[go: up one dir, main page]

WO2025184537A1 - Mitochondries bio-modifiées pour administration ciblée à des cellules - Google Patents

Mitochondries bio-modifiées pour administration ciblée à des cellules

Info

Publication number
WO2025184537A1
WO2025184537A1 PCT/US2025/017885 US2025017885W WO2025184537A1 WO 2025184537 A1 WO2025184537 A1 WO 2025184537A1 US 2025017885 W US2025017885 W US 2025017885W WO 2025184537 A1 WO2025184537 A1 WO 2025184537A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
mitochondrion
modified
antibody
azide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/017885
Other languages
English (en)
Inventor
Colwyn Ansel HEADLEY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Veterans Affairs
Leland Stanford Junior University
Original Assignee
US Department of Veterans Affairs
Leland Stanford Junior University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Veterans Affairs, Leland Stanford Junior University filed Critical US Department of Veterans Affairs
Publication of WO2025184537A1 publication Critical patent/WO2025184537A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2845—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • Mt-dys includes reduced mitochondrial DNA (mtDNA) quality, inefficient electron transport chain (ETC) activity, abnormal mitochondrial membrane potential (m), and abnormal generation of 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.
  • 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 dysregulations 7 .
  • the present disclosure provides an engineered mitochondrion comprising one or more modified phospholipids on the mitochondrial membrane (MM) of the mitochondrion.
  • the mitochondrial membrane (MM) is the outer mitochondrial membrane (OMM).
  • the mitochondrial membrane (MM) is the inner mitochondrial membrane (IMM).
  • the mitochondrial membrane (MM) is a combination of the OMM and the IMM.
  • the modified phospholipids comprise azide-modified phospholipids and/or alkyne-modified phospholipids.
  • the modified phospholipids are derived from phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines (PSs), phosphatidylinositol (PLs), phosphatidic acids (Pas), cardiolipins (CLs), or sphingomyelins (SMs).
  • PCs phosphatidylcholines
  • PEs phosphatidylethanolamines
  • PSs phosphatidylserines
  • PLs phosphatidylinositol
  • Pas phosphatidic acids
  • CLs cardiolipins
  • 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.
  • Az-Cho azido-ethyl- phosphocholine
  • Az-PE azide-modified phosphatidylethanolamine
  • Az-PS azide-modified phosphatidylserine
  • Az-PI azide-modified phosphatidylinositol
  • Az-PA azide-modified phosphatidic acid
  • Az-CL azide-
  • 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.
  • P-Cho propargyl choline
  • Ak-PE alkyne-modified phosphatidylethanolamine
  • Ak-PS alkyne-modified phosphatidylserine
  • Ak-PI alkyne- modified phosphatidylinositol
  • Ak-PA alkyne-modified phosphatidic acid
  • 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).
  • IAM-1 intercellular adhesion molecule type 1
  • LFA-1 lymphocyte function-associated antigen 1
  • the transmembrane protein is platelet endothelial cell adhesion molecule type 1 (PECAM-1, also known as CD31).
  • the cell is a vascular cell or an immune cell.
  • 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.
  • 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, a neutrophils, an 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).
  • M macrophage
  • T cell T cell
  • B cell a dendritic cell
  • DC dendritic cell
  • NK cell a neutrophils
  • eosinophil an eosinophil
  • basophil a basophil
  • mast cell a helper t cell (th1, th2, th17), a regulatory t cell (treg)
  • 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.
  • the therapeutic payload is Coenzyme Q10 (CoQ10), Mito-TEMPO, or a gene therapy agent.
  • the present disclosure provides an antibody-mitochondrion conjugate, comprising an antibody and a mitochondrion.
  • 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.
  • the antibody and the mitochondrion are covalently linked through a linker.
  • the linker comprises an azide-modified phospholipid and/or an alkyne-modified phospholipid.
  • 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-
  • the antibody and the mitochondrion are covalently linked via an azide-alkyne cycloaddition reaction.
  • the antibody binds to a transmembrane protein of a cell.
  • the transmembrane protein is intercellular adhesion molecule type 1 (ICAM-1).
  • the transmembrane protein is lymphocyte function- associated antigen 1 (LFA-1).
  • the transmembrane glycoprotein is platelet endothelial cell adhesion molecule type 1 (PECAM-1).
  • the cell is a vascular cell or an immune cell.
  • 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.
  • 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, 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).
  • M macrophage
  • T cell T cell
  • B cell a dendritic cell
  • DC dendritic cell
  • NK cell a NK cell
  • neutrophils a neutrophils
  • eosinophil a basophil
  • mast cell a helper t cell (th1, th2, th17), a regulatory t cell (treg)
  • 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.
  • CVD cardiovascular disease
  • mitochondrial dysfunction 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.
  • 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.
  • 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.
  • the antibody and the mitochondrion are covalently linked through a linker.
  • any linker that undergoes a click chemistry reaction can be applied to covalently link the antibody and the mitochondrion.
  • 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.
  • the linker is derived from phospholipids, polymers, peptides, or other biocompatible linker materials.
  • the linker comprises an azide-modified phospholipid and/or an alkyne-modified phospholipid.
  • 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
  • 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.
  • the transmembrane protein is intercellular adhesion molecule type 1 (ICAM-1, also known as CD54).
  • IAM-1 intercellular adhesion molecule type 1
  • the transmembrane protein is lymphocyte function-associated antigen 1 (LFA-1, also known as Integrin, alpha L, ITGAL, or CD11a).
  • the transmembrane glycoprotein is platelet endothelial cell adhesion molecule type 1 (PECAM-1, also known as CD31).
  • the cell is a vascular cell or an immune cell.
  • 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.
