Method for reducing mitochondrial DNA leakage

Abstract

The present application relates to a method for reducing mitochondrial DNA leakage, which method comprises localizing a nuclease to a mitochondrion to reduce mitochondrial DNA leakage.

Description

A method of reducing mitochondrial DNA leakage TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a method of reducing mitochondrial DNA leakage, thereby treating diseases and / or disorders associated therewith. BACKGROUND

[0002] Mitochondria are often referred to as the "energy factories" of cells because they play a central role in the energy metabolism process of cells. They are double-membrane organelles that contain their own genome, mitochondrial DNA (mtDNA), inside. Unlike nuclear DNA, mtDNA is naked, lacks protective histones, and its repair mechanism is imperfect. Under normal circumstances, mtDNA is responsible for encoding some key mitochondrial proteins that participate in the energy metabolism process of cells. However, when mitochondria are damaged or dysfunctional, their membrane integrity can be disrupted, leading to leakage of mtDNA into the cytoplasm. Because mtDNA has genetic material characteristics similar to prokaryotes, it can be recognized by DNA sensors in the cell, such as the cGAS-STING pathway. Once these sensors recognize exogenous or abnormal DNA, they activate a series of immune and inflammatory responses, including type I interferon response.

[0003] This inflammatory response triggered by mitochondrial DNA leakage may help cells cope with damage in the short term, but if it persists, it can lead to cell aging, tissue dysfunction, and even cell death. In the long term, this chronic inflammatory state is associated with the development of various diseases, including cardiovascular diseases. The development of these diseases may be related to various pathological mechanisms caused by mitochondrial dysfunction, such as oxidative stress, energy metabolism disorders, apoptosis, and decreased tissue repair capacity. Therefore, mitochondrial DNA leakage and the chain reaction it triggers are key links in the pathogenesis of many major diseases.

[0004] However, there is no treatment method developed targeting mitochondrial DNA leakage as a target. SUMMARY

[0005] The present application provides a method of reducing mitochondrial DNA leakage, comprising reducing mitochondrial DNA leakage in a cell of interest by localizing a nuclease to mitochondria. The present application also provides a method for delaying aging, preventing and / or treating related diseases according to the method of reducing mitochondrial DNA leakage.

[0006] The method for reducing mitochondrial DNA leakage described in the present application has one or more of the following beneficial effects: (1) can reduce mitochondrial DNA leakage caused by various factors; (2) can down-regulate downstream inflammatory pathways including the cGAS-STING pathway activated by mitochondrial DNA leakage, and the effect is better than that of pathway inhibitors; (3) can delay cell senescence induced by mitochondrial DNA leakage activation, such as cell senescence induced by cGAS-STING pathway activation; (4) remove leaked mitochondrial DNA and restore mitochondrial respiratory function, etc.

[0007] In one aspect, a method for reducing mitochondrial DNA leakage in a cell of interest, comprising localizing a nuclease to a mitochondrion.

[0008] In certain embodiments, wherein the nuclease comprises a human nuclease.

[0009] In certain embodiments, wherein the nuclease comprises a DNAse.

[0010] In certain embodiments, wherein the DNAse comprises a member of the DNase I family and / or a functional variant thereof.

[0011] In certain embodiments, wherein the DNAse comprises DNase I and / or a functional variant thereof.

[0012] In certain embodiments, wherein the DNAse comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0013] In certain embodiments, wherein the nucleic acid molecule encoding the DNAse comprises the nucleotide sequence set forth in SEQ ID NO: 4.

[0014] In certain embodiments, wherein the nuclease is localized to the mitochondrion by a mitochondrial localization sequence.

[0015] In certain embodiments, wherein the mitochondrial localization sequence is directly or indirectly linked to the nuclease.

[0016] In certain embodiments, wherein the mitochondrial localization sequence is at the N-terminus or C-terminus of the nuclease.

[0017] In certain embodiments, wherein the mitochondrial localization sequence comprises a polypeptide sequence.

[0018] In certain embodiments, wherein the mitochondrial localization sequence comprises a polypeptide sequence derived from the N-terminal region or C-terminal region of a mitochondrial protein.

[0019] In certain embodiments, wherein the mitochondrial localization sequence comprises a polypeptide sequence derived from the N-terminal region or C-terminal region of a mitochondrial membrane protein.

[0020] In certain embodiments, wherein the mitochondrial localization sequence comprises a mitochondrial localization sequence derived from cytochrome c oxidase subunit 8A.

[0021] In certain embodiments, wherein the cytochrome c oxidase subunit 8A comprises a human cytochrome c oxidase subunit 8A.

[0022] In certain embodiments, wherein the mitochondrial localization sequence comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0023] In certain embodiments, wherein the nucleic acid molecule encoding the mitochondrial localization sequence comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0024] In certain embodiments, it comprises causing the cell of interest to express a fusion protein comprising a mitochondrial localization sequence and the nuclease and / or to express a nucleic acid molecule encoding the fusion protein.

[0025] In certain embodiments, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0026] In certain embodiments, wherein the nucleic acid molecule encoding the fusion protein comprises the nucleotide sequence set forth in SEQ ID NO: 6.

[0027] In certain embodiments, it comprises introducing an isolated modified mitochondrion into a cell of interest, wherein the nuclease is localized to the modified mitochondrion.

[0028] In certain embodiments, wherein the modified mitochondrion is isolated from a cell and / or a tissue.

[0029] In certain embodiments, wherein the cell from which the modified mitochondrion is isolated comprises a somatic cell, a germ cell, and / or a stem cell.

[0030] In certain embodiments, wherein the modified mitochondrion is isolated from an induced pluripotent stem cell.

[0031] In certain embodiments, wherein the modified mitochondrion is isolated from a muscle cell, a neural cell, an endothelial cell, an epithelial cell, an embryonic cell, and / or a tumor cell.

[0032] In certain embodiments, wherein the modified mitochondrion is isolated from a cardiac cell and / or a skeletal muscle cell.

[0033] In certain embodiments, wherein the modified mitochondrion is isolated from a cardiac cell differentiated from an induced pluripotent stem cell.

[0034] In certain embodiments, wherein the modified mitochondria are isolated from a mammalian cell.

[0035] In certain embodiments, wherein the modified mitochondria are isolated from a human or mouse cell.

[0036] In certain embodiments, wherein the modified mitochondria are isolated from a healthy cell.

[0037] In certain embodiments, wherein the modified mitochondria are isolated from a cell that is syngeneic to the cell of interest.

[0038] In certain embodiments, wherein the modified mitochondria are isolated from a cell that is allogeneic to the cell of interest.

[0039] In certain embodiments, wherein the modified mitochondria are isolated from a cell that is of the same cell type as the cell of interest.

[0040] In certain embodiments, wherein the modified mitochondria are isolated from a cell that is metabolically compatible with the cell of interest.

[0041] In certain embodiments, wherein the cell of interest comprises a cell with impaired mitochondria.

[0042] In certain embodiments, wherein the cell of interest comprises a cell with activation of the cGAS-STING pathway.

[0043] In certain embodiments, wherein the cell of interest comprises a cell in a state of senescence.

[0044] In certain embodiments, wherein the cell of interest comprises a cell with ischemia-reperfusion injury, hypoxic injury, and / or exposure to an oxidant.

[0045] In certain embodiments, wherein the cell of interest is derived from a mammal.

[0046] In certain embodiments, wherein the cell of interest is derived from a human or mouse.

[0047] In certain embodiments, wherein the cell of interest comprises a muscle cell, a neural cell, an endothelial cell, an epithelial cell, an embryonic cell, and / or a tumor cell.

[0048] In certain embodiments, wherein the cell of interest comprises a cardiomyocyte, a skeletal muscle cell, a vascular endothelial cell, a kidney epithelial cell, and / or a neuroblastoma cell.

[0049] In certain embodiments, wherein the mitochondrial DNA leakage in the cell of interest is caused by ischemia-reperfusion.

[0050] In certain embodiments, wherein the mitochondrial DNA leakage in the cell of interest is caused by aging.

[0051] In certain embodiments, wherein the mitochondrial DNA leakage in the cell of interest is caused by an anti-tumor therapy.

[0052] In certain embodiments, wherein the anti-tumor therapy comprises chemotherapy and / or radiotherapy.

[0053] In certain embodiments, wherein the chemotherapeutic drug comprises a platinum-based chemotherapeutic drug and / or an anthracycline-based chemotherapeutic drug.

[0054] In certain embodiments, wherein the anthracycline-based chemotherapeutic drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin.

[0055] In certain embodiments, wherein the platinum-based chemotherapeutic drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin.

[0056] In certain embodiments, wherein the mitochondrial DNA leakage in the cell of interest is caused by hypoxia.

[0057] In certain embodiments, wherein the mitochondrial DNA leakage in the cell of interest is caused by peroxide stimulation.

[0058] In certain embodiments, wherein the peroxide comprises H2O2.

[0059] In certain embodiments, wherein the method is capable of delaying aging of the cell of interest.

[0060] In certain embodiments, wherein the method is capable of alleviating damage of the cell of interest.

[0061] In certain embodiments, wherein the method is capable of suppressing inflammatory response in the cell of interest.

