mitochondria function: The bioenergetic profiles of Az-Cho, P-Cho, and Wt-Cho treated MEFs can be examined using extracellular flux analysis (Seahorse assay). Flow cytometry panels with specific probes and antibodies can be employed to evaluate mt-ROS (MitoSOX Red), m (JC-1, Mito-tracker Orange), and mitochondria abundance / mass (Mitoview-Green & -TOMM20, and -ATP5a). Additionally, transmission electron microscopy (TEM) can be used to observe mitochondrial cristae post-OMM modifications.

[0135] OMM tagging after azide / alkyne modifications: Following treatment, mitochondria can be isolated from the different groups using differential centrifugation and preserve them in a high-sucrose mitochondrial buffer (SHE). To demonstrate the specificity of Az-Cho-mito and P- Cho-mito for SPAAC or CuAAC reactions, the isolated mitochondria can be incubated with fluorescently tagged alkynes and azides (DBCO-Cy5 and AF488-Picolyl-Azide respectively). Antibodies with alkyne or azide tags, produced using commercial conjugation kits, can also betested. Flow cytometry can be employed to quantify the frequencies and mean fluorescent intensity (MFI) of tagged mitochondria.

[0136] Mitochondrial health after CC tagging: To ensure that the mitochondria remain functional after tagging, the oxygen consumption rate (OCR) of the tagged mitochondria can be measured using the Seahorse assay.

[0137] Expected Results: Based on previous cellular cytotoxicity studies, it can be anticipated that modifying mitochondria with Az-Cho or P-Cho will not significantly alter their function at concentrations up to 250 μM11. PC is found in smaller quantities in the inner mitochondrial membrane; at higher concentrations, these modifications might reduce oxidative phosphorylation (Ox-Phos) efficiency or affect the bioenergetic processes of mouse embryonic fibroblasts (MEFs). SPAAC reactions do not require a catalyst like CuAAC reactions; I anticipate that Az-Cho-mito will be more suitable for subsequent ex vivo and in vivo experiments.

[0138] The use of copper in CuAAC reactions may increase mt- ROS and impair mitochondrial function. Adding antioxidants to the reaction buffers may help counter this. Trying different phospholipid analogs, such those for PE, is an alternative if safe level of modifications can be attained. Example 2: Efficacy of Click Chemistry Mediated Mitochondrial Transplantation in Vascular Cell Co-culture Models

[0139] This example illustrates that outer mitochondrial membrane (OMM) tagged with cell- specific antibodies can boost Mitochondrial Transplantation (mt-tfr) efficiency.

[0140] Background: Extracellular mt-tfr to recipient cells is known to occur by two methods; direct uptake of naked mitochondria and by the uptake of mitochondria within microvesicles (mEVs)7. While the mechanisms behind naked mitochondrial uptake are unclear, mEV-mediated transfer is believed to involve specific ligand-receptor interactions7.

[0141] Independent of mt-tfr, receptor-mediated uptake has also influenced the development of antibody-mediated delivery methods, where antibodies or their fragments target specific receptors on cell surfaces for cargo delivery. For example, adhesion molecules on endothelial cells (ECs) like ICAM-1 and PECAM-1 have each been previously exploited for intracellular delivery of biological materials12.

[0142] Preliminary data shows that mt-tfr can effectively mitigate dysfunctions associatedwith aging and mitochondrial dysfunction (mt-dys) (FIG. 2). In M s, centrifugation-mediatedmt-tfr was observed to promote anti-inflammatory (M2) activity in bone marrow-derived M s(BMDM) (FIGs. 2A-C). Similarly, in ECs, this approach enhanced aerobic glycolysis, cell proliferation, and mitochondrial volume in aged ECs (FIGs.2D-F). Methods:

[0143] Assessment of mitochondria uptake: The uptake of microvesicle- encapsulated mitochondria (mEVs), naked mitochondria (nkd-mito), and antibody-tagged mitochondria (ab- mito) can be evaluated by using endothelial cells (ECs) isolated from young (3-4 month) and old (18-24 month) C57BL / 6 mice. Nkd-mito and mEVs will be isolated from mouse embryonic fibroblasts (MEFs) and recovered from MEF cell culture supernatants using ultra-centrifugation. Ab-mito, tagged with -ICAM-1 and -PECAM-1( -CD31), will be preparedusing methods optimized in AIM 1. To standardize mitochondrial transfer across different groups (nkd-mito, mEVs, and ab-mito), the cardiolipin (CL) content in each sample can be determined, equated, and quantified using commercially available kits. Notably, the MEFs utilized in this example will be isolated from mito::mKate2 mice, inherently expressing far-red fluorescent mitochondria. Post 24-hour incubation of EC cultures with nkd-mito, mEVs, or ab- mito, the ECs will be washed and analyzed for mitochondrial internalization using flow cytometry and confocal microscopy.

[0144] Impact of CCMM on EC function: NO release will be quantified using the Griess assay. Endothelial cell proliferation will be determined through EdU incorporation into newlysynthesized DNA, utilizing the Click-it EdU Proliferation Kit. For cytokine profiling (IL-1 ,IL-6, IL-8, TNF- , VEGF, PDGF, TGF- , MCP-1, IL- 10), a Luminex assay can be employed,supplemented with intracellular cytokine staining for validation. Surface and intracellular markers indicative of EC health (ET-1, eNOS, VCAM-1, ICAM-1, E-selectin, PAI-1) will be characterized using flow cytometry and intracellular staining.

[0145] Co-culture experiments: the specificity of ab-mito can be assessed in co-cultures ofECs and M s. ECs from both young and old mice will be cultured under basal and pro-inflammatory conditions. Using a trans-well system, it will be investigated if mitochondrial transfer to activated endothelial cells promotes anti-inflammatory activities in both ECs andM s.Results:

[0146] For assessing mitochondria uptake in endothelial cells, the mitochondria of recipient cells were stained with tMito-tracker Deep Red, a mitochondrial membrane potential sensitive probe (FIG.3). The donor mitochondria were tagged with 2 different ligands; Azido-pycolly- 488 and Azido modified -PECAM-1, that was already fluorescently conjugated to PE. This allows for dual detection of mitochondria after delivery. The cells (endothelial cells) were incubated with the tagged mitochondria for 24h, after which they cells were fixed and visualized.

[0147] The images as shown in FIG. 3 demonstrate that donor mitochondria have a membrane potential and integrate into the recipient cell’s native mitochondrial network. This is proof of principle that antibody-tagged mitochondria can be effectively delivered to cells and are functional (i.e. have a membrane potential).

[0148] For further assessing mitochondria uptake in endothelial cells, the donor mitochondria were tagged with 2 different ligands; Azido-pycolly-488 and Azido modified - PECAM-1, that was already fluorescently conjugated to PE (FIG. 4). This allows for dual detection of mitochondria after delivery. The cells (endothelial cells) were incubated with the tagged mitochondria for 24h, after which they cells were fixed and stained with VE-Cadherin, an extracellular surface marker of endothelial cells, and Hoechst 33342 a nuclear marker.

[0149] As shown in FIG. 4, donor mitochondria are internalized. This is proof of principle that antibody-tagged mitochondria can be effectively delivered to cells.

[0150] Expected Results: An enhanced uptake of ab-mito can be expected due to targeted tagging, with potential age-related differences in uptake efficiency. The use of mito::mKate2 mice should allow for clear visualization and quantification of mitochondrial uptake using flow cytometry and confocal microscopy. a notable increase in NO production and EC proliferation are expected, especially with ab- mito due to targeted delivery. It is also expected that ab-mito will demonstrate higher specificity and efficiency in mitochondrial transfer, potentially leading to enhanced anti-inflammatory responses in both cell types.

[0151] In case of non-specific uptake, other selective targeting ligands on ab-mito can be investigated. In the event of no anti-inflammatory benefits, the concentration of mitochondria delivered can be modified.Example 3: Specificity and Efficacy of Click Chemistry Mediated Mitochondrial Transplantation in an In Vivo Mouse Model of Cardiovascular Disease (CVD)

[0152] This example illustrates that tail vein infusion-mediated mt-tfr in C57BL / 6 mice which have undergone abdominal aortic aneurysm (AAA) injury can mitigate inflammation and decrease AAA growth.