  • 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, 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).
  • M macrophage
  • T cell T cell
  • B cell a dendritic cell
  • DC dendritic cell
  • NK cell a NK cell
  • neutrophils a neutrophils
  • eosinophil a basophil
  • mast cell a helper t cell (th1, th2, th17), a regulatory t cell (treg)
  • the antibody-mitochondrion conjugate comprises an anti-PECAM-1 ( -PECAM-1) antibody bound to a mitochondrion, and the cell is a vascular cell.
  • the vascular cell is an endothelial cell (EC).
  • the antibody-mitochondrion conjugate comprises an anti-LFA-1 ( - LFA-1) antibody bound to a mitochondrion, and the cell is an immune cell.
  • M s depicts both mitochondria with 1-Azidoethyl-choline (Az-Cho-mito) and mitochondria with propargyl choline (P-Cho-mito) are taggable to macrophages (M s).
  • FIG. 2 depicts mitochondrial transplantation (mt-tfr) modulates macrophage (M ) and endothelial cell (EC) function.
  • 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 dual detection of mitochondria after delivery.
  • the cells endothelial cells
  • the images demonstrate that donor mitochondria have colocalize and seemingly integrate into the recipient cell’s native mitochondrial network.
  • 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.
  • PE R-phycoerythrin
  • 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.
  • FIG. 5 depicts mitochondrial dysfunction (mt-dys) in cells and tissue isolated from mice with surgically induced abdominal aortic aneurysms (AAA).
  • ETC electron transport chain
  • FIG. 6 depicts impact of mito-transfer with P-cho mito on mitochondrial ROS production in macrophages (M s).
  • M s macrophages
  • 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.
  • MitoSOX 5 uM
  • 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.
  • 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).
  • FIG. 1 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).
  • MFI of Mitoview Green MTV-G
  • 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), and antibody-conjugated mitochondria ( -CD31 mito-tag).
  • 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.
  • 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, -CD31 antibody (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) endothelial cells (ECs) treated with or without interferon gamma (IFN ), naked mitochondria (nkd), or - CD31 mito-tags (mito-tags).
  • IFN interferon gamma
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • mtDNA mitochondrial DNA
  • 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.
  • CC a highly selective biochemical reaction involving azide and alkyne functional groups, can be harnessed to tag cellular components like phospholipids 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.
  • 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.
  • compositions of engineered mitochondria or antibody- mitochondrion conjugates for targeted delivery to cells, tissue, and organs are provided.
  • 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.
  • the subject is an aged individual. II.
  • 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.
  • 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.
  • 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.
  • antibody means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope.
  • 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.
  • 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.
  • subject refers 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.
  • 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.
  • 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.
  • 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.
  • 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]
  • 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).
  • 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.
  • an effective amount depends on a variety of factors including the distribution profile of a therapeutic 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.
  • a therapeutic agent e.g., engineered mitochondria or antibody-mitochondrion conjugates
  • 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.
  • pharmacological parameters e.g., half-life in the body
  • other factors such as age and gender, etc.
  • 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
  • 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.
  • the carrier is an agent that facilitates the delivery of the engineered mitochondria or the antibody-mitochondrion conjugates to a target cell or tissue.
  • the carrier is an agent that facilitates the delivery of the engineered mitochondria or the antibody-mitochondrion conjugates to a target cell or tissue.
  • 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.
  • 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 cells 8 . This work strongly supports the use of mt-tfr as a strategy for extending health span.
  • Extracellular delivery of mitochondria 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.
  • 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.
  • ATP adenosine triphosphate
  • the central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs cycle, and oxidative phosphorylation.
  • 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.
  • mtFASII mitochondrial fatty acid synthesis
  • the present disclosure provides an engineered mitochondrion comprising one or more modified phospholipids on the mitochondrial membrane (MM) of the mitochondria.
  • the one or more modified phospholipids are on the outer mitochondrial membrane (OMM).
  • the one or more modified phospholipids are on the inner mitochondrial membrane (IMM).
  • 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 IMM accessibility 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.
  • detergents e.g., saponin, digitonin
  • 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.
  • the engineered mitochondrion comprises one or more modified phospholipids on both the OMM and IMM.
  • 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.
  • Tagging the IMM with therapeutic payloads allows for the precise conjugation of drugs to mitochondria.
  • 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 an antioxidant agent
  • 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.
  • CRISPR/Cas9-mRNA can be conjugated to the modified phospholipids on the IMM via click chemistry, enabling mitochondrial genome editing.
  • 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.