[0062] In certain embodiments, wherein the method is capable of suppressing downstream pathways activated by mitochondrial DNA leakage.

[0063] In certain embodiments, the downstream pathway comprises the cGAS-STING pathway.

[0064] In certain embodiments, wherein the method is capable of preserving the respiratory capacity of mitochondria.

[0065] In certain embodiments, wherein the method is in vitro and / or ex vivo.

[0066] In another aspect, the present application provides a modified mitochondrion, wherein a nuclease is localized to the mitochondrion.

[0067] In certain embodiments, wherein the nuclease is located at the outer membrane of the mitochondria.

[0068] In certain embodiments, wherein the nuclease comprises a human nuclease.

[0069] In certain embodiments, wherein the nuclease comprises a DNA nuclease.

[0070] In certain embodiments, wherein the DNA nuclease comprises a member of the DNase I family and / or a functional variant thereof.

[0071] In certain embodiments, wherein the DNA nuclease comprises DNase I and / or a functional variant thereof.

[0072] In certain embodiments, wherein the DNA nuclease comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0073] In certain embodiments, wherein the nucleic acid molecule encoding the DNA nuclease comprises the nucleotide sequence set forth in SEQ ID NO: 4.

[0074] In certain embodiments, wherein the nuclease is located at the mitochondria by a mitochondrial localization sequence.

[0075] In certain embodiments, wherein the mitochondrial localization sequence is directly or indirectly linked to the nuclease.

[0076] In certain embodiments, wherein the mitochondrial localization sequence is located at the N-terminus or C-terminus of the nuclease.

[0077] In certain embodiments, wherein the mitochondrial localization sequence comprises a polypeptide sequence.

[0078] In certain embodiments, wherein the mitochondrial localization sequence comprises a polypeptide sequence derived from the N-terminal region or C-terminal region of a mitochondrial protein.

[0079] In certain embodiments, wherein the mitochondrial localization sequence comprises a polypeptide sequence derived from the N-terminal region or C-terminal region of a mitochondrial membrane protein.

[0080] In certain embodiments, wherein the mitochondrial localization sequence comprises a mitochondrial localization sequence derived from cytochrome c oxidase subunit 8A (COX8A).

[0081] In certain embodiments, wherein the cytochrome c oxidase subunit 8A comprises human cytochrome c oxidase subunit 8A.

[0082] In certain embodiments, wherein the mitochondrial localization sequence comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0083] In certain embodiments, wherein the nucleic acid molecule encoding the mitochondrial localization sequence comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0084] In certain embodiments, wherein the mitochondrion is isolated from a cell or tissue.

[0085] In certain embodiments, wherein the mitochondrion is isolated from a somatic cell, a germ cell, and / or a stem cell.

[0086] In certain embodiments, wherein the mitochondrion is isolated from an induced pluripotent stem cell.

[0087] In certain embodiments, wherein the mitochondrion is isolated from a muscle cell, a neural cell, an endothelial cell, an epithelial cell, an embryonic cell, and / or a tumor cell.

[0088] In certain embodiments, wherein the mitochondrion is isolated from a cardiac cell and / or a skeletal muscle cell.

[0089] In certain embodiments, wherein the mitochondrion is isolated from an induced pluripotent stem cell differentiated cardiac cell.

[0090] In certain embodiments, wherein the mitochondrion is isolated from a mammalian cell.

[0091] In certain embodiments, wherein the mitochondrion is isolated from a human or mouse cell.

[0092] In certain embodiments, wherein the modified mitochondrion is isolated from a healthy cell.

[0093] In another aspect, the present application provides a fusion protein comprising a mitochondrial localization sequence and a nuclease.

[0094] In certain embodiments, wherein the nuclease comprises a human nuclease.

[0095] In certain embodiments, wherein the nuclease comprises a DNAse.

[0096] In certain embodiments, wherein the DNAse comprises a member of the DNase I family and / or a functional variant thereof.

[0097] In certain embodiments, wherein the DNAse comprises DNase I and / or a functional variant thereof.

[0098] In certain embodiments, wherein the DNAse comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0099] In certain embodiments, the nucleic acid molecule encoding the DNA enzyme comprises the nucleotide sequence set forth in SEQ ID NO: 4.

[0100] In certain embodiments, the nucleic acid enzyme is localized to the mitochondria by a mitochondrial localization sequence.

[0101] In certain embodiments, the mitochondrial localization sequence is directly or indirectly linked to the nucleic acid enzyme.

[0102] In certain embodiments, the mitochondrial localization sequence is located at the N-terminus of the nucleic acid enzyme.

[0103] In certain embodiments, the mitochondrial localization sequence comprises a polypeptide sequence.

[0104] In certain embodiments, the mitochondrial localization sequence comprises a polypeptide sequence derived from an N-terminal region or a C-terminal region of a mitochondrial protein.

[0105] In certain embodiments, the mitochondrial localization sequence comprises a polypeptide sequence derived from an N-terminal region or a C-terminal region of a mitochondrial membrane protein.

[0106] In certain embodiments, the mitochondrial localization sequence comprises a mitochondrial localization sequence derived from cytochrome c oxidase subunit 8A (COX8A).

[0107] In certain embodiments, the cytochrome c oxidase subunit 8A comprises a human cytochrome oxidase subunit 8A.

[0108] In certain embodiments, the mitochondrial localization sequence comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0109] In certain embodiments, the nucleic acid molecule encoding the mitochondrial localization sequence comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0110] In certain embodiments, it comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0111] In certain embodiments, a nucleotide sequence encoding the same comprises the nucleotide sequence set forth in SEQ ID NO: 6.

[0112] In another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein described herein.

[0113] In certain embodiments, it comprises the nucleotide sequence set forth in SEQ ID NO: 6.

[0114] In certain embodiments, it further comprises an expression regulatory element.

[0115] In certain embodiments, wherein the expression regulatory element comprises a promoter and / or an enhancer, wherein the promoter is located upstream of the nucleotide sequence encoding the fusion protein.

[0116] In certain embodiments, it further comprises a polynucleotide signal sequence, which is located downstream of the nucleotide sequence encoding the fusion protein.

[0117] In certain embodiments, it comprises DNA and / or RNA.

[0118] In certain embodiments, it comprises mRNA.

[0119] In certain embodiments, it further comprises a 5' cap, a 5' untranslated region, and a 3' untranslated region.

[0120] In certain embodiments, it comprises at least one modified nucleotide.

[0121] In certain embodiments, it further comprises AAV inverted terminal repeats (ITRs), which are located upstream and downstream of the nucleotide sequence encoding the fusion protein.

[0122] In certain embodiments, wherein the AAV ITRs are derived from AAV9.

[0123] In certain embodiments, it further comprises lentiviral long terminal repeat (LTR) sequences, which are located upstream and downstream of the nucleotide sequence encoding the fusion protein.

[0124] In another aspect, the present application provides a vector comprising the nucleic acid molecule described herein.

[0125] In certain embodiments, it is a viral vector or a polynucleotide vector.

[0126] In certain embodiments, it is a plasmid.

[0127] In certain embodiments, it is a viral vector, and the viral vector comprises an AAV vector and a lentiviral vector.

[0128] In certain embodiments, wherein the AAV vector is an AAV9 vector.

[0129] In another aspect, the present application provides a modified cell comprising the mitochondrion described herein, the fusion protein described herein, the nucleic acid molecule described herein, and / or the vector described herein.

[0130] In certain embodiments, it comprises a somatic cell, a germ cell, and / or a stem cell.

[0131] In some embodiments, it comprises an induced pluripotent stem cell.

[0132] In some embodiments, it comprises a muscle cell, a neural cell, an endothelial cell, an epithelial cell, an embryonic cell, and / or a tumor cell.

[0133] In some embodiments, wherein the cell comprises a cardiomyocyte and / or a skeletal muscle cell.

[0134] In some embodiments, it comprises a cardiomyocyte differentiated from an induced pluripotent stem cell.

[0135] In some embodiments, it comprises a mammalian cell.

[0136] In some embodiments, it comprises a human or a mouse cell.

[0137] In some embodiments, it comprises a healthy cell.

[0138] In another aspect, the present application provides a method of preparing a modified mitochondrion, comprising preparing a modified cell described herein and isolating a mitochondrion produced by the modified cell.

[0139] In another aspect, the present application provides a pharmaceutical composition comprising a mitochondrion described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein.

[0140] In some embodiments, it further comprises a pharmaceutically acceptable carrier.

[0141] In some embodiments, it comprises a nucleic acid molecule described herein and a carrier for delivering the nucleic acid molecule.

[0142] In some embodiments, wherein the nucleic acid molecule comprises an mRNA, and the carrier for delivering the nucleic acid molecule comprises a lipid nanoparticle (LNP).

[0143] In another aspect, the present application provides use of a mitochondrion described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein in the preparation of a reagent.

[0144] In some embodiments, wherein the reagent is for delaying aging.

[0145] In some embodiments, wherein the delaying aging comprises delaying cardiac aging, vascular aging, muscle aging, kidney aging, and / or nervous system aging.

[0146] In certain embodiments, wherein the delaying aging comprises reducing the expression level of a marker of aging.