[0153] Background: Abdominal aortic aneurysm (AAA), a CVD closely linked to aging, is characterized by the irreversible ballooning of the infrarenal aorta3, 13-18. Persistent pro-inflammatory immune cell (M & T cells) activity19-25, vascular cell (ECs & VSMCs)inflammation, and oxidative stress are key drivers of aortic wall deterioration and AAA growth3, 13-16. Despite its severity, effective treatments for AAA growth or rupture are currently unavailable. Recent studies, including those by Vats et al.26, indicate a link between AAA and mtDNA defects. Vats et al. found increased mtDNA mutations, particularly in the D-loop region's MT-TAS2 subregion, in AAA patients compared to healthy individuals26. These mutations, known to disrupt mtDNA replication, are also associated with several cancers and Marfan Syndrome, a condition sharing features with AAA like aortic wall deterioration and cellular inflammation16, 27-29. It has been shown that significant downregulation in mitochondrial genes in AAA-induced mice, indicating decreased mtDNA transcription during aneurysm growth (FIG. 5A). Additionally, single-cell RNA sequencing of infrarenal tissuefrom AAA-mice revealed downregulated mitochondrial-related genes in M s and ECs (FIGs.5B-D). Furthermore, P-cho-Az 647 mito were transplanted into macrophages, after which the levels of mitochondrial superoxide production, another indicator of mitochondrial function / dysfunction was examined using the fluorescent probe MitoSOX (5 uM) and quantified by flow cytometry using the PE channel. The macrophages were incubated at 37 C with MitoSOX for ~25 mins. As shown in FIG. 6, both concentrations of mitochondria delivered to macrophages (10 and 50 ug), significantly decreased MitoROS production. These data indicate that P-cho-Az 647 mito can modulate the production of mitoROS, which may be useful in reducing cellular oxidative stress and dysfunction.

[0154] Additionally, there's growing evidence supporting in vivo mitochondrial targeting for cardiovascular health preservation. In mice, overexpression of mitochondrial DNA quality control genes (e.g., TWINKLE, TFAM) has enhanced mtDNA copy number and reduced dysfunction due to mitoROS, delaying vascular aging and improving heart function. Mt-tfr has shown promise in ischemic rabbit hearts, enhancing ATP production and angiogenesis withoutinducing inflammation. Aim 3 will assess the potential of CCMM to inhibit AAA aneurysm growth in mice. Methods:

[0155] AAA Induction and tracking: 3-month-old male C57BL / 6 mice will undergo laparotomy. Their infrarenal aortic segment will be perfused with porcine pancreatic elastase (PPE) to induce AAA injury, following established protocols30. Aortic dilation due to PPE will be tracked via ultrasound at days 0, 3, 5, 7, 14, 21 and 28 post surgery, and aortic diameter measurements will be obtained at multiple locations along the aorta, including the suprarenal and infrarenal segments.

[0156] Treatment Groups will consist of PPE mice receiving either saline, a bolus of ab- mitochondria (~300 μg) isolated from MEFs, or mEVs isolated from MEF, via tail vein injection. Control groups undergoing sham-PPE surgery will additionally receive mitochondria to establish baseline effects of mt-tfr. Mitochondrial infusions will occur on days 1, 8, 15, and 24 post surgery.

[0157] Localization of Mitochondria: Fluorescently labeled (GFP or RFP tagged) mitochondria will be used allowing visualization and assessment of their distribution within the AAA injury site or specific cells of interest.

[0158] Inflammation Assessment: Plasma and tissue levels of cytokines and proteins (IL-6,IL- 1 , TNF , IFN , MMP2 / 9, MCP-1, and TGF- ) will be quantified to gauge systemic andaortic inflammation in AAA mice.

[0159] Cellular Infiltration: Tissue samples will be collected at specific time points (3, 7, 14, 21, and 28 days) and processed for immunohistochemistry to evaluate cellular infiltration,particularly M and T-cells, using specific antibodies against cell-specific markers (e.g., F4 / 80for M , CD3 for T-cells).

[0160] Expected Results: Based on preliminary data and supporting literature, it is expected that mt-tfr will at least decrease inflammation in AAA mice, and at best, stunt aneurysmal growth.

[0161] Mitochondrial fluorescent tags / proteins are usually expressed on nuclear genes and after mt-tfr, the mitoGFP signal could be lost. As an alternative method to track donor mitochondria in vivo, mtDNA of donor mitochondria can be tagged with the molecule 6-O-Propynyl-dG (PdG), which can be visualized via click-chemistry31. Mt-tfr in macrophages or T cells can be done ex vivo and these cells can then be adoptively transferred into AAA mice to confirm physiological changes in vivo. No current AAA mouse model fully recapitulates AAA disease seen in humans32, and it may be necessary to perform mt-tfr in a second model of AAA such as the angiotensin II model (Ang-II) model33. The Ang-II model requiresatherosclerotic-susceptible strain (apolipoprotein E deficient; apoE / ) mice as compared towild-type C57BL / 6 mice used in the PPE model32, 33. Depending on the results from these studies, single cell sequencing and analysis of AAA tissue can be performed, to better understand how systemic delivery of mitochondria impacted AAA injury in mice. If no changes are observed in AAA mice, it may also signify to increase the number of mitochondria delivered, which can be adjusted as experiments progress.

[0162] The successful completion of this project promises to establish a groundbreaking and accurate method for mt-tfr ex vivo and in vivo. As a bonus, this method also provides a new transient way to fluorescently label mitochondria for extracellular, intracellular and in vivo studies. This proof of principle work will lay the foundation for future work in the feasibility of mt-tfr in other models of CVD, such as peripheral artery disease and atherosclerosis. Example 4. Methods For Developing and Delivering the Antibody-Mitochondrion Conjugates

[0163] This example illustrates a broad protocol for developing and delivering the antibody- mitochondrion conjugates. Generation and Isolation of Taggable Mitochondria (t-mito)

[0164] Azido / Alkyne-modified mitochondria can be generated either in vitro or in vivo. For in vitro generation, the source of donor mitochondria (i.e., donor cells) is incubated with the appropriate alkyne and azide-modified phospholipids for anywhere between 30 mins to 72 hrs. For in vivo generation of taggable mitochondria, mice or specified animals should be dosed with non-toxic levels of the appropriate alkyne and azide-modified phospholipid components. It is also possible to use ratios of azide and alkyne analogues to increase the complexity of potential tagging sites. Mitochondria can be from autologous or allogeneic donors.

[0165] Regardless of the method used to generate the taggable mitochondria (t-mito), these mitochondria must then be isolated for downstream use. The taggable mitochondria (t-mito) generated in vivo can be isolated from any cell or tissue that normally contains mitochondria.

[0166] Mitochondria can be isolated using commercial kits (example here) or via manual cell dissociation in a homogenization / mitochondrial buffer, SHE buffer [250 mM sucrose, 20 mM HEPES, 2 mM EGTA, 10 mM KCl, 1.5 mM MgCl2, and 0.1% defatted bovine serum albumin (BSA)], containing a complete MiniTab protease inhibitor cocktail. Other buffers that preserve mitochondrial membrane potential and functionality can also be used.

[0167] For manual isolation, typically, after gentle / mechanical homogenization, the cell homogenates will be centrifuged at 800-1200 g for 3-5 min to remove cellular debris. The cell homogenate can be re-homogenized a few times to increase the mitochondria yield in the recovered supernatant. The resulting supernatant is recovered and centrifuged at 8-10k x g for 3-5 min to pellet the isolated mitochondria. These t-mito are maintained in a buffer that preserves their functionality / membrane potential (i.e., SHE buffer or others).