  • 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.
  • the modified phospholipids are azide-modified phospholipids.
  • 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).
  • PAs phosphatidic acids
  • 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 engineered mitochondrion links to the antibody and/or the therapeutic payload via an azide-alkyne cycloaddition reaction.
  • 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. More description of anti-transmembrane antibodies is disclosed below in next section.
  • AMCs Antibody-mitochondrion conjugates
  • the present disclosure provides an antibody-mitochondrion conjugate (AMC, or Ab-MC) comprising an antibody and a mitochondrion.
  • the AMC comprises a functional mitochondrion.
  • the AMC comprises a mitochondrial membrane potential.
  • the antibody and the mitochondrion of the AMC are covalently or non-covalently linked.
  • the antibody is non-covalently linked to the mitochondrion.
  • 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.
  • the antibody is covalently linked to the mitochondrion.
  • the antibody is linked to the mitochondrion through a linker.
  • 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]
  • 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.
  • transmembrane protein refers to a type of integral membrane protein that spans the entirety of the cell membrane.
  • 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.
  • Mac-1 macrophage adhesion ligand-1
  • LFA-1 leukocyte function associated antigen-1
  • 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).
  • 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.
  • 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).
  • the transmembrane protein is platelet endothelial cell adhesion molecule-1 (PECAM-1).
  • 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.
  • the linker is a click chemistry linker.
  • any linker that undergoes a click chemistry reaction can be applied to link the antibody and the mitochondrion.
  • 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.
  • the linker is derived from phospholipids, polymers, peptides, or other biocompatible linker materials.
  • the linker comprises an azide-modified phospholipid.
  • linkers known in the art good for antibody-drug conjugates can be also useful in the present disclosure.
  • Such linkers can be up to 30 carbon atoms in length.
  • 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.
  • a linker may comprise one or more linker components.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • Cleavable linkers may optionally further comprise one or more additional components such as self-immolative and self-elimination groups, stretchers or hydrophilic moieties.
  • 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).
  • PABC p-aminobenzyloxycarbonyl
  • PABE p-aminobenzyl ether
  • MED methylated ethylene diamine
  • 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.
  • 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.
  • 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.
  • 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).
  • SPBD polyethylene glycol
  • mPEG stretchers monomethoxy polyethylene glycol
  • AD adipate
  • 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.
  • 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).
  • 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.
  • the click reactive functional group includes, without limitation, an azide group, a nitrone group or an alkyne group.
  • 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.
  • the alkyne group is a dibenzocyclooctyne (DBCO) group or a difluorooctyne (DIFO) group.
  • 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.
  • the engineered mitochondria and/or the antibody-mitochondrion conjugates can be delivered to a target cell.
  • 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.
  • the engineered mitochondrion or the antibody-mitochondrion conjugate is delivered to a vascular cell.
  • 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.
  • EC endothelial cell
  • VSMC vascular smooth muscle cell
  • a smooth muscle cell a cardiomyocyte
  • a pericyte a pericyte
  • fibroblast a fibroblast
  • cardiac fibroblast a cardiac fibroblast
  • an endothelial progenitor cell a pacemaker cell
  • an adventitial cell an adventitial cell.
  • the engineered mitochondrion or the antibody-mitochondrion conjugate is delivered to an immune cell.
  • 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, 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).
  • M macrophage
  • T cell T cell
  • B cell a dendritic cell
  • DC dendritic cell
  • NK cell a NK cell
  • neutrophils a neutrophils
  • eosinophil a basophil
  • mast cell a helper t cell (th1, th2, th17), a regulatory t cell (treg)
  • the engineered mitochondria and/or the antibody-mitochondrion conjugates can be delivered to a subject in need for disease treatment.
  • 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-associated abnormalities.
  • 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.
  • Non-limiting exemplary target diseases are listed below, as breakdown by systems of the body.
  • 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.
  • CAD coronary artery disease
  • DCM dilated cardiomyopathy
  • HCM hypertrophic cardiomyopathy
  • HF heart failure
  • ischemic heart disease ischemic heart disease
  • MI myocardial infarction
  • arrhythmias arrhythmias
  • atherosclerosis atherosclerosis
  • 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.
  • ALS amyotrophic lateral sclerosis
  • MS multiple sclerosis
  • epilepsy stroke, migraine, neuropathies (such as diabetic neuropathy),
  • 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 biliary cholangitis, Dermatomyositis, Polymyositis, Wegener's granulomatosis (Granulomatosis with polyangiitis), and Chronic fatigue syndrome (CFS), COVID-19.
  • SLE Systemic lupus ery
  • 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.
  • COPD Chronic Obstructive Pulmonary Disease
  • Asthma Pulmonary Fibrosis
  • Pulmonary Hypertension Cystic Fibrosis
  • Cystic Fibrosis Acute Respiratory Distress Syndrome (ARDS), Lung Cancer, Bronchiectasis, Respiratory Syncytial Virus (RSV) infection, and Tuberculosis.
  • ARDS Acute Respiratory Distress Syndrome
  • RSV Respiratory Syncytial Virus
  • Tuberculosis [0101] Digestive: Crohn's Disease, Ulcerative Colitis, Hepatic Steatosis (Non-Al
  • 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.