[0147] In certain embodiments, wherein the marker of aging comprises p21, p16INK4a, p53, telomere length, and / or a DNA damage marker.

[0148] In certain embodiments, wherein the agent is used for preventing and / or treating a disease associated with mitochondrial damage.

[0149] In certain embodiments, wherein the mitochondrial damage comprises mitochondrial DNA leakage.

[0150] In certain embodiments, wherein the agent is used for preventing and / or treating an ischemia-related disease.

[0151] In certain embodiments, wherein the agent is used for alleviating side effects of an anti-tumor therapy.

[0152] In certain embodiments, wherein the side effects of the anti-tumor therapy comprise mitochondrial damage. According to the use described herein, wherein the side effects of the anti-tumor therapy comprise mitochondrial DNA leakage.

[0153] In certain embodiments, wherein the anti-tumor therapy comprises chemotherapy and / or radiotherapy.

[0154] In certain embodiments, wherein the chemotherapeutic drug comprises a platinum-based chemotherapeutic drug and / or an anthracycline-based chemotherapeutic drug.

[0155] In certain embodiments, wherein the anthracycline-based chemotherapeutic drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin.

[0156] In certain embodiments, wherein the platinum-based chemotherapeutic drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin.

[0157] In certain embodiments, wherein the agent is used for treating a cardiovascular disease.

[0158] In certain embodiments, wherein the agent is used for treating ischemia-reperfusion injury and / or hypoxic injury.

[0159] In certain embodiments, wherein the agent is used for down-regulating an inflammatory response and / or treating an inflammatory disease.

[0160] In certain embodiments, wherein the inflammatory response and / or inflammatory disease comprises an inflammatory response and / or inflammatory disease mediated by or associated with cGAS-STING pathway activation.

[0161] In certain embodiments, wherein the inflammatory disease is selected from the group consisting of a kidney inflammatory disease, a liver inflammatory disease, a lung inflammatory disease, a heart inflammatory disease, a spleen-related inflammatory disorder, and an inflammatory disorder in smooth muscle tissue.

[0162] In certain embodiments, wherein the kidney inflammatory disease comprises acute kidney injury (AKI), chronic kidney disease (CKD), systemic lupus erythematosus- associated nephritis, IgA nephropathy, interstitial nephritis, and / or diabetic nephropathy.

[0163] In certain embodiments, wherein the liver inflammatory disease comprises nonalcoholic steatohepatitis, drug-induced liver injury, viral hepatitis, alcoholic liver disease, liver fibrosis, and / or cirrhosis.

[0164] In certain embodiments, wherein the lung inflammatory disease comprises acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bacterial or viral infection- associated pneumonia, pneumoconiosis, and / or pulmonary fibrosis.

[0165] In certain embodiments, wherein the heart inflammatory disease comprises doxorubicin-induced cardiotoxicity, myocardial ischemia-reperfusion injury, myocarditis, and / or chronic heart failure.

[0166] In certain embodiments, wherein the spleen-related inflammatory disorder comprises systemic lupus erythematosus (SLE)-associated spleen disorder, spleen immune activation accompanying autoimmune diseases, and / or infection-induced spleen immune activation.

[0167] In certain embodiments, wherein the inflammatory disorder in smooth muscle tissue comprises inflammatory response in vascular smooth muscle cells, chronic inflammatory disorder in airway smooth muscle, and / or gastrointestinal smooth muscle.

[0168] In certain embodiments, wherein the inflammatory disorder in smooth muscle tissue comprises atherosclerosis and / or arterial injury.

[0169] In another aspect, the present application provides a method of delaying aging, comprising administering to a subject an effective amount of the mitochondrion described herein, the fusion protein described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the pharmaceutical composition described herein.

[0170] In certain embodiments, wherein the delaying aging comprises delaying cardiac aging, vascular aging, muscle aging, kidney aging, and / or nervous system aging.

[0171] In certain embodiments, wherein the delaying aging comprises reducing the expression level of an aging marker.

[0172] In certain embodiments, wherein the senescence markers comprise p21, p16INK4a, p53, telomere length, and / or DNA damage markers.

[0173] In another aspect, the present application provides a method of preventing and / or treating a disease, comprising administering to a subject an effective amount of the mitochondria described herein, the fusion protein described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the pharmaceutical composition described herein.

[0174] In certain embodiments, wherein the disease comprises a disease associated with mitochondrial damage.

[0175] In certain embodiments, wherein the mitochondrial damage comprises mitochondrial DNA leakage.

[0176] In certain embodiments, wherein the disease comprises an ischemia-related disease.

[0177] In certain embodiments, wherein the disease comprises a side effect of an anti-tumor therapy.

[0178] In certain embodiments, wherein the side effect of an anti-tumor therapy comprises mitochondrial damage.

[0179] In certain embodiments, wherein the side effect of an anti-tumor therapy comprises mitochondrial DNA leakage.

[0180] In certain embodiments, wherein the anti-tumor therapy comprises chemotherapy and / or radiotherapy.

[0181] In certain embodiments, wherein the chemotherapeutic drug comprises a platinum-based chemotherapeutic drug and / or an anthracycline-based chemotherapeutic drug.

[0182] In certain embodiments, wherein the anthracycline-based chemotherapeutic drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin.

[0183] In certain embodiments, wherein the platinum-based chemotherapeutic drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin.

[0184] In certain embodiments, wherein the disease comprises a cardiovascular disease.

[0185] In certain embodiments, wherein the disease comprises ischemia-reperfusion injury and / or hypoxic injury.

[0186] In certain embodiments, wherein the disease comprises an inflammatory response and / or an inflammatory disease.

[0187] In certain embodiments, wherein the inflammatory response and / or inflammatory disease comprises or is associated with activation of the cGAS-STING pathway.

[0188] In certain embodiments, wherein the inflammatory disease is selected from the group consisting of a kidney inflammatory disease, a liver inflammatory disease, a lung inflammatory disease, a heart inflammatory disease, a spleen-related inflammatory disorder, and an inflammatory disorder in smooth muscle tissue.

[0189] In certain embodiments, wherein the kidney inflammatory disease comprises acute kidney injury (AKI), chronic kidney disease (CKD), systemic lupus erythematosus-associated nephritis, IgA nephropathy, interstitial nephritis, and / or diabetic nephropathy.

[0190] In certain embodiments, wherein the liver inflammatory disease comprises nonalcoholic steatohepatitis, drug-induced liver injury, viral hepatitis, alcoholic liver disease, liver fibrosis, and / or cirrhosis.

[0191] In certain embodiments, wherein the lung inflammatory disease comprises acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bacterial or viral infection-associated pneumonia, pneumoconiosis, and / or pulmonary fibrosis.

[0192] In certain embodiments, wherein the heart inflammatory disease comprises doxorubicin-induced cardiotoxicity, myocardial ischemia-reperfusion injury, myocarditis, and / or chronic heart failure.

[0193] In certain embodiments, wherein the spleen-related inflammatory disorder comprises systemic lupus erythematosus (SLE)-associated spleen disorder, spleen immune activation accompanying autoimmune diseases, and / or infection-induced spleen immune activation.

[0194] In certain embodiments, wherein the inflammatory disorder in smooth muscle tissue comprises an inflammatory response in vascular smooth muscle cells, chronic inflammatory disorder in airway smooth muscle, and / or gastrointestinal smooth muscle.

[0195] In certain embodiments, wherein the inflammatory disorder in smooth muscle tissue comprises atherosclerosis and / or arterial injury.

[0196] In another aspect, the present application provides use of the mitochondrion of the present application, the fusion protein of the present application, the nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application in delaying aging.

[0197] In certain embodiments, wherein the delaying aging comprises heart aging, vascular aging, muscle aging, kidney aging, and / or nervous system aging.

[0198] In certain embodiments, wherein the delaying aging comprises reducing the expression level of a marker of aging.

[0199] In certain embodiments, wherein the marker of aging comprises p21, p16INK4a, p53, telomere length, and / or a DNA damage marker.

[0200] In another aspect, the present application provides a use of the mitochondrion of the present application, the fusion protein of the present application, the nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application in treating and / or preventing a disease.

[0201] In certain embodiments, wherein the disease comprises a disease associated with mitochondrial damage.

[0202] In certain embodiments, wherein the mitochondrial damage comprises mitochondrial DNA leakage.

[0203] In certain embodiments, wherein the disease comprises an ischemia-related disease.

[0204] In certain embodiments, wherein the disease comprises a side effect of an anti-tumor therapy.

[0205] In certain embodiments, wherein the side effect of an anti-tumor therapy comprises mitochondrial damage.

[0206] In certain embodiments, wherein the mitochondrial damage comprises mitochondrial DNA leakage.

[0207] In certain embodiments, wherein the anti-tumor therapy comprises chemotherapy and / or radiotherapy.

[0208] In certain embodiments, wherein the chemotherapeutic drug comprises a platinum-based chemotherapeutic drug and / or an anthracycline-based chemotherapeutic drug.

[0209] In certain embodiments, wherein the anthracycline-based chemotherapeutic drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin.

[0210] In certain embodiments, wherein the platinum-based chemotherapeutic drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin. In certain embodiments, wherein the disease comprises a cardiovascular disease.