[0168] The isolated t-mito can then be incubated with their respective / reciprocal azido / alkyne-tagged antibody, which will then undergo Click Chemistry reactions (CC). CC refers to highly efficient and selective chemical reactions, notably between azide and alkyne groups, through two methods: copper-catalyzed (CuAAC) and strain-promoted (SPAAC). Generation of Antibody-Tags for Mitochondria (Ab-tags)

[0169] Non-azido / alkyne-conjugated antibodies can be either commercially purchased or generated in house. Non-limiting example of such kits to modify antibodies include SiteClick™ Antibody Azido Modification Kit (Invitrogen, S20026) and SiteClick™ sDIBO Alkyne Kits for Antibody Labeling (Invitrogen, C20031). To extend the length of the final antibody-mitochondria conjugate, azido- / alkyne- modified antibodies can be further modified with respective linkers that contain the appropriate alkyne / azide side chain / side group capable of undergoing conjugation with t-mito.

[0170] By proxy it is also possible to label mitochondria with fluorescent tags, enzymes or molecules in a similar manner, that can be multiplexed for future experiments involving mitochondrial transplantation. As disclosed herein, any biomolecule / conjugate that is readily accessible / reactive to click chemistry or chemistries with azide and alkyne side groups, can react with the taggable mitochondria. Generation of Antibody-Mitochondria Conjugates (Ab-MCs).

[0171] The appropriate azide or alkyne t-mito are incubated with the reciprocal alkyne- or azide-modified antibodies, and cycloaddition reactions are carried out (i.e., copper-catalyzed(CuAAC) and strain-promoted (SPAAC)). The reactions can be carried out in any buffer that preserves mitochondrial membrane potential / function, such as the SHE buffer. The reactions can be carried out at room temperature up to 37°C, or any maximum temperature that does not damage mitochondria or denature the antibody and linkers used. Reaction / incubation times can vary depending on the amount of mitochondria and antibodies (and different numbers of antibodies).

[0172] CuAAC - A source of copper (I) ions is essential as the catalyst; copper (II) salts are reduced in situ to copper(I) with reducing agents like sodium ascorbate. Using ligands like tris(benzyltriazolylmethyl)amine (TBTA) can enhance the reaction's rate and reduce production of reactive oxygen species, which may damage mitochondria. Commercial kits (e.g., Click-iT™ Plus Alexa Fluor™ 647 Picolyl Azide Toolkit, Invitrogen, C10643) can also be used to facilitate this process. SPAAC - This reaction does not require any additional reagents.

[0173] The final Ab-mitochondria conjugates (Ab-MCs) can be delivered to cells and animals thereafter. Delivery of Antibody-Mitochondria Conjugates (Ab-MCs)

[0174] In vitro – Ab-MCs can be applied directly to primary or immortalized cell cultures.

[0175] In vivo - Potential routes of administration for Ab-MCs include intravenous (IV), subcutaneous (SC), intramuscular (IM), intraperitoneal (IP), intrathecal (IT), topical, intra- articular, intraocular, inhalation, or oral administrations.

[0176] Ab-MCs may be delivered in solvent / vehicles like buffered saline solutions to maintain physiological pH, normal saline (0.9% sodium chloride) to match blood osmolarity, 5% dextrose in water as an alternative solvent and energy source, human serum albumin to stabilize the formulation and match plasma oncotic pressure, added electrolytes like potassium chloride and sodium phosphate to replicate plasma's electrolyte balance, stabilizers and excipients such as sucrose, mannitol, and trehalose to maintain antibody stability, non-ionic surfactants like polysorbate 80 or 20 to prevent aggregation, or other biocompatible solvents that maintain Ab-MCs integrity (i.e. antibody function, and mitochondrial function).Example 5. Internalization of -CD31-Mito-Tags into Endothelial Cells (ECs)

[0177] This example illustrates the characterization of -CD31-mito-tags internalization into endothelial cells (ECs).

[0178] The internalization of -CD31-mito-tags into endothelial cells were further confirmed through Z-depth images. The Z-depth images provide a detailed three-dimensional analysis of the localization and distribution of fluorescently tagged cellular components, including VE- Cadherin, -PECAM-1-tagged mitochondria, and their merged interactions within endothelial cells. These images capture the spatial organization of the targeted mitochondria and their relationship with endothelial structures, clearly demonstrating internalization and targeted delivery. The Z-depth projection of -CD31-mito-tags shows their distribution at depths ranging from 3 m to approximately 4 m. The Z-depth projection of VE-Cadherin shows its distribution at depths ranging from 1 m to 2 m. VE-Cadherin staining is predominantly at the cell surface. Its surface localization establishes the framework for distinguishing extracellular versus intracellular compartments. The Merged Z-depth projection of -CD31- mito-tags and VE-Cadherin shows that the -CD31-mito-tags are a depth beneath VE- Cadherin. This indicates that the mito-tags have successfully penetrated the endothelial cell layers, confirming their internalization and cytoplasmic localization. Mitochondria are therefore clearly observed within the cytoplasmic regions below the VE-Cadherin layer, substantiating successful mitochondrial delivery and retention within the cells.

[0179] We compared the magnitude of mitochondrial transplantation using unmodified mitochondria (naked mitochondria or nkd-mito) vs -CD31 mito-tags in ECs. To Do this, ECs were exposed to the same quantities of extracellular ndk-mito and -CD31 mito-tags for 4 hrs. After which, the ECs were washed with 1x sterile PBS and subsequently cultured for 24h at 37 C. After 24 h, the ECs exposed to either ndk-mito or -CD31-PE-Mito were fixed with 4% PFA, and nuclei counterstained with Hoechst 33342. In some experiments, ECs were pre- stained with Mito-tracker Deep Red to determine the proximity of the donor mitochondria to the recipient cell’s mitochondrial network. As shown in FIG.7, ECs exposed to -CD31 mito- tags (FIGs. 7C and 7D) had significantly higher amounts of fluorescently labelled mitochondria (punctates) than ECs exposed to ndk-mito (FIGs.7A and 7B).

[0180] We further quantified the number of punctate / dots in 4-7 confocal images of endothelial cells (ECs) treated with non-tagged mitochondria (nkd-mito) and -CD31 mito- tags. As shown in FIG.8A, cells treated with mito-tags exhibited a significantly higher numberof particles per nucleus, averaging 310.05 punctates, compared to 27.67 punctates in cells treated with nkd-mito. FIG. 8B further demonstrated that the -CD31 mito-tags occupied significantly more cellular area (3.4%) compared to nkd-mito (0.3%). These results indicate that mito-tags facilitate markedly enhanced mitochondrial uptake into cells compared to the passive delivery observed with nkd-mito. This highlights the potential of -CD31 mito-tags as a reliable and efficient tool for targeted mitochondrial delivery to cells that undergo endocytosis.

[0181] FIG. 9A presents the Thresholded Overlap Coefficient values, which ranged from 0.79 to 0.85, confirming a high degree of colocalization between -CD31 mito-tags and the recipient cell’s mitochondrial network. FIG. 9B displays the Random Pearson Costes 2D values, which ranged from 20 to 42.8, demonstrating varying levels of mitochondrial colocalization with endothelial markers. To assess statistical confidence, the anti-correlated p-values were log-transformed (-log p-value), revealing a moderate positive correlation (R² =0.5377) between colocalization efficiency and statistical significance. Linear regressionanalysis of Pearson Costes 2D values against -log (anti-correlated p-values) showed thathigher colocalization values correspond to stronger statistical confidence in mitochondrial targeting.

[0182] These findings collectively indicate that the observed colocalization is non-random and results from PECAM-1 (CD31)-mediated mitochondrial delivery to endothelial cells. The increased cellular integration and enhanced uptake efficiency of -CD31 mito-tags, as shown in FIG.8, combined with the high colocalization levels observed in FIG.9, support the use of mito-tags as a robust and efficient tool for targeted mitochondrial delivery.

[0183] The specificity of the -CD31-PE mito-tags was further evaluated through co-culture experiments involving endothelial cells (ECs, CD31+) and vascular smooth muscle cells (VSMCs, CD31-), with results visualized using confocal microscopy (FIG.10) and quantified by flow cytometry (FIG.11).