  • Thyroid Disorders such as Hashimoto's Thyroiditis and Graves' Disease
  • PCOS Polycystic Ovary Syndrome
  • Obesity Obesity
  • Metabolic Syndrome Osteoporosis
  • Addison's Disease Addison's Disease
  • Pituitary Adenomas affecting hormone production, regulation, and energy metabolism.
  • Musculoskeletal Osteoarthritis, Rheumatoid Arthritis, Muscular Dystrophies, Fibromyalgia, Osteoporosis, Sarcopenia (age-related muscle loss), Lupus Erythematosus, Ankylosing Spondylitis, Paget's Disease of Bone, and Tendinopathy.
  • 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.
  • the present disclosure provides a composition comprising the engineered mitochondrion or the antibody-mitochondrion conjugate described herein.
  • 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.
  • 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.
  • 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 the patient. In any event, the methods provide a sufficient quantity of the engineered mitochondrion or the antibody-mitochondrion conjugate to effectively treat the patient.
  • 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.
  • the composition may be administered by insufflation.
  • 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.
  • 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.
  • 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.
  • kits Materials and reagents to carry out the various methods of the present disclosure can be provided in kits to facilitate execution of the methods.
  • kit includes a combination of articles that facilitates a process, assay, analysis, or manipulation.
  • kits of the present disclosure find utility in a wide range of applications including, for example, diagnostics, prognostics, therapy, and the like.
  • Kits can contain chemical reagents as well as other components.
  • 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.
  • 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.
  • Methods for Treatment [0117]
  • 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.
  • CVD cardiovascular disease
  • 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.
  • the pharmaceutical composition comprises an antibody-mitochondrion conjugate, comprising an antibody and a mitochondrion.
  • the antibody and the mitochondrion are covalently linked through a linker.
  • the antibody and the mitochondrion are covalently linked via an azide-alkyne cycloaddition reaction.
  • 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.
  • administering a pharmaceutical composition comprising an engineered mitochondrion or antibody-mitochondrion conjugate can reduce reactive oxygen species (ROS) levels and alleviate oxidative stress (e.g., reduce IFN -induced mitochondrial superoxide production). In some embodiments, the administration of the pharmaceutical composition disclosed herein can further enhance glycolysis and promote endothelial regeneration.
  • ROS reactive oxygen species
  • the administration of the pharmaceutical composition disclosed herein modulates cellular energy metabolism, reprogram metabolism to meet energy demands, 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.
  • the engineered mitochondrion or antibody-mitochondrion conjugate disclosed herein can be used to prevent or treat inflammatory conditions.
  • 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.
  • 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.
  • the subject is an aged individual.
  • 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.
  • the antibody-mitochondrion conjugate is an anti-LFA-1 antibody conjugated with a mitochondrion, useful for treating immune dysfunctions in a subject.
  • the subject is an aged individual.
  • 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.
  • the antibody and the mitochondrion are covalently linked through a linker.
  • the linker comprises an azide-modified phospholipid and/or an alkyne-modified phospholipid.
  • 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 variant thereof.
  • Az-Cho azido-ethyl-phosphocholine
  • Az-PE azide-modified phosphatidylethanolamine
  • Az-PS azide-modified phosphatidylserine
  • Az-PI azide- modified phosphatidylinositol
  • 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.
  • P-Cho propargyl choline
  • Ak-PE alkyne-modified phosphatidylethanolamine
  • Ak-PS alkyne-modified phosphatidylserine
  • Ak-PI alkyne-modified phosphatidylinositol
  • Ak-PA alkyne-modified phosphatidic acid
  • the antibody and the mitochondrion are covalently linked via an azide-alkyne cycloaddition reaction.
  • the cell surface protein is selected from the antibody targets listed in Table 1.
  • the cell surface protein is a transmembrane protein.
  • the transmembrane protein is intercellular adhesion molecule type 1 (ICAM-1).
  • the transmembrane protein is lymphocyte function- associated antigen 1 (LFA-1).
  • the transmembrane glycoprotein is platelet endothelial cell adhesion molecule type 1 (PECAM-1).
  • the cell is selected from the target cells listed in Table 3.
  • the cell is a vascular cell.
  • 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.
  • the vascular cell is an endothelial cell (EC) or a vascular smooth muscle cell (VSMC).
  • the cell is an immune cell.
  • 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, 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).
  • M macrophage
  • T cell T cell
  • B cell a dendritic cell
  • DC dendritic cell
  • NK cell a NK cell
  • neutrophils a neutrophils
  • eosinophil a basophil
  • mast cell a helper t cell (th1, th2, th17), a regulatory t cell (treg)
  • the immune cell is a macrophage (M ), a T cell, a B cell, or a dendritic cell (DC).
  • M macrophage
  • DC dendritic cell
  • the targeted delivery of the engineered mitochondrion or antibody-mitochondrion conjugate disclosed herein can improve systemic circulation and ensure precise mitochondrial targeting.
  • 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.
  • 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.
  • 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).
  • 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).
  • immune cells e.g., CD45+ cells.
  • Example 1 Extracellular Tagging Capacity of Bioengineered Mitochondria
  • This example illustrates a safe level of azide and alkyne modifications on the outer mitochondrial membrane (OMM) does not impair mitochondrial function.
  • OMM outer mitochondrial membrane
  • CuAAC copper-catalyzed
  • SPAAC strain-promoted