[0211] In certain embodiments, wherein the disease comprises ischemia-reperfusion injury and / or hypoxic injury.

[0212] In certain embodiments, wherein the disease comprises an inflammatory response and / or an inflammatory disease.

[0213] In certain embodiments, wherein the inflammatory response and / or inflammatory disease comprises or is associated with activation of the cGAS-STING pathway.

[0214] In certain embodiments, wherein the inflammatory disease is selected from the group consisting of a kidney inflammatory disease, a liver inflammatory disease, a lung inflammatory disease, a heart inflammatory disease, a spleen-related inflammatory disorder, and an inflammatory disorder in smooth muscle tissue.

[0215] In certain embodiments, wherein the kidney inflammatory disease comprises acute kidney injury (AKI), chronic kidney disease (CKD), systemic lupus erythematosus-associated nephritis, IgA nephropathy, interstitial nephritis, and / or diabetic nephropathy.

[0216] In certain embodiments, wherein the liver inflammatory disease comprises nonalcoholic steatohepatitis, drug-induced liver injury, viral hepatitis, alcoholic liver disease, liver fibrosis, and / or cirrhosis.

[0217] In certain embodiments, wherein the lung inflammatory disease comprises acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bacterial or viral infection-associated pneumonia, pneumoconiosis, and / or pulmonary fibrosis.

[0218] In certain embodiments, wherein the heart inflammatory disease comprises doxorubicin-induced cardiotoxicity, myocardial ischemia-reperfusion injury, myocarditis, and / or chronic heart failure.

[0219] In certain embodiments, wherein the spleen-related inflammatory disorder comprises systemic lupus erythematosus (SLE)-associated splenic disorder, autoimmune disease- associated splenic immune activation, and / or infection-induced splenic immune activation.

[0220] In certain embodiments, wherein the inflammatory disorder in smooth muscle tissue comprises an inflammatory response in vascular smooth muscle cells, chronic inflammatory disorder in airway smooth muscle, and / or gastrointestinal smooth muscle.

[0221] In certain embodiments, wherein the inflammatory disorder in smooth muscle tissue comprises atherosclerosis and / or arterial injury.

[0222] Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description in conjunction with the drawings. The illustrative embodiments described in the detailed description are intended to show examples of the present application. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the inventive concept. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive. Attached Figure Description

[0223] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0224] Figures 1A-1B illustrate the construction of a lentiviral vector that localizes a nuclease to mitochondria as described in this application. Figure 1A shows the lentiviral vector expression cassette that localizes a nuclease to mitochondria, and Figure 1B shows the lentiviral vector (Lenti-mtDNase I) that expresses a nuclease localized to mitochondria.

[0225] Figure 2 shows that the nuclease described in this application is overexpressed in mitochondria, and the co-localization of DNase I and TOM20 increases in cells infected with Lenti-mtDNase I.

[0226] Figure 3 shows the content of mitochondrial DNA in the cytoplasm after cell infection by the lentivirus DNase I (mtDNase I) described in this application, which is a mitochondrial nuclease.

[0227] Figure 4 shows that the mitochondrial nuclease DNase I (mtDNase I) described in this application can reduce the activation of the cGAS-STING pathway in hypoxic-reperfused cells.

[0228] Figures 5A-5B show that the mitochondrial nuclease DNase I (mtDNase I) described in this application can reduce mitochondrial superoxide (MitoSOX, Figure 5A) and increase membrane potential (TMRM, Figure 5B) in hypoxic-reperfused cells.

[0229] Figure 6 shows that the mitochondrial nuclease DNase I (mtDNase I) described in this application can alleviate myocardial aging induced by ischemia-reperfusion or doxorubicin injury. Using p21 as an aging marker, mtDNase I can significantly reduce the increase in the proportion of p21-positive cardiomyocytes caused by ischemia-reperfusion or doxorubicin injury.

[0230] Figures 7A-7B show that the mitochondrial nuclease DNase I (mtDNase I) described in this application can reduce the cardiac ejection fraction induced by ischemia-reperfusion or doxorubicin injury (Figure 7A) and shorten the fractional decrease (Figure 7B).

[0231] Figure 8 shows the mouse phenotype of cardiac fibrosis induced by ischemia-reperfusion or doxorubicin induced by the mitochondrial nuclease DNase I (mtDNase I) described in this application.

[0232] Figure 9 shows the enlargement of mitochondrial volume and damage to mitochondrial cristae caused by ischemia-reperfusion, or the mitochondrial vacuolation and sarcomere rupture in the heart of mice with chronic heart failure induced by doxorubicin. The mitochondrial nuclease DNase I (mtDNase I) treatment described in this application can alleviate the damage to mitochondria and sarcomeres caused by ischemia-reperfusion or doxorubicin.

[0233] Figure 10 shows that the cardiomyocyte contractile function of doxorubicin mice was significantly reduced, as evidenced by decreased cardiomyocyte contraction speed, relaxation speed, and contraction percentage. Treatment with the mitochondrial nuclease DNase I (mtDNase I) described in this application significantly improved doxorubicin-induced cardiomyocyte contractile dysfunction.

[0234] Figure 11 shows that DNase I is located in mitochondria and retains its DNA-degrading activity after extraction.

[0235] Figure 12 shows the well plate layout design used to evaluate the effect of mtDNase I expression on doxorubicin-induced cGAS-STING pathway activation under mitochondrial transplantation conditions.

[0236] Figures 13A-13B show that transplanting extracted mtDNase I mitochondria into wild-type cells (+mtDNase mito) and cell-mediated expression of DNase I in mitochondria can effectively reduce Sting expression (Figure 13A) and γH2AX positivity rate caused by doxorubicin damage (Figure 13B).

[0237] Figure 14 shows the experimental results of the effects of mitochondrial transplantation on cell morphology and apoptosis under cisplatin-induced injury conditions.

[0238] Figures 15A-15C show the results of experiments on the protective effect of transplanting mitochondria with mtDNase I in an AKI mouse model. Figures 15A and 15C show the changes in body weight of mice in different treatment groups, and Figure 15B shows the ratio of kidney mass to tibia length, used to assess overall health and the degree of kidney damage after treatment.

[0239] Figures 16A-16C show the results of the protective effect of transplanting mitochondria with mtDNase I in an acute doxorubicin mouse model. Figure 16A shows the effect on the expression of cGAS-STING pathway-related proteins in liver tissue, Figure 16B shows the effect on the expression of cGAS-STING pathway-related proteins in kidney tissue, and Figure 16C shows the effect on the expression of cGAS-STING pathway-related proteins in lung tissue. Detailed Implementation

[0240] The following detailed description of the application is provided as an example of the embodiments of the application and is not intended to limit the application to the described embodiments, but rather to enable a person skilled in the art to make and use the application. Other advantages and effects of the application will be apparent to those skilled in the art from the contents of the present specification.

[0241] Definitions of terms

[0242] In the present application, the term "mitochondrial DNA leakage" generally refers to the phenomenon that DNA in mitochondria (mtDNA) escapes from mitochondria into the cytoplasm. Mitochondria are important organelles in cells responsible for energy metabolism, and have their own genetic material mtDNA. Normally, mtDNA is confined within mitochondria, but when mitochondria are damaged or stressed, it can lead to mtDNA leakage into the cytoplasm. Once mtDNA enters the cytoplasm, it can be recognized by the immune system of the cell as foreign DNA, thereby activating inflammatory responses and innate immune pathways. In the present application, the term "downstream pathways activated by mitochondrial DNA leakage" generally refers to a series of signaling and response pathways within the cell triggered after mitochondrial DNA leaks from mitochondria into the cytoplasm. These pathways include but are not limited to immune responses, inflammatory responses, and apoptosis, etc. The downstream pathways activated by mitochondrial DNA leakage include but are not limited to the activation of cGAS and its downstream signals, TLR-9 and its downstream signals, and / or inflammasome. For example, the downstream pathways activated by mitochondrial DNA leakage include but are not limited to the cGAS-STING signaling pathway. Mitochondrial leaked DNA can activate cGAS and its downstream signals, causing pro-inflammatory cytokine secretion, type I interferon response, and interferon-stimulated gene expression. Mitochondrial leaked DNA can activate TLR9 and its downstream signals, thereby causing pro-inflammatory cytokine, neutrophil chemotactic factor, and matrix metalloproteinase secretion, and inducing type I interferon response. Mitochondrial leaked DNA can activate NLRP3, NLRC4 inflammasome, AIM2 inflammasome, thereby causing caspase-1 activation, inducing interleukin-1β and interleukin-18 secretion. Mitochondrial leakage also leads to decreased mitochondrial respiratory function. Mitochondrial respiratory function generally refers to the process of energy production by mitochondria. Decreased mitochondrial respiratory function generally refers to a decrease in the efficiency of energy production by mitochondria, for example, decreased mitochondrial respiratory function can include decreased ATP generation rate, decreased oxygen consumption rate, excessive reactive oxygen species (ROS) generation, decreased respiratory control ratio (RCR), and / or decreased mitochondrial enzyme activity, etc. Mitochondrial DNA leakage can be identified and quantified by various biological detection techniques, for example, mitochondrial DNA leakage can be detected by fluorescence staining, real-time fluorescent quantitative PCR, imaging techniques, etc. In addition, mitochondrial DNA leakage can also be indirectly detected by evaluating the biological processes in which mitochondrial DNA leakage is involved. For example, mitochondrial DNA leakage can activate the cGAS-STING pathway, and the level of activation of the cGAS-STING pathway can be detected to indirectly detect mitochondrial DNA leakage.