[0184] As shown in FIG. 10, the merged image (FIG. 10E) provided qualitative evidence that mito-tags are effectively localized within endothelial cells (ECs), with the PE fluorescence (FIG. 11B) indicating the presence of labeled mitochondria. This pattern contrasted with the minimal fluorescence observed in non-target vascular smooth muscle cells (FIG.10D), further emphasizing the specificity of the mito-tags.

[0185] In parallel, flow cytometry analysis quantitatively supported the aforementioned qualitative findings. As shown in FIG.11, nearly all ECs exhibit significant uptake of -CD31- PE mito-tags, while VSMCs demonstrate minimal internalization.

[0186] Collectively, the confocal imaging (FIG. 10) and flow cytometry data (FIG. 11) demonstrated that -CD31-PE mito-tags not only target ECs effectively but also achieve precise delivery and uptake. This finding supported the broad potential of mito-tags as a robust tool for targeted mitochondrial delivery.

[0187] We also evaluated, by confocal imaging, the selective uptake of -CD31-PE mito- tags by bone marrow-derived macrophages (BMDMs) relative to vascular smooth muscle cells (VSMCs). The analysis highlighted some level of mitochondrial internalization in BMDMs but minimal uptake in VSMCs. As shown in FIG.12, minimal fluorescence from -CD31-PE mito-tags was observed in the VSMCs (FIG. 12B), indicating negligible uptake of mitochondria by this cell type. These results supported the conclusion that -CD31 is not expressed, or is expressed at very low levels, on VSMCs, preventing effective mitochondrial targeting. Example 6. Functional Impact of Internalized Mito-Tags on Endothelial Cells (ECs)

[0188] This example illustrates the functional impact of -CD31-mito-tags internalization into endothelial cells (ECs).

[0189] To evaluate the functional impact of mito-tags on endothelial cells (ECs), bioenergetics and mitochondrial abundance were assessed approximately 24 hours after exposure to mito-tags. The analysis compared aged ECs treated with mito-tags to untreated controls, as well as to ECs exposed to nkd-mitochondria or mito-tags loaded with damaged mitochondria in specific instances. Impact of -CD31 mt-tag on mitochondrial abundance (baseline)

[0190] As shown in FIG. 13A, endothelial cells (ECs) treated with -CD31 mito-tags exhibited a significant increase in mitochondrial abundance, as measured by mean fluorescence intensity (MFI) of FITC-TOMM20, compared to untreated control cells (*p<0.05). However, treatment with -CD31 mito-tags containing damaged mitochondria (mt-tag-dmg) did not result in a similar increase, indicating that the functional integrity of the mitochondria is criticalfor enhancing mitochondrial abundance in ECs. These findings underscored the specificity and functional efficacy of intact mito-tags in augmenting mitochondrial content.

[0191] FIG. 13B illustrates that treatment with -CD31 mito-tags significantly enhanced mitochondrial content in both young (Y) and old (O) endothelial cells (ECs), as measured by relative mean fluorescence intensity (MFI) of Mitoview Green (MTV-G), compared to untreated controls (Y-ctrl and O-ctrl) and cells treated with non-targeted mitochondria (Y-ndk and O-ndk). The observed increase (**p<0.01) highlighted the efficacy of mito-tags in facilitating targeted mitochondrial delivery and retention in ECs, irrespective of the cell age, with a notably higher mitochondrial load compared to passive mitochondrial uptake. General reactive oxygen species (ROS) production (Old Mu-AoEC)

[0192] FIG. 14 illustrates the relative mean fluorescence intensity (MFI) of DCF-DA, a fluorescent probe that detects reactive oxygen species (ROS), under different experimental conditions. The conditions included Control, -CD31 (antibody only), naked mitochondria (nk-mito), and antibody-conjugated mitochondria ( -CD31 mito-tag). ROS levels were detectedroughly 24 hours post-treatment. The data provided insights into ROS levels in response to mitochondrial treatments and PECAM1-mediated targeting. As shown in FIG. 14, when the baseline DCF-DA signal was normalized to 1.0, representing the untreated condition orbaseline ROS levels (Control), cells treated with PECAM1 antibodies ( -CD31) showed asignificant reduction in ROS levels compared to the control (p < 0.01). Compared to control, naked mitochondria (nk-mito) did not significantly impact basal levels of ROS in aged ECs,while the ROS levels in cells treated with Ab-MC ( -CD31 mito-tag) were significantly lowerthan those with nkd-mito, p < 0.01, indicating the importance of antibody-mediated targeting in minimizing oxidative stress. The results indicate that -CD31 mito-tag are the most effective at reducing ROS levels. Seahorse XF Real-Time ATP Rate Assay

[0193] The Seahorse XF Real-Time ATP rate assay (FIG. 15) highlighted the bioenergetic improvements achieved through -CD31 mito-tag treatment in aged aortic endothelial cells (O- AoECs). As shown in FIG. 15A, basal ATP production measurements revealed a significant decrease in mitochondrial ATP generation in -CD31-mito-tag treated cells compared to untreated controls, as shown by the smaller light grey component in the ATP production graph. As shown in FIG. 15B, the energetic map illustrated the shift toward glycolysis in -CD31- mito-tag treated ECs. In contrast, untreated AoECs demonstrated a greater dependence onOxidative Phosphorylation. As shown in FIG. 15C, the ATR Rate Index measurements showed a lower mitoATP / glycoATP ratio in -CD31-mito-tag treated O-AoECs. These results show -CD31-mito-tags modulated cellular energy metabolism in aged endothelial cells. Mito-Tags in Mitigating Oxidative Stress in Inflammation-Driven Pathologies

[0194] To evaluate the functional impact of mito-tags on endothelial cells (ECs), aged ECswere treated with IFN for 24 hours prior to the addition of -CD31-mito-tags. ROS levelswere assessed approximately 24 hours after mito-tag exposure. The analysis compared IFN -pretreated aged ECs treated with mito-tags to untreated controls, as well as to ECs exposed to nkd-mitochondria.

[0195] FIG. 16A highlighted the relative mean fluorescence intensity (MFI) of DCF-DA, a reactive oxygen species (ROS) indicator, under different experimental conditions involvingIFN treatment, -CD31 antibody (control), naked mitochondria (nkd-mt), and -CD31 mito-tags. Key Observations from FIG. 16A: 1. IFN increased ROS production. In the presenceof IFN , a significant increase in ROS levels was observed compared to untreated controls, asshown by elevated DCF-DA MFI. This was consistent with the pro-inflammatory role of IFNin promoting oxidative stress. 2. Control -CD31 antibody alone showed no effect. The presence of -CD31 antibody alone slightly altered ROS levels. 3. Nkd-mt alone showed no effect. The addition of naked / unmodified mitochondria (nkd-mito) did not significantlyattenuate IFN -induced ROS. 4. -CD31 mito-tags significantly mitigated ROS. The -CD31mito-tags reduced ROS levels more effectively than any other treatmennt, bringing the DCF- DA MFI closer to baseline levels seen in untreated cells. In conclusion, the -CD31 mito-tags showed a robust capacity to counteract ROS accumulation under inflammatory conditions, outperforming naked mitochondria. This indicated the enhanced efficacy of targeted mitochondrial delivery in restoring redox balance. These findings further supported the therapeutic potential of mito-tags in mitigating oxidative stress in inflammation-driven pathologies. Mito-Tags in Mitigating Oxidative Stress in Aging-Related Inflammatory Conditions

[0196] To evaluate the functional impact of mito-tags on endothelial cells (ECs), bioenergetics and mitoROS were assessed approximately 24 hours after exposure to mito-tags. The analysis compared aged ECs treated with mito-tags to untreated controls, as well as to ECs exposed to nkd-mitochondria or mito-tags loaded with damaged mitochondria in specific instances.