  • mouse fibroblasts 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.
  • SHE mitochondrial buffer
  • 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 median fluorescent intensity (MFI) of DBCO-Cy5+ M s (FIGs. 1A-F).
  • MFI median fluorescent intensity
  • Z-stacks Confocal orthogonal projections (Z-stacks) of M s with Az-Cho-mito also confirmed the presence of the DBCO- Cy5 signal.
  • 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 ⁇ M, or using an unsupplemented DMEM (Wt- Cho) as a control.
  • P-Cho propargyl choline
  • Wt- Cho unsupplemented DMEM
  • 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.
  • TEM transmission electron microscopy
  • 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).
  • 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 be tested.
  • Flow cytometry can be employed to quantify the frequencies and mean fluorescent intensity (MFI) of tagged mitochondria.
  • 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.
  • 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 .
  • OMM outer mitochondrial membrane
  • mt-tfr Mitochondrial Transplantation
  • mEV-mediated transfer is believed to involve specific ligand-receptor interactions 7 .
  • 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.
  • adhesion molecules on endothelial cells (ECs) like ICAM-1 and PECAM-1 have each been previously exploited for intracellular delivery of biological materials 12 .
  • mt-dys mitochondrial dysfunction
  • 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 prepared using methods optimized in AIM 1.
  • the cardiolipin (CL) content in each sample can be determined, equated, and quantified using commercially available kits.
  • the MEFs utilized in this example will be isolated from mito::mKate2 mice, inherently expressing far-red fluorescent mitochondria.
  • 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.
  • Co-culture experiments the specificity of ab-mito can be assessed in co-cultures of ECs and M s. ECs from both young and old mice will be cultured under basal and pro- inflammatory conditions.
  • FIG. 3 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).
  • 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.
  • donor mitochondria are internalized. This is proof of principle that antibody-tagged mitochondria can be effectively delivered to cells.
  • 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.
  • Example 3 Specificity and Efficacy of Click Chemistry Mediated Mitochondrial Transplantation in an In Vivo Mouse Model of Cardiovascular Disease (CVD)
  • CVD In Vivo Mouse Model of Cardiovascular Disease
  • 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.
  • AAA abdominal aortic aneurysm
  • Background Abdominal aortic aneurysm (AAA), a CVD closely linked to aging, is characterized by the irreversible ballooning of the infrarenal aorta 3, 13-18 .
  • Treatment Groups will consist of PPE mice receiving either saline, a bolus of ab- mitochondria ( ⁇ 300 ⁇ g) 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.
  • 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.
  • 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 and aortic inflammation in AAA mice.
  • 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., F4/80 for M , CD3 for T-cells).
  • cell-specific markers e.g., F4/80 for M , CD3 for T-cells.
  • 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.
  • Mitochondrial fluorescent tags/proteins are usually expressed on nuclear genes and after mt-tfr, the mitoGFP signal could be lost.
  • mtDNA of donor mitochondria can be tagged with the molecule 6-O- Propynyl-dG (PdG), which can be visualized via click-chemistry 31 .
  • PdG 6-O- Propynyl-dG
  • 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 humans 32 , and it may be necessary to perform mt-tfr in a second model of AAA such as the angiotensin II model (Ang-II) model 33 .
  • Ang-II angiotensin II model
  • the Ang-II model requires atherosclerotic-susceptible strain (apolipoprotein E deficient; apoE / ) mice as compared to wild-type C57BL/6 mice used in the PPE model 32, 33 .
  • atherosclerotic-susceptible strain apolipoprotein E deficient; apoE /
  • apoE / apolipoprotein E deficient mice
  • 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.
  • 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.
  • the source of donor mitochondria i.e., donor cells
  • the appropriate alkyne and azide-modified phospholipids for anywhere between 30 mins to 72 hrs.
  • 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.
  • 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.
  • 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)
  • BSA defatted bovine serum albumin
  • 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).
  • the isolated t-mito can then be incubated with their respective/reciprocal azido/alkyne-tagged antibody, which will then undergo Click Chemistry reactions (CC).
  • Non-azido/alkyne-conjugated antibodies can be either commercially purchased or generated in house.
  • kits to modify antibodies include SiteClickTM Antibody Azido Modification Kit (Invitrogen, S20026) and SiteClickTM sDIBO Alkyne Kits for Antibody Labeling (Invitrogen, C20031).
  • 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.
  • linkers that contain the appropriate alkyne/azide side chain/side group capable of undergoing conjugation with t-mito.
  • 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)).
  • CuAAC copper-catalyzed
  • SPAAC strain-promoted
  • 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).
  • 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-iTTM Plus Alexa FluorTM 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.
  • TBTA tris(benzyltriazolylmethyl)amine
  • Ab-MCs 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.
  • IV intravenous
  • SC subcutaneous
  • IM intramuscular
  • IP intraperitoneal
  • T intrathecal
  • topical intra- articular
  • intraocular intraocular
  • inhalation or oral administrations.
  • 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).
  • 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
  • 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.
  • ECs exposed to either ndk-mito or -CD31-PE-Mito were fixed with 4% PFA, and nuclei counterstained with Hoechst 33342.
  • ECs were pre- stained with Mito-tracker Deep Red to determine the proximity of the donor mitochondria to the recipient cell’s mitochondrial network.
  • 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).
  • 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.