[0243] In the present application, the term "nuclease" generally refers to a protein or fragment thereof that is capable of cleaving a phosphodiester bond between deoxyribonucleotides. The nucleases described in the present application can include non-specific nucleases. The nucleases described in the present application can include endonucleases. The nucleases described in the present application can include nucleases that cleave double stranded and / or single stranded DNA. The nucleases described in the present application can include nucleases of various origins, for example, including nucleases derived from mammals such as primates (e.g., humans). The nucleases described in the present application can not contain their naturally occurring localization domains.

[0244] In the present application, the term "DNase I family" generally refers to a class of DNases, DNase I family members include DNase I, DNase1L1, DNase 1L2, and DNase1L3. In the present application, the term "DNase I" can include DNase I and functionally active fragments thereof. DNase I is a non-specific endonuclease that is capable of cleaving phosphodiester bonds in DNA molecules, degrading double stranded or single stranded DNA. The DNase I described in the present application can include DNase I of any origin, for example, including DNase I derived from mammals such as primates (e.g., humans). The Uniprot accession number for human DNase I is P24855. DNase I can include "full-length", unprocessed, as well as any form of DNase I resulting from processing in cells.

[0245] In the present application, "functionally active fragment" generally refers to a fragment that has a partial region of a full-length protein or nucleic acid, but retains or partially retains the biological activity or function of the full-length protein or nucleic acid. The DNase I functionally active fragment can retain or partially retain its function of cleaving phosphodiester bonds. For example, the DNase I described in the present application can not contain its naturally occurring localization domains.

[0246] In the present application, the term "functional variant of DNase I" generally refers to a polypeptide or protein that has one or more activities of naturally occurring DNase I. The functional variant of DNase I can have substantially the same amino acid sequence as a naturally occurring DNase I sequence, or is encoded by a substantially identical nucleotide sequence. "Substantially identical" generally refers to when two sequences share at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or 80% sequence identity, or at least 90% sequence identity, or at least 95%, 96%, 97%, or 98% sequence identity, or at least 99% sequence identity, when optimally aligned, for example, when using the program GAP or BESTFIT and using default gap weight.

[0247] In the present application, the terms "mitochondrial localization sequence" and "mitochondrial targeting sequence (MTS)" can be used interchangeably, and generally refer to an amino acid sequence or a nucleotide sequence encoding the same, which is capable of directing a polypeptide or a protein to mitochondria. The mitochondrial localization sequence can include an amino acid sequence and / or a nucleotide sequence which is capable of targeting a substance that does not naturally exist in mitochondria to mitochondria. Depending on the targeting position of the mitochondrial localization sequence, the mitochondrial localization sequence described in the present application can include a mitochondrial matrix localization sequence, a mitochondrial inner membrane localization sequence, a mitochondrial membrane spanning localization sequence, and / or a mitochondrial outer membrane localization sequence, as long as it is capable of localizing to mitochondria. In some embodiments, the mitochondrial localization sequence includes a mitochondrial outer membrane localization sequence. In the present application, the term "mitochondrial outer membrane localization sequence" generally refers to an amino acid sequence which is capable of directing a polypeptide or a protein to the outer membrane of mitochondria. The mitochondrial localization sequence described in the present application can be a naturally occurring sequence or an artificially designed sequence, for example, mitochondrial membrane proteins generally have a mitochondrial localization sequence to localize to the mitochondrial membrane, and the mitochondrial localization sequence described in the present application can include a fragment having a function of targeting mitochondria in a mitochondrial membrane protein and / or a variant thereof.

[0248] In the present application, the term "polypeptide" generally refers to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The term can also include amino acid polymers that are modified, for example, by the addition of sugar residues to form a glycoprotein, or by phosphorylation.

[0249] In the present application, the term "mitochondrial membrane protein" generally refers to a protein present on the mitochondrial membrane. The mitochondrial membrane is divided into the inner membrane and the outer membrane, and the mitochondrial membrane protein can include a mitochondrial outer membrane protein, a mitochondrial inner membrane protein, and a mitochondrial membrane spanning protein. The mitochondrial membrane protein generally has a domain that helps it to be correctly localized to mitochondria, and such a domain is usually located at the N-terminus or the C-terminus of the mitochondrial protein.

[0250] In the present application, the term "cytochrome c oxidase subunit 8A" and "COX8A" can be used interchangeably, and generally refers to cytochrome c oxidase subunit 8A and / or a gene encoding the same, which is widely used to target molecules to mitochondria through a protein import mechanism. Cytochrome c oxidase subunit 8A is a component of cytochrome c oxidase. The cytochrome oxidase subunit 8 described in the present application includes cytochrome oxidase subunit 8 of various origins, for example, cytochrome oxidase subunit 8 derived from mammals such as primates (e.g., humans). The Uniprot accession number of human cytochrome c oxidase subunit 8A is P10176. The mitochondrial localization sequence derived from cytochrome c oxidase subunit 8A can comprise a polypeptide sequence at the N-terminus thereof, for example, a polypeptide sequence comprising the N-terminal 29 amino acids thereof.

[0251] In the present application, the term "fusion protein" generally refers to a polypeptide or protein composed of two or more polypeptides. The two or more polypeptides are generally not combined in a natural state, but can form a continuous polypeptide by direct combination or indirectly through a peptide linker / spacer. In the present application, the fusion protein described can include a protein composed of a mitochondrial localization sequence and a nuclease.

[0252] In the present application, the term "isolated" generally refers to a substance removed from its original or primary environment (e.g., a natural environment if it naturally exists). For example, when used to describe mitochondria, it generally refers to a mitochondria structure isolated from a cell, which has been separated from the cytoplasm and is no longer in the native environment within the cell.

[0253] In the present application, the term "modified" generally refers to a changed state or structure. For example, when describing a modified mitochondria or cell, the modified mitochondria or cell includes a mitochondria or cell that has a substance, structure, function or property that does not exist in a natural state. The modification can be achieved by genetic engineering, chemical modification, physical treatment, introduction of exogenous substances, etc.

[0254] In the present application, when used to describe the source of biological material, it generally refers to the source of biological material from the same species or the same biological individual. For example, the homology includes the case of "autologous". In the present application, the term "allogeneic" generally refers to the source of biological material from the same species but different individuals.

[0255] In the present application, the term "healthy cell" generally refers to a cell that exhibits normal morphology, normal cell function, and normal cell growth. For example, a healthy cell can include a cell that does not show any pathology. A healthy cell can be obtained from a tissue sample that does not have any pathological characteristics, for example, extracted from a tissue, a body fluid, or an organ of a healthy donor. Whether a cell is healthy or not can be detected by various methods, including assessment of cell function, structure, metabolic state, and gene expression status, for example, methods for detecting whether a cell is healthy or not include, but are not limited to, cell viability and survival rate detection, cell apoptosis and necrosis detection, cell proliferation detection, cell metabolic state detection, oxidative stress detection, cell structure and morphology detection, and gene and protein expression detection.

[0256] In the present application, the term "mitochondrial-damaged cell" generally refers to a cell with impaired mitochondrial function and / or structure, which can affect the normal physiological function of the cell. For example, a mitochondrial-damaged cell can exhibit mitochondrial respiratory dysfunction. Mitochondrial damage can manifest as mitochondrial DNA mutation, mitochondrial membrane potential decrease, ATP synthesis decrease, oxidative stress increase, mitochondrial membrane structure damage, mitochondrial DNA leakage, etc. In the present application, the mitochondrial damage includes mitochondrial DNA leakage. Mitochondrial damage can be detected by various methods, including assessment of mitochondrial function, structure, genomic integrity, and metabolic products produced, for example, methods for detecting whether a mitochondrion is damaged or not include, but are not limited to, mitochondrial membrane potential detection, reactive oxygen species detection, ATP content detection, mitochondrial DNA detection, mitochondrial respiratory function detection, mitochondrial morphology detection, protein detection, and apoptosis detection.

[0257] In the present application, the term "metabolic level" generally refers to the ability and intensity of metabolic activity. The metabolic level of a cell described in the present application can include, but is not limited to, mitochondrial respiratory function, cellular oxidative state, mitochondrial morphological structure, mitochondrial number, mitochondrial membrane potential, mitochondrial oxygen consumption, and / or mitochondrial superoxide level. In the present application, the term "metabolic level match" generally refers to similar or identical metabolic levels. The metabolic level match between two cells or cell populations generally refers to similarity or consistency in metabolic level indicators. For example, the similarity or consistency can include a difference in metabolic level indicators between two cells or cell populations within ±30%, within ±25%, within ±20%, within ±15%, within ±10%, within ±5%, or closer. For example, the similarity or consistency can include a difference in mitochondrial membrane potential within ±10%, a difference in mitochondrial number within ±15%, a difference in ROS level within ±20%, a difference in oxygen consumption within ±15%, and / or a difference in superoxide level within ±20%, etc.