[0197] FIG. 16B showed the mean fluorescence intensity (MFI) of MitoSOX, an indicator of mitochondrial superoxide production, across various experimental groups involving young(Y) and old (O) endothelial cells (ECs) treated with or without interferon gamma (IFN ), nakedmitochondria (nkd), or -CD31 mito-tags (mito-tags). Key Observations from FIG. 16B: 1. baseline superoxide levels. Young control cells (Y-ctrl) exhibited lower MitoSOX MFI compared to old control cells (O-ctrl), indicating higher baseline mitochondrial superoxidelevels in aged cells. 2. IFN -Induced Oxidative Stress. Treatment with IFN significantlyincreased mitochondrial superoxide levels in both young and old ECs (Y-IFN and O-IFN, respectively). 3. Mitigation by mitochondrial treatments. In young cells, both nkd-mito (Y-IFN-nkd) and mito-tags (Y-IFN-mt-tag) reduced superoxide levels compared to IFN treatmentalone, with mito-tags showing a more pronounced effect. In old cells, -CD31 mito-tags (O-IFN-mt-tag) effectively lowered superoxide levels compared to IFN treatment, whereas nkd-mitochondria (O-IFN-nkd) provided only a modest reduction.4. Aged ECs responded better to mito-tags. The reduction in mitochondrial superoxide by mito-tags was more significant in old cells (O-IFN-mt-tag) compared to young cells, suggesting enhanced efficacy of targeted delivery in mitigating oxidative stress in aged ECs. In summary, the data demonstrated that -CD31 mito-tags were more effective than nkd-mitochondria in reducing IFN -inducedmitochondrial superoxide production, with enhanced benefits observed in aged endothelial cells. This highlighted their therapeutic potential in mitigating oxidative stress, particularly in aging-related inflammatory conditions. Mitochondrial bioenergetic assay under inflammatory conditions

[0198] To evaluate the functional impact of mito-tags on endothelial cells (ECs), bioenergetics were assessed approximately 24 hours after exposure to mito-tags. The analysis compared aged ECs treated with mito-tags to untreated controls, as well as to ECs exposed to nkd-mitochondria or mito-tags loaded with damaged mitochondria in specific instances. Theanalysis compared IFN -pretreated aged ECs treated with mito-tags to untreated controls, aswell as to ECs exposed to nkd-mitochondria.

[0199] Mitochondrial bioenergetics were assessed under inflammatory conditions inducedby IFN to evaluate the therapeutic effects of naked mitochondria (nkd-Mito) and -CD31antibody-conjugated mitochondria ( -CD31-mito-tag). As shown in FIG. 17A, basalmitochondrial respiration, as indicated by oxygen consumption rates (OCR), was notsignificantly reduced in IFN -stimulated cells. Treatment with -CD31 did not impact basalOCR. Treatment with nkd-mito or -CD31-mito-tags significantly decreased basal respiration. As shown in FIGs.17B and 17E, no significant change in maximal mitochondrial respiration, as well as non-mitochondrial OCR was observed across treatment groups. As shown in FIGs. 17C and 17D, spare respiratory capacity was highest in ECs treated with nkd-mito or -CD31- mito-tags, while mito-ATP levels were lowest in these group. Basal and Compensatory Glycolysis Assessments

[0200] We further evaluated the basal and compensatory glycolysis in the study. Basal glycolysis is the rate of glycolysis in cells at rest, while compensatory glycolysis is the rate of glycolysis when mitochondrial respiration is inhibited. In the basal glycolysis assessment, asshown in FIG. 18A, IFN treatment alone led to a notable increase in basal glycoPERcompared to the control. Both naked mitochondria (nkd-mito) and antibody-conjugatedmitochondria ( -CD31 mito-tags) further enhanced basal glycoPER, with -CD31 mito-tagsshowing the greatest increase. This suggests that mitochondrial treatments, particularly - CD31 mito-tags, may augment glycolysis even under inflammatory conditions, potentially as part of a metabolic reprogramming to meet energy demands.

[0201] In the compensatory glycolysis assessment, as shown in FIG. 18B, unlike basal glycolysis, compensatory glycolysis levels were relatively consistent across all treatmentgroups, including IFN , nkd-Mito, and Ab-MC. This indicates that while mitochondrialtreatments influence baseline glycolysis, they do not significantly alter the cell's capacity to shift toward glycolysis when mitochondrial function is perturbed. Proliferation of Young and Old Mouse Aortic Endothelial Cells (AoECs)

[0202] We further evaluated the impact of mitochondrial transfer on endothelial cell (EC) proliferation in young (2–3 months) and old (18–22 months) mouse aortic endothelial cells (AoECs). The experiments assessed the percentage of proliferating ECs, measured as Ki67+ EdU+ APC+ ECs, at different time points post-mitochondrial transfer using flow cytometry.

[0203] Endothelial cells (ECs) were treated with mitochondria for 2–3 hours, followed by two PBS washes and an additional 22-hour incubation to complete a 24-hour treatment period. To label newly synthesized DNA, ECs were exposed to 10 M EdU for 2 hours. Intracellular staining for Ki67 and EdU (Az647) was then conducted using flow cytometry to identify proliferating cells 24 hours post-EdU addition. Analysis involved sub-gating on Ki67+ EdU+ cells to detect recently proliferated ECs.

[0204] In the single time point (48 hours post-mitochondrial transfer) experiment, as shown in FIG. 19A, mitochondrial transfer significantly increased the percentage of Ki67+ EdU+ cells compared to untreated controls in young AoECs (p < 0.0001). This indicates that mitochondrial transfer promotes proliferation in young endothelial cells. As shown in FIG. 19B, while the proliferation response was less pronounced than in young cells, a significant increase in Ki67+ EdU+ cells was observed compared to untreated controls (p < 0.001) in old AoECs. This suggests that mitochondrial transfer can partially restore the proliferative capacity of aged ECs.

[0205] In the experiment 2: Two Time Points (48 and 96 Hours Post-Mitochondrial Transfer), as shown in FIG.20A, proliferation remained significantly elevated at both 48 hours (p < 0.001) and 96 hours (p < 0.001) post-transfer in young AoECs, indicating sustained pro- proliferative effects of mitochondrial delivery in young ECs. No significant decline in proliferation was observed between time points, suggesting continued mitochondrial function. As shown in FIG.20B, proliferation significantly increased at both 48 hours (p < 0.01) and 96 hours (p < 0.01) compared to untreated controls in old AoECs. The sustained response indicates that mitochondrial transfer effectively promotes cellular proliferation even in aged endothelial cells, albeit at a lower magnitude than in young cells. In conclusion, mitochondrial transfer significantly enhances the proliferation of endothelial cells in both young and old AoECs. Young cells exhibit a robust and sustained proliferative response, while aged cells show a more modest but significant restoration of proliferative capacity. These findings suggest that mitochondrial transfer has therapeutic potential for promoting endothelial regeneration, particularly in the context of aging. Proliferation of mtDNA-deficient rho-null endothelial cells (Rho ECs)

[0206] We also evaluated the effects of mitochondrial transfer on the proliferation of mtDNA-deficient rho-null endothelial cells (Rho ECs), which rely on exogenous uridine for survival and proliferation. These experiments assess the retention and functionality of mitochondrial tags (mito-tags) and their impact on promoting cellular proliferation, as measured by the percentage of Ki67+ EdU+ APC+ ECs using flow cytometry.

[0207] Rho endothelial cells (ECs) were treated with mitochondria for 2–3 hours, followed by two PBS washes and an additional ~22-hour incubation to complete a 24-hour treatment period. To label newly synthesized DNA, cells were pulsed with 10 M EdU for 2 hours. Flow cytometry was performed 24 hours post-EdU addition, with Ki67 and EdU staining used toquantify recent proliferation. Proliferation was further evaluated in the context of the cells' inability to replicate without exogenous uridine, emphasizing the importance of mitochondrial functionality.

[0208] In the Experiment 1: Single Time Point (48 Hours Post-Mitochondrial Transfer), as shown in FIG. 21A, Rho ECs treated with mito-tags showed a significant increase in the percentage of proliferating cells (p < 0.0001) compared to untreated controls. This suggests that mito-tags restore mitochondrial functionality, enabling Rho ECs to overcome their dependency on uridine for proliferation.