  • 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).
  • MFI mean fluorescence intensity
  • 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 critical for enhancing mitochondrial abundance in ECs.
  • 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).
  • MFI mean fluorescence intensity
  • MTV-G Mitoview Green
  • MFI mean fluorescence intensity
  • -CD31 antibody only
  • naked mitochondria nk- mito
  • -CD31 mito-tag antibody-conjugated mitochondria
  • ROS levels were detected roughly 24 hours post-treatment.
  • the data provided insights into ROS levels in response to mitochondrial treatments and PECAM1-mediated targeting.
  • FIG. 14 when the baseline DCF-DA signal was normalized to 1.0, representing the untreated condition or baseline ROS levels (Control), cells treated with PECAM1 antibodies ( -CD31) showed a significant reduction in ROS levels compared to the control (p ⁇ 0.01).
  • FIG. 15 highlighted the bioenergetic improvements achieved through -CD31 mito-tag treatment in aged aortic endothelial cells (O- AoECs).
  • O- AoECs aged aortic endothelial cells
  • 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.
  • FIG. 15B the energetic map illustrated the shift toward glycolysis in -CD31- mito-tag treated ECs.
  • untreated AoECs demonstrated a greater dependence on Oxidative Phosphorylation.
  • 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.
  • FIG. 15B the energetic map illustrated the shift toward glycolysis in -CD31- mito-tag treated
  • FIG. 16A highlighted the relative mean fluorescence intensity (MFI) of DCF-DA, a reactive oxygen species (ROS) indicator, under different experimental conditions involving IFN treatment, -CD31 antibody (control), naked mitochondria (nkd-mt), and -CD31 mito- tags.
  • MFI mean fluorescence intensity
  • ROS reactive oxygen species
  • MFI mean fluorescence intensity
  • Mitochondrial bioenergetic assay under inflammatory conditions 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.
  • the analysis compared IFN -pretreated aged ECs treated with mito-tags to untreated controls, as well as to ECs exposed to nkd-mitochondria.
  • Mitochondrial bioenergetics were assessed under inflammatory conditions induced by IFN to evaluate the therapeutic effects of naked mitochondria (nkd-Mito) and -CD31 antibody-conjugated mitochondria ( -CD31-mito-tag).
  • nkd-Mito naked mitochondria
  • -CD31-mito-tag -CD31 antibody-conjugated mitochondria
  • FIG. 17A basal mitochondrial respiration, as indicated by oxygen consumption rates (OCR), was not significantly reduced in IFN -stimulated cells.
  • OCR oxygen consumption rates
  • Treatment with -CD31 did not impact basal OCR.
  • Treatment with nkd-mito or -CD31-mito-tags significantly decreased basal respiration.
  • FIGs.17B and 17E no significant change in maximal mitochondrial respiration, as well as non-mitochondrial OCR was observed across treatment groups.
  • FIGs.17B and 17E no significant change in maximal mitochondrial respiration, as well as non-mitochondrial OCR was observed across
  • 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, as shown in FIG. 18A, IFN treatment alone led to a notable increase in basal glycoPER compared to the control.
  • Rho endothelial cells 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.
  • 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 to quantify recent proliferation. Proliferation was further evaluated in the context of the cells' inability to replicate without exogenous uridine, emphasizing the importance of mitochondrial functionality.
  • Experiment 1 Single Time Point (48 Hours Post-Mitochondrial Transfer), as shown in FIG.
  • 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.
  • 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.
  • FIG. 22 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.
  • 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 the specificity and distribution of injected mitochondria in the experimental groups.
  • 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.
  • 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.
  • -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.
  • Example 8 Ex Vivo Study of -CD11a Mito-Tag Delivery
  • This example illustrates the uptake of -CD11a-tagged mitochondria by aged splenocytes in an ex vivo study.
  • 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.
  • 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) was determined 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.
  • MFI mean fluorescence intensity
  • the flow cytometry scatter plot of the nkd-mito uptake by aged splenocytes 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.
  • 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.
  • 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.
  • modified cardiolipin (CL) and outer membrane phospholipids 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.
  • 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 ⁇ M. Through natural lipid trafficking pathways, the modified phospholipids integrate into the inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) over 4-24 hours.
  • IMM inner mitochondrial membrane
  • OOMM outer mitochondrial membrane
  • 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.
  • 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.
  • 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.
  • PE or PC bioorthogonal-modified OMM phospholipids
  • 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
  • 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.
  • 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.
  • live-cell imaging Mitsubishi colocalization
  • lipidomics lipidomics
  • Seahorse XF metabolic analysis validate mitochondrial function and therapeutic effects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Zoology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente divulgation concerne des mitochondries modifiées et des conjugués anticorps-mitochondries pour une administration ciblée à des cellules. La présente divulgation concerne en outre des méthodes d'utilisation des mitochondries modifiées et des conjugués anticorps-mitochondries pour le traitement d'une maladie cardiovasculaire (MCV) ou d'un dysfonctionnement mitochondrial (mt-dys) chez un sujet.
PCT/US2025/017885 2024-03-01 2025-02-28 Mitochondries bio-modifiées pour administration ciblée à des cellules Pending WO2025184537A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463560421P 2024-03-01 2024-03-01
US63/560,421 2024-03-01