[0258] In the present application, the term "anti-tumor therapy" generally refers to a therapeutic approach that is capable of killing or inhibiting the growth of tumor cells. Anti-tumor therapies are intended to eliminate or control tumor cells, but these therapies can also have side effects on non-tumor cells or organs, leading to damage and senescence of cells or tissues. For example, anti-tumor therapies include anti-tumor therapies that have cardiotoxicity. In the present application, the term "chemotherapy" or "chemotherapeutic" generally refers to a therapeutic approach involving a chemotherapeutic agent. Chemotherapeutic agents can include standard chemotherapeutic drugs (which generally attack any rapidly dividing cells), targeted therapeutics, and immunomodulators. In the present application, the term "radiotherapy" generally refers to a therapeutic approach that utilizes high-energy radiation (such as X-rays, gamma rays, or proton beams) to kill or inhibit the growth and division of tumor cells.

[0259] In the present application, the term "inflammatory response" generally refers to a biological response to a harmful stimulus (such as a pathogen, a damaged cell, an irritant, etc.). The inflammatory response described herein can include the recognition of a harmful stimulus by a pattern recognition receptor, the activation of an inflammatory pathway, the production of inflammatory mediators, and / or the recruitment of inflammatory cells.

[0260] In the present application, the term "mitochondrial respiratory capacity" generally refers to the efficiency and capacity of mitochondria to generate ATP through the process of oxidative phosphorylation. Mitochondrial respiratory capacity reflects the level of oxygen consumption and the functional state of the electron transport chain required by mitochondria in the process of oxidizing substrates to generate ATP. Mitochondrial respiratory capacity can be assessed by, but is not limited to, the detection of oxygen consumption rate, ATP production, and / or membrane potential of mitochondria.

[0261] In the present application, the terms "nucleic acid," "nucleic acid molecule," "polynucleotide," and "oligonucleotide" generally refer to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) and include naturally occurring (adenine, guanine, cytosine, uracil, and thymine), non-naturally occurring, and modified nucleic acids. The terms are not limited as to the length of the polymer (e.g., number of monomers). The nucleic acids can be single-stranded or double-stranded, and generally contain 5'-3' phosphodiester bonds, although in some cases nucleotide analogs can have other linkages. The monomers are often referred to as nucleotides. The term "nucleotide sequence" as used herein generally refers to a specific sequence of nucleotides of a nucleic acid molecule.

[0262] In the present application, the term "expression regulatory element" generally refers to a DNA sequence that exerts regulatory effects during the process of gene expression. For example, the expression regulatory element includes a promoter and / or an enhancer.

[0263] In the present application, the term "polyadenylation signal sequence" or "polyA signal sequence" generally refers to a nucleotide sequence used to induce cleavage and polyadenylation of the primary transcript of a particular nucleotide sequence segment.

[0264] In the present application, the terms "5'" and "3'" are conventional expressions used to describe the features of nucleotide sequences, and relate to the position of genetic elements and / or the direction of events such as RNA polymerase transcription or ribosomal translation, which proceed in a 5' to 3' direction (5' to 3'). Synonyms are upstream (5') and downstream (3'). Usually, DNA sequences, genetic maps, vector maps and RNA sequences are drawn from left to right in the 5' to 3' direction, or, alternatively, the 5' to 3' direction is indicated by an arrow symbol, wherein the arrowhead points in the 3' direction. Therefore, when following this conventional usage, 5' (upstream) means that a genetic element is placed to the left hand side, whereas 3' (downstream) means that a genetic element is placed to the right hand side.

[0265] In the present application, the term "mRNA" generally refers to an RNA transcript that has been processed to remove introns and is capable of being translated into a polypeptide. An mRNA can be composed of five major parts: a cap structure at the 5' end, a 5' untranslated region (5' UTR) that is associated with translation efficiency, an open reading frame (ORF) region that encodes a protein, a 3' UTR that is associated with translation efficiency, and a poly A tail composed of multiple adenosine monophosphates that is associated with mRNA stability and translation efficiency. For example, the open reading frame that encodes a protein can comprise expression control elements and a gene of interest operably linked thereto. In certain embodiments, the gene of interest comprises a nucleotide sequence that encodes a fusion protein described herein.

[0266] In the present application, the term "vector" generally refers to a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector. In the present application, the term "viral vector" generally refers to a nucleic acid vehicle that has been constructed based on a viral genome and is capable of carrying foreign nucleotide sequences. Typically, a viral vector is capable of self-replication and / or expression of its contained genes (endogenous and exogenous) in a suitable host cell. A viral vector can comprise the genome of a whole, wild-type virus, or a mutated or modified viral genome.

[0267] In the present application, the term "AAV" or "AAV serotype" generally refers to the ten naturally occurring and available adeno-associated viruses, as well as artificial AAVs, and can be used to refer to the virus itself or a derivative thereof. Unless otherwise indicated, the AAV capsids, ITRs, and other selected AAV components described herein can be readily selected from any AAV. In the present application, the terms "AAV vector," "AAV," and "adeno-associated viral vector" are used interchangeably to generally refer to a vector derived from at least a portion of an adeno-associated viral genome. The ends of the AAV genome are generally flanked by inverted terminal repeats (ITRs), and the term "ITR" or "inverted terminal repeat" refers to a segment of nucleic acid sequence found in AAV and / or recombinant AAV that can form a T-shaped palindromic structure required for completion of the AAV lytic and latent life cycle. Techniques for producing AAV vectors are standard in the art, and include providing a cell with a polynucleotide to be delivered, a rep and cap gene, and an AAV genome to be packaged with helper virus functions.

[0268] In the present application, the term "lentivirus" generally refers to a type of retrovirus. The term "lentiviral vector" generally refers to a vector derived from at least a portion of a lentiviral genome. A lentiviral vector can comprise non-coding sequences from one or more proteins of a lentivirus, such as HIV-1. The term "long terminal repeat" or "LTR" refers to the sequence of hundreds of base pairs at each end of the DNA synthesized by reverse transcription of retroviral RNA. A "lentiviral transfer vector" can comprise, for example, a heterologous nucleic acid sequence to be transferred into a cell, and can further comprise, for example, one or more lentiviral genes or portions thereof. A "lentiviral packaging vector" can comprise one or more genes encoding lentiviral proteins or portions thereof. For example, a lentiviral envelope protein can comprise a gene encoding an env protein or portion thereof. A host cell can be transfected with a transfer vector and one or more packaging vectors to produce virus, which in turn is used to infect a target cell to express one or more genes of interest contained within the heterologous nucleic acid sequence within the target cell. In certain embodiments, the gene of interest comprises a nucleotide sequence encoding a fusion protein described herein.

[0269] In the present application, the term "cell" can include a single cell, a cell line, and / or a cell culture. A cell can include the cell and its progeny. The progeny can not necessarily be identical to the parent cell (in morphology or in genomic

[0270] In the present application, the term "senescence" can include senescence at the level of cells, tissues, organs, systems, and biological individuals, as well as diseases and / or conditions caused by senescence. Senescence can be detected by a variety of methods, including detection of morphological changes, detection of DNA damage markers, detection of cell cycle arrest markers, detection of senescence-associated secretory phenotype (SASP) markers, detection of anti-apoptotic markers, in vivo assays for senescence, and the like. General features of senescence are chronic DNA damage response (DDR) activation, cell cycle arrest, enhanced secretion of pro-inflammatory and tissue remodeling factors, anti-apoptosis, metabolic alterations, and endoplasmic reticulum stress. Cellular senescence generally refers to a permanent state of division arrest of a cell. This process is often accompanied by changes in gene expression and a decline in cellular functions, such as mitochondrial dysfunction, which is one of the hallmarks of cellular senescence. Senescent cells often exhibit characteristic structural changes, including cell enlargement and flattening, lysosome and mitochondria accumulation, nuclear changes, and plasma membrane alterations. Nuclear changes can be observed by heterochromatin markers, including H3K9me3 and HP1y. Lysosomal content in senescent cells can be determined by beta-galactosidase activity (SA-beta-gal) assay. Markers of DNA damage in senescent cells include phosphorylated histone H2AX (yH2AX) and phosphorylated p53. Cell cycle protein-dependent kinase inhibitors (CDKis) drive irreversible cell cycle arrest, and major CDKi markers include CDKN2A (pl6INK4a), CDKN2B (pl5), and CDKN1A (p21), and BrdU / EdU incorporation assays can also be used to determine cell cycle arrest. Components of SASP specifically include some interleukins (IL1, IL1a, IL1b, IL6, IL7, IL8, IL13, and IL15), chemokines (BLC, CXCL1, CXCL2, CXCL3, MCP1, MCP2, MCP4, MIP-1A, MIP-1B, MIP-3A, HCC-4, eotaxin, eotaxin-3, TECK, ENA-78, I-309, and I-TAC), some inflammatory markers (such as TGF-beta, INF-gamma, and MIF), growth factors and regulators, proteases, and receptors and ligands. BCL-XL, BCL-W, p21, PAI-2, and HSP90 all play a role in the survival of senescent cells and are also considered markers of senescence. In addition, TRAIL-R4 / DCR2 is a common marker of senescence. Manifestations of mitochondrial dysfunction in senescent cells include, but are not limited to, an increase in the number of mitochondrial ROS and a decrease in mitochondrial membrane potential.