[0209] In the Experiment 2: Two Time Points (48 and 96 Hours Post-Mitochondrial Transfer), as shown in FIG. 21B, Rho ECs treated with mito-tags demonstrated significantly enhanced proliferation compared to controls (p < 0.01) at 48 hours, while proliferation remained elevated (p < 0.01) at 96 hours, indicating that mito-tags are retained and remain functional over time. While the proliferation response was slightly lower at 96 hours than at 48 hours, the sustained increase supports the long-term impact of mt-tag delivery. It is worth noting that the dependency of Rho ECs on exogenous uridine underscores the critical role of functional mitochondria in supporting cellular bioenergetics and proliferation. In conclusion, these results demonstrate that mitochondrial transfer using mito-tags restores the proliferative capacity of mtDNA-deficient Rho ECs, enabling them to overcome uridine dependency. The sustained proliferative response at 48 and 96 hours post-transfer highlights the retention and functionality of mito-tags. These findings strongly suggest that mito-tags hold promise as a therapeutic tool for restoring mitochondrial function in cells with compromised bioenergetics. Example 7. In Vivo Study of -CD31 Mito-Tag Delivery

[0210] This example illustrates -CD31-tagged mitochondrion delivery in a mouse study.

[0211] The in vivo imaging data (FIG. 22) demonstrate the biodistribution of MitoTracker Deep Red-stained mitochondria 24 hours after injection into mice. The images were acquired using a 1-second scan on an IVIS in vivo imager, comparing three groups: no injection(control), naked mitochondria (nkd-mt), and -CD31-tagged mitochondria ( -CD31 mt-tag).These results provide insights into the targeting efficiency and retention of mitochondria delivered via antibody tagging. As shown in FIG.22A, the control no injection group shows no significant fluorescent signal, confirming the absence of background fluorescence from MitoTracker Deep Red in untreated animals. This provides a baseline for assessing thespecificity and distribution of injected mitochondria in the experimental groups. As shown in FIG. 22B, fluorescent signals are localized primarily near the injection site of the mouse injected with nkd-mt (naked mitochondria), indicating that naked mitochondria lack significant systemic distribution or targeting ability. The limited signal suggests rapid clearance or poor retention of mitochondria in circulation without a targeting mechanism. As shown in FIG. 22C, fluorescent signals are prominently distributed throughout the vascular and endothelial regions in the mouse tail injected with -CD31 mt-tag (Antibody-Tagged Mitochondria), particularly in areas associated with PECAM1-expressing endothelial cells. Compared to naked mitochondria, -CD31 mt-tagged mitochondria demonstrate enhanced systemic circulation and targeted delivery to endothelial compartments. The increased fluorescent intensity and broader distribution highlight the efficiency of antibody-mediated targeting for precise mitochondrial delivery. Isolated Aortic Tissue stained for ECs and Donor Mitochondria

[0212] Confocal images of isolated aortic tissue show donor mitochondria (MitoTracker Deep Red-stained) in endothelial cells (ECs) stained for PECAM1 and nuclei. In the nkd-mt condition (FIG. 23A), sparse fluorescence indicates limited uptake or retention of mitochondria in PECAM1-positive ECs, suggesting nonspecific delivery. In contrast, the mito- tag condition (FIG. 23B) displays significantly increased fluorescence co-localized with PECAM1-positive regions, demonstrating enhanced specificity and retention of donor mitochondria. These results highlight the superior efficiency of antibody-tagged mitochondria for targeted delivery to endothelial cells, supporting their therapeutic potential for vascular dysfunction. Example 8. Ex Vivo Study of -CD11a Mito-Tag Delivery

[0213] This example illustrates the uptake of -CD11a-tagged mitochondria by aged splenocytes in an ex vivo study.

[0214] The data evaluate the uptake of -CD11a mito-tagged mitochondria compared to naked mitochondria (nkd-mito) by splenocytes isolated from aged mice in an ex vivo system. Splenocytes from aged mice (20-month-old C57BL / 6) were isolated and cryopreserved until use. Approximately 100k splenocytes were incubated with either ndk or -CD11a mito-tags for ~2 hours. Following incubation, the splenocytes were stained with -CD45 antibodies,washed three times with PBS, and fixed with 4% PFA. Mitochondrial volume (mito-vol) wasdetermined using mitochondria isolated from 10 MEF cells per 300 L.

[0215] The analysis combines flow cytometry scatter plots with quantified metrics for mitochondrial uptake, including the percentage of mitochondria-positive cells and mean fluorescence intensity (MFI), across increasing mitochondrial volumes.

[0216] A control condition (no mitochondria) shows negligible mitochondrial-positive cells (0.17% in Q2-UR), serving as a baseline for comparison. The flow cytometry scatter plot of the nkd-mito uptake by aged splenocytes (FIGs. 24A and 25A), shows that only a minimal percentage (1.63%) of splenocytes were positive for the mitochondrial signal (Q2-UR), indicating poor uptake of naked mitochondria. The scatter plot shows a dense population of cells negative for mitochondrial signal (Q2-LR), consistent with the limited efficiency of nkd- mito delivery. The flow cytometry scatter plot of the -CD11a Mito-Tag uptake by aged splenocytes (FIGs. 24B and 25B), shows a dramatic increase in mitochondrial-positive splenocytes is observed (57.40% in Q2-UR), reflecting significantly enhanced uptake compared to nkd-mito. The targeting mechanism via -CD11a improves specific interaction with splenocytes, likely through CD11a-binding receptors. -CD11a mito-tag significantly increases mito-transplantation

[0217] As shown in FIG. 24C, at all tested volumes (5, 25, 50 L), -CD11a mito-tags significantly increase the percentage of mitochondrial-positive splenocytes compared to nkd- mito (p < 0.0001). The dose-dependent increase highlights the scalability and effectiveness of -CD11a tagging for enhancing mitochondrial delivery. As shown in FIGs.24D and 25C, the mean fluorescence intensity (MFI) of mitochondrial signal per cell is significantly higher in the -CD11a mito-tag group than in the nkd-mito group at all volumes. This indicates that - CD11a tagging not only increases the number of cells interacting with mitochondria but also the quantity of mitochondria internalized per cell. The flow data also suggest that mito-tags increase the amount of mitochondria internalized / cell at higher concentrations. In conclusion, the results demonstrate that -CD11a mito-tags significantly enhance the uptake and internalization of mitochondria by splenocytes in a dose-dependent manner compared to naked mitochondria. The increased percentage of mitochondrial-positive cells and higher MFI per cell confirm the efficacy of the targeting strategy. These findings suggest that -CD11a mito- tags could be a powerful tool for delivering mitochondria to immune cells, with potentialapplications in immunomodulation and treatment of mitochondrial dysfunction in various pathologies.

[0218] We further evaluated the uptake of -CD31-tagged mitochondria by aged splenocytes in confocal microscopy. As shown in FIGs.26A-C, these confocal (cross sectional) slices of CD45+ (FIG.26B) splenocytes show that -CD11a mito-tags (FIG.26C) are within the cells and not on the surface, indicative of successful internalization. The colocalization (FIG.26A) of the mito-tag signal with the intracellular regions of the CD45+ splenocytes, rather than the cell membrane, confirms that the mito-tags were effectively taken up by the cells. This finding highlights the specificity and efficacy of -CD11a mito-tags for facilitating mitochondrial delivery directly into CD45+ Cells (Immune Cells). Example 9. Click Chemistry Mediated Mitochondrial Transplantation by Tagging the IMM With Therapeutic Payloads

[0219] This example illustrates that inner mitochondrial membrane (IMM) tagged with cell- specific antibodies or nanoparticles for Mitochondrial Transplantation (mt-tfr).

[0220] By introducing modified cardiolipin (CL) and outer membrane phospholipids into live cells, isolating mitochondria after incorporation, and performing controlled drug conjugation, this strategy could enable fusion-mediated therapeutic release directly into the recipient cell’s native mitochondrial network.

[0221] The process begins with the synthesis of azide- or alkyne-functionalized cardiolipin (CL) and phosphatidylethanolamine (PE) or phosphatidylcholine (PC). These modified phospholipids are introduced into live cell cultures at concentrations of 10-100 μM. Through natural lipid trafficking pathways, the modified phospholipids integrate into the inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) over 4-24 hours.