Publications (1)

Publication Number Publication Date
WO2025184537A1 true WO2025184537A1 (fr) 2025-09-04

Family

ID=95065580

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/017885 Pending WO2025184537A1 (fr) 2024-03-01 2025-02-28 Mitochondries bio-modifiées pour administration ciblée à des cellules

Country Status (1)

Country Link
WO (1) WO2025184537A1 (fr)

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
WO2002088172A2 (fr) 2001-04-30 2002-11-07 Seattle Genetics, Inc. Composes pentapeptidiques et leurs utilisations
US7375078B2 (en) 2004-02-23 2008-05-20 Genentech, Inc. Heterocyclic self-immolative linkers and conjugates
US7521541B2 (en) 2004-09-23 2009-04-21 Genetech Inc. Cysteine engineered antibodies and conjugates
US7553816B2 (en) 2001-09-24 2009-06-30 Seattle Genetics, Inc. p-amidobenzylethers in drug delivery agents
US8455622B2 (en) 2006-12-01 2013-06-04 Seattle Genetics, Inc. Variant target binding agents and uses thereof
US9000130B2 (en) 2010-06-08 2015-04-07 Genentech, Inc. Cysteine engineered antibodies and conjugates
US20180057610A1 (en) * 2016-01-15 2018-03-01 Children's Medical Center Corporation Therapeutic Use of Mitochondria and Combined Mitochondrial Agent
US20220160782A1 (en) * 2019-02-15 2022-05-26 Children`S Medical Center Corporation Treating Heart Failure
US20220395531A1 (en) * 2019-04-15 2022-12-15 Children`S Medical Center Corporation Aerosolized compositions comprising mitochondria and methods of use thereof
WO2023237788A1 (fr) * 2022-06-10 2023-12-14 Cellvie Ag Mitochondries en tant que plateforme d'administration ciblée
WO2023237789A1 (fr) * 2022-06-10 2023-12-14 Cellvie Ag Composition et procédé de cryoconservation de mitochondries

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
WO2002088172A2 (fr) 2001-04-30 2002-11-07 Seattle Genetics, Inc. Composes pentapeptidiques et leurs utilisations
US7553816B2 (en) 2001-09-24 2009-06-30 Seattle Genetics, Inc. p-amidobenzylethers in drug delivery agents
US7375078B2 (en) 2004-02-23 2008-05-20 Genentech, Inc. Heterocyclic self-immolative linkers and conjugates
US7521541B2 (en) 2004-09-23 2009-04-21 Genetech Inc. Cysteine engineered antibodies and conjugates
US8455622B2 (en) 2006-12-01 2013-06-04 Seattle Genetics, Inc. Variant target binding agents and uses thereof
US9000130B2 (en) 2010-06-08 2015-04-07 Genentech, Inc. Cysteine engineered antibodies and conjugates
US20180057610A1 (en) * 2016-01-15 2018-03-01 Children's Medical Center Corporation Therapeutic Use of Mitochondria and Combined Mitochondrial Agent
US20220160782A1 (en) * 2019-02-15 2022-05-26 Children`S Medical Center Corporation Treating Heart Failure
US20220395531A1 (en) * 2019-04-15 2022-12-15 Children`S Medical Center Corporation Aerosolized compositions comprising mitochondria and methods of use thereof
WO2023237788A1 (fr) * 2022-06-10 2023-12-14 Cellvie Ag Mitochondries en tant que plateforme d'administration ciblée
WO2023237789A1 (fr) * 2022-06-10 2023-12-14 Cellvie Ag Composition et procédé de cryoconservation de mitochondries