[0271] Organ or system aging generally refers to extensive deterioration and functional decline in structure and function. For example, heart aging includes, but is not limited to, cardiac myocyte hypertrophy, cardiac myocyte reduction, cardiac myofibrosis, abnormal cardiac electrical activity, cardiac myocyte mitochondrial dysfunction, ventricular wall thickening, coronary artery hardening, weakened cardiac contractility, diastolic dysfunction, cardiac vascular functional decline, increased inflammation and oxidative stress, shortened telomere length in cardiac myocytes, reduced number and function of cardiac myocytes, etc. For example, kidney aging includes, but is not limited to, glomerulosclerosis (e.g., thickening of the basement membrane of the glomerulus, reduction of glomerular capillary tuft, etc.), tubule atrophy and interstitial fibrosis, renal interstitial cell hyperplasia, renal vascular lesions (e.g., renal vascular hardening, atherosclerosis, and vascular lumen stenosis, etc.), reduced kidney function (e.g., reduced glomerular filtration rate, reduced urine concentrating ability, etc.). For example, vascular aging includes, but is not limited to, arterial hardening, vascular endothelial dysfunction (e.g., decreased anti-coagulation and anti-inflammatory functions of endothelial cells, reduced nitric oxide production, etc.), vascular smooth muscle cell changes (e.g., vascular smooth muscle cell proliferation and migration, smooth muscle cell functional abnormalities, etc.), fibrosis (e.g., vascular stiffness and decreased elasticity), microvessel rarefaction (e.g., reduced number and diameter of microvessels), increased levels of inflammatory cells (e.g., macrophages) and inflammatory factors in the vascular wall, increased vascular wall permeability, vascular calcification, hemodynamic changes (e.g., increased vascular resistance), and reduced vascular regenerative capacity, etc. For example, muscle aging includes, but is not limited to, reduced muscle mass (e.g., reduced skeletal muscle mass, muscle fiber thinning), decreased muscle strength, muscle fiber composition changes (e.g., reduced number of fast muscle fibers (type II fibers), increased proportion of slow muscle fibers (type I fibers)), muscle fat infiltration (e.g., increased fat tissue between muscle fibers and within muscle fibers), deterioration of neuromuscular junction, increased inflammation and oxidative stress, increased levels of apoptosis and autophagy, protein metabolism imbalance (e.g., reduced muscle protein synthesis and increased breakdown), reduced muscle regenerative capacity, reduced local blood flow to muscle, and increased collagen fibers in muscle tissue, etc. For example, nervous system aging includes, but is not limited to, loss of neurons, deterioration of axons and dendrites of neurons (e.g., atrophy of axons and dendrites of neurons, reduced dendritic spines, etc.), reduced synaptic density of neurons, changes in glial cells (e.g., over-activation of microglia, dysfunction of astrocytes, etc.), myelin degeneration, aggregation of abnormal proteins (e.g., beta-amyloid and tau proteins), impaired integrity of the blood-brain barrier, changes in neurotransmitter levels and receptor expression, neuroinflammation, mitochondrial dysfunction of neural cells, etc. Aging at the level of a biological individual generally refers to the state of overall health level, including decreased physical fitness, reduced cognitive ability, and increased susceptibility to diseases.

[0272] The aging described herein can include physiological aging and / or pathological aging, the former refers to the physiological deterioration process after the mature period, and the latter is the change caused by various external factors including various diseases. For example, the aging described herein can include aging caused by side effects of a certain confirmed disease therapy on the body. For example, the aging described herein can include aging caused by anti-tumor therapy. For example, the aging described herein can include aging caused by chemotherapy and / or radiotherapy. The aging described herein can include aging caused by aging-inducing treatment and / or aging-inducing agent. For example, the aging-inducing treatment can include but is not limited to radiation, up-regulation or overexpression of aging-promoting genes, and / or down-regulation or knockout of aging-inhibiting genes. For example, the aging-inducing agent can include chemical toxins, cytotoxins, etc. The aging-inducing treatment and / or aging-inducing agent can be known in the art. For example, the aging described herein can include aging caused by doxorubicin.

[0273] In the present application, the term "delaying aging" generally refers to delaying the deterioration process of an organism, an organ, a tissue or a cell in structure and function, and maintaining its normal physiological function. In the present application, the term "delaying aging" includes delaying the time of occurrence of an aging state (for example, relative to the normal aging process), alleviating and / or reducing the aging state, preventing the aging state and / or reversing the aging state. For example, the delaying aging includes alleviating and / or reducing one or more aging phenotypes. For example, the alleviating aging includes reducing the expression of one or more aging markers. The aging marker generally refers to a biological indicator that appears in the aging process and can reflect the aging state or is related to the aging process. For example, the aging marker can include cell cycle inhibitory proteins such as p21, p16INK4a, p53, etc., which are related to cell cycle arrest and aging. For example, the aging marker can include β-galactosidase activity, and the increase of β-galactosidase activity is generally related to cell aging. For example, the aging marker can include telomere length, and the decrease of telomere length is related to aging, and when the telomere length is lower than a certain threshold, the cell will enter the aging or apoptosis state. The reduction of the expression level of the telomere length described herein can include the reduction of the degree of the decrease of the telomere length. For example, the aging marker can include the degree of DNA damage, and the marker of DNA damage can include γH2AX, 53BP1, etc. The reduction of the expression level of the degree of DNA damage described herein can include the reduction of the expression level of the marker of DNA damage.

[0274] In the present application, the term "disease associated with mitochondrial damage" generally refers to any disease or disorder mediated by or associated with mitochondrial damage. For example, in a patient with a disease associated with mitochondrial damage, mitochondrial damage can be detected in the disease site. In the present application, the disease associated with mitochondrial damage includes any disease or disorder associated with mitochondrial DNA leakage. In the present application, the disease associated with mitochondrial damage can also include any disease or disorder mediated by or associated with downstream pathways activated by mitochondrial damage. In the present application, the disease associated with mitochondrial damage can also include any disease or disorder mediated by or associated with downstream pathways activated by mitochondrial DNA leakage. For example, the disease associated with mitochondrial damage includes any disease or disorder mediated or involved by immune response and / or inflammatory response activated by mitochondrial DNA leakage. For example, the disease associated with mitochondrial DNA leakage includes any disease or disorder mediated by or associated with cGAS-STING pathway activation. For example, in a patient with a disease associated with mitochondrial DNA leakage, mitochondrial DNA leakage can be detected in cells of the disease site. For example, in a patient with a disease associated with mitochondrial DNA leakage, activation of cGAS-STING pathway can be detected in cells of the disease site. In the present application, the disease associated with mitochondrial damage includes, but is not limited to, cardiovascular disease, side effects of anti-tumor therapy, ischemia-related disease, hypoxic damage, inflammatory response and / or inflammatory disease. The disease associated with mitochondrial damage described in the present application also includes diseases and pathological conditions that benefit from alleviating mitochondrial damage. The disease associated with mitochondrial DNA leakage described in the present application also includes diseases and pathological conditions that benefit from inhibiting mitochondrial DNA leakage.Diseases associated with mitochondrial damage can be found in the prior art: Ding Wanyue, et al. Mitochondrial DNA leakage triggers inflammation in age-related cardiovascular diseases, Front. Cell Dev. Biol., 2024 Feb 15: 12: 1287447, Russell, Oliver M., et al. Mitochondrial Diseases: Hope for the Future, Cell, 2020 Apr 2; 181(1): 168-188, Yi Shiau Ng, et al. Mitochondrial disease in adults: recent advances and future promise, The Lancet Neurology, 2021 Jul; 20(7): 573-584.

[0275] In the present application, the term “cardiovascular disease” generally refers to a disease or disorder related to the heart or blood vessels. In the present application, the cardiovascular disease includes, but is not limited to, arteriosclerosis; atherosclerosis; stroke; ischemia-related diseases; endothelial dysfunction; peripheral vascular disease; coronary artery disease, coronary heart disease; myocardial infarction; cerebral infarction and restenosis; thrombosis; hypertension and angina, etc.

[0276] In the present application, the term “ischemia-related disease” generally refers to any disease involving ischemia. The ischemia described in the present application can include a reduction in blood flow to an organ and / or tissue. The reduction in blood flow can be caused by any suitable mechanism, including partial or complete occlusion (blockage), narrowing (constriction), and / or leakage / breakage of one or more blood vessels supplying blood to the organ and / or tissue, etc. The ischemia-related disease described in the present application can also include damage caused by ischemia. For example, the ischemia-related disease includes ischemia-reperfusion injury.

[0277] In the present application, the term “ischemia-reperfusion injury” generally refers to damage resulting from restoring blood flow to a region of tissue or organ that has previously experienced insufficient blood flow due to an ischemic event. Ischemia-reperfusion injury can be caused, for example, by a natural event (e.g., restoration of blood flow after myocardial infarction), trauma, or by one or more surgical or other therapeutic interventions that restore blood flow to tissue or organs that have already experienced compromised blood supply. The ischemia-reperfusion injury described in the present application includes ischemia-reperfusion injury of various organs and / or various systems, for example, ischemia-reperfusion injury of the heart, kidney, liver, lung, and / or brain.