[0222] Following successful lipid incorporation, mitochondria are isolated from live cells via differential centrifugation, or other established methods of mitochondrial isolation. The isolated mitochondria undergo transient permeabilization of the OMM using low-dose digitonin (0.002-0.01%) or hypotonic buffer treatment, allowing controlled access to the IMM while maintaining membrane potential and structural integrity. OMM permeabilization is validated using membrane integrity assays, JC-1 or TMRM staining to assess IMM potential, and cytochrome c retention assays to confirm selective OMM disruption.

[0223] With IMM phospholipids now accessible, click chemistry-based drug conjugation is performed to functionalize the mitochondria with therapeutic payloads. A bio-orthogonal drug or gene therapy agent functionalized with a complementary click moiety is introduced, allowing precise covalent attachment to the functionalized IMM phospholipids. Examples include DBCO-CoQ10 for mitochondrial bioenergetic support, Azide-MitoTEMPO for oxidative stress protection, and Alkyne-CRISPR / Cas9-mRNA for mitochondrial genome editing. The reaction occurs under mild aqueous conditions at 37°C, pH 7.4, for 2-4 hours, followed by sequential washes to remove unreacted conjugates. Drug loading efficiency is confirmed via fluorescent drug-tagging (e.g., FITC, Cy5), HPLC, and LC-MS lipidomics analysis.

[0224] To facilitate targeted cellular uptake, the OMM of the drug-loaded mitochondria is functionalized with receptor-targeting moieties. This is achieved by conjugating bioorthogonal-modified OMM phospholipids (PE or PC) to targeting ligands, such as anti- PECAM-1, anti-LFA-1, or anti-ICAM-1 antibodies for immune and endothelial cell targeting, mitochondria-binding peptides (e.g., Szeto-Schiller (SS) peptides) for membrane fusion-based uptake, or lipophilic anchoring tags (e.g., PEGylated phospholipids, biotin-streptavidin conjugates) for enhanced cell interaction. Successful OMM functionalization is validated using flow cytometry, immunoblotting, and microscopy-based ligand detection.

[0225] Once fully modified, the mito-tagged, drug-loaded mitochondria are introduced into recipient cells, where receptor-mediated endocytosis, macropinocytosis, or direct membrane fusion enables efficient mitochondrial uptake. Once inside, the mitochondria fuse with the endogenous mitochondrial network, facilitating the redistribution of functionalized IMM lipids across the entire mitochondrial population. This results in localized release of the conjugated drug within native mitochondria, maximizing therapeutic efficacy while preventing off-target cytosolic diffusion.

[0226] To confirm successful mitochondrial uptake and functional integration, mass spectrometry may be used to track the presence of conjugated drugs within native mitochondria post-fusion, while live-cell imaging (MitoTracker colocalization), lipidomics, and Seahorse XF metabolic analysis validate mitochondrial function and therapeutic effects. By optimizing the workflow to incorporate clickable phospholipids in live cells before mitochondrial isolation, this approach may provide highly specific, efficient mitochondrial drug loading and delivery, making it a scalable and clinically relevant strategy for mitochondrial-targeted therapies inneurodegeneration, cancer, metabolic diseases, and beyond. Additionally, variations of this workflow may be used to deliver synthetic mitochondrial DNA or other mitochondrial gene editing tools to cells. V. References: 1) Vaduganathan, M., et al., J Am Coll Cardiol, 2022. 2) Benjamin, E.J., et al., Circulation, 2019. 3) Aggarwal, S., et al., Exp Clin Cardiol, 2011. 4) Choke, E., et al., Eur J Vasc Endovasc Surg, 2005. 5) Weiss, N., et al., Vasa, 2014. 6) Mirel, L.B., et al., Medical Expenditure Panel Survey (US), 2001. 7) Liu, Z., et al., Cell Biosci, 2022. 8) Headley, C.A., et al., Adv Sci (Weinh), 2023. 9) Bauer, D., et al., Nat Protoc, 2023. 10) Funai, K., et al., Curr Opin Cell Biol, 2020. 11) Jao, C.Y., et al., Chembiochem, 2015. 12) Muro, S., et al., J Cell Sci, 2003. 13) Altobelli, E., et al., Int J Environ Res Public Health, 2018. 14) Golledge, J., Nat Rev Cardiol, 2019. 15) Liu, B., et al., Am J Physiol Heart Circ Physiol, 2020. 16) Sakalihasan, N., et al., Nat Rev Dis Primers, 2018. 17) Forester, N.D., et al., Immunology, 2005. 18) Suh, M.K., et al., J Vasc Surg, 2020. 19) Yuan, Z., et al., Front Immunol, 2020. 20) Li, H., et al., J Immunol Res, 2018. 21) Sagan, A., et al., Front Immunol, 2019. 22) Da le, M.A., et al., Arterioscler Thromb Vasc Biol, 2015. 23) Cheng, Z., et al., J Transl Med, 2018. 24) Raffort, J., et al., Nat Rev Cardiol, 2017. 25) Sharma, A.K., et al., Circulation, 2012. 26) Vats, S., et al., J Am Heart Assoc, 2023. 27) Isselbacher, E.M., Circulation, 2005.28) Yap, Z.J., et al., J Card Surg, 2021. 29) Mathur, A., et al., J Transl Int Med, 2016. 30) Azuma, J., et al., J Vis Exp, 2009. 31) Venkatesham, A., et al., Molecules, 2019. 32) Golledge, J., et al., Br J Pharmacol, 2020. 33) Cao, R.Y., et al., Front Pharmacol, 2010.

[0227] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. An engineered mitochondrion comprising one or more modified phospholipids on the mitochondrial membrane (MM) of the mitochondrion, wherein the modified phospholipids comprise azide-modified phospholipids and / or alkyne-modified phospholipids.

2. The engineered mitochondrion of claim 1, wherein the mitochondrial membrane (MM) is the outer mitochondrial membrane (OMM), the inner mitochondrial membrane (IMM), or a combination thereof.

3. The engineered mitochondrion of claim 1 or 2, wherein the modified phospholipids are derived from phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines (PSs), phosphatidylinositol (PLs), phosphatidic acids (PAs), cardiolipins (CLs), sphingomyelins (SMs), or a combination thereof.

4. The engineered mitochondrion of any one of claims 1- 3, wherein the azide-modified phospholipids comprise azido-ethyl-phosphocholine (Az-Cho), azide-modified phosphatidylethanolamine (Az-PE), azide-modified phosphatidylserine (Az-PS), azide- modified phosphatidylinositol (Az-PI), azide-modified phosphatidic acid (Az-PA), azide- modified cardiolipin (Az-CL), azide-modified sphingomyelin (Az-SM), and / or any variant thereof.

5. The engineered mitochondrion of any one of claims 1- 3, wherein the alkyne-modified phospholipids comprise propargyl choline (P-Cho), alkyne-modified phosphatidylethanolamine (Ak-PE), alkyne-modified phosphatidylserine (Ak-PS), alkyne- modified phosphatidylinositol (Ak-PI), alkyne-modified phosphatidic acid (Ak-PA), alkyne- modified cardiolipin (Ak-CL), alkyne-modified sphingomyelin (Ak-SM), and / or any variant thereof.

6. The engineered mitochondrion of any one of claims 1-5, wherein the engineered mitochondrion is linked to an antibody and / or a therapeutic payload through the modified phospholipids.

7. The engineered mitochondrion of claim 6, wherein the engineered mitochondrion is linked to the antibody and / or the therapeutic payload via an azide-alkyne cycloaddition reaction.

8. The engineered mitochondrion of claim 6 or 7, wherein the antibody binds to a transmembrane protein of a cell.

9. The engineered mitochondrion of claim 8, wherein the transmembrane protein is intercellular adhesion molecule type 1 (ICAM-1) or lymphocyte function-associated antigen-1 (LFA-1).

10. The engineered mitochondrion of claim 8, wherein the transmembrane protein is platelet endothelial cell adhesion molecule type 1 (PECAM-1).