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
AGARD ET AL.: "A Comparative Study of Bioorthogonal Reactions with Azides", ACS CHEM. BIOL., vol. 1, no. 10, 2006, pages 644 - 648, XP002522847, DOI: 10.1021/CB6003228
AMSBERRY ET AL., J. ORG. CHEM., vol. 55, 1990, pages 5867 - 5877
AXUP ET AL., PNAS, vol. 109, 2012, pages 16101 - 16106
CINDY Y. JAO ET AL: "Biosynthetic Labeling and Two-Color Imaging of Phospholipids in Cells", CHEMBIOCHEM, vol. 16, no. 3, 13 January 2015 (2015-01-13), Hoboken, USA, pages 472 - 476, XP055538122, ISSN: 1439-4227, DOI: 10.1002/cbic.201402149 *
DE GRAAFET, CURR. PHARM. DES., vol. 8, 2002, pages 1391 - 1403
DUBOWCHIK ET AL., TETRAHEDRON LETTERS, vol. 38, 1997, pages 5257 - 60
FRISCH ET AL., BIOCONJUGATE CHEM, vol. 7, 1996, pages 180 - 186
GAI MEIYU ET AL: "A bio-orthogonal functionalization strategy for site-specific coupling of antibodies on vesicle surfaces after self-assembly", POLYMER CHEMISTRY, vol. 11, no. 2, 14 October 2019 (2019-10-14), Cambridge, pages 527 - 540, XP055834449, ISSN: 1759-9954, DOI: 10.1039/C9PY01136F *
HOFER ET AL., BIOCHEMISTRY, vol. 48, 2009, pages 12047 - 12057
JEWETT. JOHN C.BERTOZZI, CAROLYN. R.: "Cu-free click cycloaddition reactions in chemical biology", CHEM SOC REV, vol. 39, no. 4, 2010, pages 1272 - 1279, XP002659163, DOI: 10.1039/b901970g
LEE DONG-YUP ET AL: "Strategies for targeted gene delivery using lipid nanoparticles and cell-derived nanovesicles", NANOSCALE ADVANCES, vol. 5, no. 15, 1 January 2023 (2023-01-01), pages 3834 - 3856, XP093267763, ISSN: 2516-0230, DOI: 10.1039/D3NA00198A *
MACKENZIE ET AL.: "Strain-promoted cycloadditions involving nitrones and alkynes-rapid tunable reactions for biorthogonal labeling", CURRENT OPINION IN CHEMICAL BIOLOGY, vol. 21, 2014, pages 81 - 88, XP093074345, DOI: 10.1016/j.cbpa.2014.05.023
RODRIGUES ET AL., CHEMISTRY BIOLOGY, vol. 2, 1995, pages 223 - 227
SUN MINGCHU ET AL: "Mitochondrial transplantation as a novel therapeutic strategy for cardiovascular diseases", JOURNAL OF TRANSLATIONAL MEDICINE, vol. 21, no. 1, 25 May 2023 (2023-05-25), XP093267672, ISSN: 1479-5876, Retrieved from the Internet <URL:https://link.springer.com/article/10.1186/s12967-023-04203-6/fulltext.html> DOI: 10.1186/s12967-023-04203-6 *
TAMURA TOMONORI ET AL: "Organelle membrane-specific chemical labeling and dynamic imaging in living cells", NATURE CHEMICAL BIOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 16, no. 12, 21 September 2020 (2020-09-21), pages 1361 - 1367, XP037295475, ISSN: 1552-4450, [retrieved on 20200921], DOI: 10.1038/S41589-020-00651-Z *
TOKI ET AL., J. ORG. CHEM., vol. 67, 2002, pages 1866 - 1872
WALKER, M. A., J. ORG. CHEM., vol. 60, 1995, pages 5352 - 5355
WU, PNAS, vol. 106, 2009, pages 3000 - 3005
ZIMMERMAN ET AL., BIOCONJ. CHEM., vol. 25, 2014, pages 351 - 361

Similar Documents

Publication Publication Date Title
Moon et al. Anti-PD-L1 peptide-conjugated prodrug nanoparticles for targeted cancer immunotherapy combining PD-L1 blockade with immunogenic cell death
Li et al. Sono/photodynamic nanomedicine‐elicited cancer immunotherapy
Kuai et al. High-density lipoproteins: nature’s multifunctional nanoparticles
Xiong et al. Pursuing specific chemotherapy of orthotopic breast cancer with lung metastasis from docking nanoparticles driven by bioinspired exosomes
Yang et al. Targeting small molecule drugs to T cells with antibody-directed cell-penetrating gold nanoparticles
CA2680206C (fr) Systemes de distribution de nanoparticules pour des peptides d&#39;integration de la membrane
Karatas et al. A nanomedicine transports a peptide caspase-3 inhibitor across the blood–brain barrier and provides neuroprotection
Dakwar et al. Delivery of proteins to the brain by bolaamphiphilic nano-sized vesicles
EP3261677B1 (fr) Transplantation ciblée de mitochondries dans des hépatocytes
EP3238746B1 (fr) Lipoprotéine haute densité et administration de médicament au segment postérieur de l&#39; oeil par instillation oculaire de ladite lipoprotéine haute densité fusionnée à un peptide cytophile
WO2015038924A1 (fr) Nanoparticules paramagnétiques modifiées pour la délivrance ciblée de produits thérapeutiques et procédés associés
Dave et al. Delivery of mitochondria-containing extracellular vesicles to the BBB for ischemic stroke therapy
Clayton et al. Sustained and specific in vitro inhibition of HIV-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes
US20170332910A1 (en) Modified paramagnetic nanoparticles for targeted delivery of therapeutics and methods thereof
CN112048002A (zh) 一种靶向t细胞的酶敏感纳米体系及其制备方法和应用
Zhang et al. Nanointegrative in situ reprogramming of tumor-intrinsic lipid droplet biogenesis for low-dose radiation-activated ferroptosis immunotherapy
Wöll et al. Sortagged anti-EGFR immunoliposomes exhibit increased cytotoxicity on target cells
Moon et al. Engineered nanoparticles for enhanced antitumoral synergy between macrophages and T cells in the tumor microenvironment
JP2022524753A (ja) 電荷修飾グロビンを含む抗腫瘍細胞
WO2025184537A1 (fr) Mitochondries bio-modifiées pour administration ciblée à des cellules
US20150017098A1 (en) Carrier that targets fucosylated molecule-producing cells
Zou et al. Sequential rocket-mode bioactivating ticagrelor prodrug nanoplatform combining light-switchable diphtherin transgene system for breast cancer metastasis inhibition
WO2023237842A1 (fr) Microbulles lipidiques pour la delivrance ciblee d&#39;actifs
Sarisozen et al. Intracellular delivery of proteins and peptides
Yang et al. Novel Globo H targeting antibody–drug conjugate with binding specificity and anti-tumor efficacy in multiple cancer types

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25713445

Country of ref document: EP

Kind code of ref document: A1