11. The engineered mitochondrion of any one of claims 8-10, wherein the cell is a vascular cell or an immune cell.

12. The engineered mitochondrion of claim 11, wherein the vascular cell is selected from the group consisting of an endothelial cell (EC), a vascular smooth muscle cell (VSMC), a smooth muscle cell, a cardiomyocyte, a pericyte, a fibroblast, a cardiac fibroblast, an endothelial progenitor cell, a pacemaker cell, and an adventitial cell.

13. The engineered mitochondrion of claim 11, wherein the immune cell isselected from the group consisting of a macrophage (M ), a T cell, a B cell, a dendritic cell(DC), a NK cell, a neutrophils, a eosinophil, a basophil, a mast cell, a helper t cell (th1, th2, th17), a regulatory t cell (treg), a memory B cell, a plasma cell, a follicular dendritic cell, amonocyte, an innate lymphoid cell (ilc), and a gamma delta t cell ( t cell).

14. The engineered mitochondrion of any one of claims 6-13, wherein the engineered mitochondrion comprises one or more modified phospholipids on the OMM linked to the antibody and one or more modified phospholipids on the IMM linked to the therapeutic payload.

15. The engineered mitochondrion of claim 16, wherein the therapeutic payload is Coenzyme Q10 (CoQ10), Mito-TEMPO, or a gene therapy agent.

16. An antibody-mitochondrion conjugate, comprising an antibody and a mitochondrion, wherein the antibody and the mitochondrion are covalently linked through a linker.

17. The antibody-mitochondrion conjugate of claim 16, wherein the linker comprises an azide-modified phospholipid and / or an alkyne-modified phospholipid.

18. The antibody-mitochondrion conjugate of claim 17, wherein the azide- modified phospholipid comprises azido-ethyl-phosphocholine (Az-Cho), azide-modified phosphatidylethanolamine (Az-PE), azide-modified phosphatidylserine (Az-PS), azide- modified phosphatidylinositol (Az-PI), azide-modified phosphatidic acid (Az-PA), azide- modified cardiolipin (Az-CL), azide-modified sphingomyelin (Az-SM), and the alkyne- modified phospholipid comprises propargyl choline (P-Cho), alkyne-modified phosphatidylethanolamine (Ak-PE), alkyne-modified phosphatidylserine (Ak-PS), alkyne- modified phosphatidylinositol (Ak-PI), alkyne-modified phosphatidic acid (Ak-PA), alkyne- modified cardiolipin (Ak-CL), alkyne-modified sphingomyelin (Ak-SM), and / or any variant thereof.

19. The antibody-mitochondrion conjugate of any one of claims 16-18, wherein the antibody and the mitochondrion are covalently linked via an azide-alkyne cycloaddition reaction.

20. The antibody-mitochondrion conjugate of any one of claims 16-19, wherein the antibody binds to a transmembrane protein of a cell.

21. The antibody-mitochondrion conjugate of claim 20, wherein the transmembrane protein is intercellular adhesion molecule type 1 (ICAM-1) or lymphocyte function-associated antigen-1 (LFA-1).

22. The antibody-mitochondrion conjugate of claim 20, wherein the transmembrane glycoprotein is platelet endothelial cell adhesion molecule type 1 (PECAM-1).

23. The antibody-mitochondrion conjugate of any one of claims 20-22, wherein the cell is a vascular cell or an immune cell.

24. The antibody-mitochondrion conjugate of claim 23, wherein the vascular cell is selected from the group consisting of an endothelial cell (EC), a vascular smooth muscle cell (VSMC), a smooth muscle cell, a cardiomyocyte, a pericyte, a fibroblast, a cardiac fibroblast, an endothelial progenitor cell, a pacemaker cell, and an adventitial cell.

25. The antibody-mitochondrion conjugate of claim 23, wherein theimmune cell is selected from the group consisting of a macrophage (M ), a T cell, a B cell, adendritic cell (DC), a NK cell, a neutrophils, a eosinophil, a basophil, a mast cell, a helper t cell (th1, th2, th17), a regulatory t cell (treg), a memory B cell, a plasma cell, a folliculardendritic cell, a monocyte, an innate lymphoid cell (ilc), and a gamma delta t cell ( t cell).

26. A composition comprising the engineered mitochondrion of any one of claims 1-15 or the antibody-mitochondrion conjugate of any one of claims 16-25.

27. A pharmaceutical composition comprising the composition of claim 26 and a pharmaceutically acceptable carrier.

28. A kit for treating a subject comprising the pharmaceutical composition of claim 27, wherein the subject has a cardiovascular disease (CVD) or mitochondrial dysfunction.

29. A method of treating a cardiovascular disease (CVD) or mitochondrial dysfunction in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 27.

30. The method of claim 29, wherein the subject is an aged individual.

31. A method of delivering a mitochondrion to a cell, comprising: a) generating an antibody-mitochondrion conjugate by covalently linking the mitochondrion to an antibody, wherein the antibody recognizes and binds to a transmembrane protein of the cell; and b) contacting the cell with the antibody-mitochondrion conjugate, wherein the antibody binds to the transmembrane protein of the cell, thereby delivering the mitochondrion to the cell.

32. The method of claim 31, wherein the antibody and the mitochondrion are covalently linked through a linker.

33. The method of claim 32, wherein the linker comprises an azide-modified phospholipid and / or an alkyne-modified phospholipid.

34. The method of claim 33, wherein the azide-modified phospholipid comprises azido-ethyl-phosphocholine (Az-Cho), azide-modified phosphatidylethanolamine (Az-PE), azide-modified phosphatidylserine (Az-PS), azide-modified phosphatidylinositol (Az-PI), azide-modified phosphatidic acid (Az-PA), azide-modified cardiolipin (Az-CL), azide-modified sphingomyelin (Az-SM), and the alkyne-modified phospholipid comprises propargyl choline (P-Cho), alkyne-modified phosphatidylethanolamine (Ak-PE), alkyne- modified phosphatidylserine (Ak-PS), alkyne-modified phosphatidylinositol (Ak-PI), alkyne- modified phosphatidic acid (Ak-PA), alkyne-modified cardiolipin (Ak-CL), alkyne-modified sphingomyelin (Ak-SM), and / or any variant thereof.

35. The method of any one of claims 31-34, wherein the antibody and the mitochondrion are covalently linked via an azide-alkyne cycloaddition reaction.

36. The method of any one of claims 31-35, wherein the transmembrane protein is intercellular adhesion molecule type 1 (ICAM-1) or lymphocyte function-associated antigen-1 (LFA-1).

37. The method of any one of claims 31-35, wherein the transmembrane glycoprotein is platelet endothelial cell adhesion molecule type 1 (PECAM-1).

38. The method of any one of claims 31-37, wherein the cell is a vascular cell or an immune cell.

39. The method of claim 38, wherein the vascular cell is selected from the group consisting of an endothelial cell (EC), a vascular smooth muscle cell (VSMC), a smooth muscle cell, a cardiomyocyte, a pericyte, a fibroblast, a cardiac fibroblast, an endothelial progenitor cell, a pacemaker cell, and an adventitial cell.

40. The method of claim 38, wherein the immune cell is selected from the group consisting of a macrophage (M ), a T cell, a B cell, a dendritic cell (DC), a NK cell, aneutrophils, a eosinophil, a basophil, a mast cell, a helper t cell (th1, th2, th17), a regulatory t cell (treg), a memory B cell, a plasma cell, a follicular dendritic cell, a monocyte, an innate lymphoid cell (ilc), and a gamma delta t cell ( t cell).

41. The method of any one of claims 31-37, wherein the antibody- mitochondrion conjugate comprises an anti-PECAM-1 antibody bound to a mitochondrion, andwherein the cell is a vascular cell, and optionally wherein the vascular cell is an endothelial cell (EC).

42. The method of any one of claims 31-37, wherein the antibody- mitochondrion conjugate comprises an anti-LFA-1 antibody bound to a mitochondrion, and wherein the cell is an immune cell.

Images