[0278] In the present application, the term "hypoxic injury" generally refers to damage to tissues or cells due to insufficient supply of oxygen. In the present application, the hypoxic injury can include injury resulting from restoration of oxygen supply to a region of tissue or organ that has previously experienced insufficient oxygen supply due to hypoxia. The hypoxic injury described in the present application includes hypoxic injury of various organs and / or various systems, such as hypoxic injury of heart, kidney, liver, brain, lung, brain, and / or muscle.

[0279] In the present application, the term "inflammatory response" generally refers to the physiological and immune response of an organism to tissue damage, pathogen infection, or other stimulating factors. The inflammatory response involves a series of complex biological processes, including but not limited to activation of blood vessels, increase in vascular permeability, recruitment and activation of leukocytes, release of cytokines and chemical mediators, and subsequent tissue damage repair mechanisms. The assessment of inflammatory response can be performed by methods such as determination of cytokine levels, counting of immune cells, pathological examination of tissue sections, or quantitative analysis of inflammatory-related gene expression. The inflammatory response described in the present application includes acute and chronic inflammatory responses, as well as tissue damage and repair processes caused by inflammatory responses. The inflammatory response described in the present application includes inflammatory responses mediated by and / or associated with mitochondrial damage. For example, the inflammatory response described in the present application includes inflammatory responses mediated by and / or associated with mitochondrial DNA leakage. For example, the inflammatory response described in the present application includes inflammatory responses mediated by and / or associated with downstream pathways activated by mitochondrial DNA leakage. For example, the inflammatory response described in the present application includes inflammatory responses mediated by or associated with activation of the cGAS-STING pathway.

[0280] In the present application, the term "inflammatory disease" generally refers to diseases and / or conditions characterized by persistent or excessive inflammatory responses. Inflammatory diseases can be local or systemic, and can be triggered by a variety of factors, including but not limited to infection, autoimmune response, allergic reaction, physical or chemical damage. Inflammatory diseases can be acute or chronic. The inflammatory disease described in the present application includes inflammatory diseases mediated by and / or associated with mitochondrial damage. For example, the inflammatory disease described in the present application includes inflammatory diseases mediated by and / or associated with mitochondrial DNA leakage. For example, the inflammatory disease described in the present application includes inflammatory diseases mediated by and / or associated with downstream pathways activated by mitochondrial DNA leakage. For example, the inflammatory disease described in the present application includes inflammatory diseases mediated by or associated with activation of the cGAS-STING pathway.

[0281] In the present application, the term "cGAS-STING pathway" generally refers to a cytosolic DNA recognition and innate immune response-related signaling pathway, which is mainly mediated by cGAS (cyclic GMP-AMP synthase) and STING (stimulator of interferon genes) in mammalian cells. This pathway plays an important role in physiological or pathological processes such as pathogenic infection, cell damage, autoimmune response, and anti-tumor immunity. When double-stranded DNA abnormally appears in the cytoplasm (such as exogenous DNA, viral DNA, or abnormal leakage of cellular DNA), cGAS can recognize and bind to the DNA, catalyze the synthesis of signal molecule cGAMP, and then activate the STING protein, triggering downstream signal cascade reactions, including TBK1 activation, IRF3 phosphorylation and nuclear translocation, and then inducing the expression of type I interferon (IFN-β) and various pro-inflammatory factors (such as TNF-α, IL-6, etc.), starting innate immune response and inflammatory response. Mitochondrial DNA (mtDNA) leakage is one of the important endogenous factors that induce abnormal activation of the cGAS-STING pathway. As a semi-autonomous organelle, mitochondria have similar characteristics to bacteria (no modification, rich in CpG, etc.), and under conditions of cell stress, damage, aging, drug stimulation, etc., damaged mitochondria can release mtDNA into the cytoplasm, which is recognized by cGAS and activates downstream STING signals, thereby triggering pathological processes such as inflammation, apoptosis, or tissue damage.

[0282] In the present application, the term "mediated by activation of the cGAS-STING pathway" generally refers to the occurrence or aggravation of a certain physiological or pathological process, at least partially caused by the activation of the cGAS-STING signaling pathway in cells. For example, inflammatory response mediated by activation of the cGAS-STING pathway generally refers to the expression of type I interferon, pro-inflammatory factors (such as TNF-α, IL-6), etc. after the DNA (such as mitochondrial DNA) present in the cytoplasm is recognized by cGAS and activates the STING pathway, thereby causing inflammation at the cellular or tissue level. Inflammatory diseases mediated by activation of the cGAS-STING pathway generally refer to diseases whose pathological mechanisms have been clearly related to the activation of the cGAS-STING signaling pathway, and are involved in tissue damage or dysfunction through the above inflammatory response.

[0283] In this application, the term "related to cGAS-STING pathway activation" generally refers to a physiological or pathological process that is mechanistically linked to the activation of the cGAS-STING pathway, accompanied by activation phenomena, or where the condition tends to worsen under the activated state of this pathway. For example, an inflammatory response related to cGAS-STING pathway activation usually refers to an inflammatory state in which elevated expression of cGAS, STING, downstream interferon, or inflammatory factors is detected during the inflammatory process, suggesting the involvement of the cGAS-STING pathway but not necessarily being the sole driving factor. Inflammatory diseases related to cGAS-STING pathway activation generally refer to diseases in which existing literature or experimental models show elevated cGAS-STING pathway expression and enhanced activation markers in diseased tissues or cells, or where symptoms can be alleviated by genetic knockout / drug inhibition of this pathway.

[0284] In this application, the terms "disease mediated by cGAS-STING pathway activation" or "disease associated with cGAS-STING pathway activation" can occur in multiple tissue or organ systems. Specifically, such diseases may include, but are not limited to, pathological processes such as inflammatory lesions, immune-related damage, organ dysfunction, or fibrosis occurring in tissues such as the heart, liver, spleen, lungs, kidneys, skeletal muscle, or smooth muscle.

[0285] In the present application, the term "cGAS-STING pathway activation" generally refers to a state that abnormal cytosolic DNA (e.g. DNA derived from pathogens or mitochondria) is recognized by cGAS to synthesize cGAMP, which in turn activates STING protein and initiates downstream signaling pathway. This activation state can be identified and verified by one or more of the following detection objects and methods: elevated phosphorylation level of key proteins, including but not limited to phosphorylated STING, phosphorylated TBK1, phosphorylated IRF3, etc., which are usually detected by Western blot, flow cytometry, or immunofluorescence staining, etc.; elevated expression level of downstream signaling molecules, including but not limited to type I interferons (e.g. IFN-β), interferon-stimulated genes (ISGs) (e.g. ISG15, CXCL10, etc.), proinflammatory cytokines (e.g. TNF-α, IL-6), etc., which can be detected by qPCR, ELISA, Western blot or RNA-seq, etc.; elevated cGAMP content, as a direct reflection of cGAS activity, which can be determined by LC-MS / MS or specific ELISA method; changes in subcellular localization of STING protein, such as translocation of STING from endoplasmic reticulum to Golgi apparatus, etc., which are usually observed by immunofluorescence microscopy imaging. In some cases, at least 10%, at least 20%, at least 30% or at least 50% increase in at least one of the above indicators relative to the negative control group or the basal level can be considered as the cGAS-STING pathway being activated. In special experimental design, positive control (e.g. stimulant treatment group known to activate cGAS-STING) can also be used as a reference standard. In the present application, the term "pharmaceutical composition" generally refers to a preparation in a form that is effective for the biological activity of the active ingredient, and which does not contain additional components that are unacceptable toxic to the subject to which the preparation is to be administered. The pharmaceutical composition can also include one or more pharmaceutically acceptable carriers. The acceptable components of the pharmaceutical composition are preferably non-toxic to the recipient at the used dose and concentration. The pharmaceutical composition of the present application includes but is not limited to liquid, frozen and lyophilized compositions.

[0286] In the present application, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art. Any conventional medium or agent can be considered for use in the pharmaceutical composition of the present application, unless incompatible with the active compound.

[0287] In the present application, the term "preventing and / or treating" includes not only preventing and / or treating a disease, but also generally includes preventing the onset of the disease, slowing or reversing the progression of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated therewith, and / or preventing further increases in the severity of the disease and / or any symptoms associated therewith, preventing, reducing, or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to the treated patient.

[0288] In the present application, the term "subject" or "individual" or "animal" or "patient" or "subject in need thereof" are used interchangeably herein to refer to a subject, such as a mammalian subject, in need of administration of a pharmaceutical composition of the present application. Animal subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, yellow cattle, dairy cattle, and the like, for example, humans.

[0289] In the present application, the terms "effective amount" or "effective dose" are used interchangeably and generally refer to an amount sufficient to achieve, at least partially, an intended therapeutic effect.

[0290] In the present application, the term "comprising" generally means including, embracing, containing, or having, and in certain embodiments, also means "consisting of" or "consisting of."

[0291] In the present application, the term "about" generally means within 0.5-10% above or below the indicated numerical value, such as within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the indicated numerical value.

[0292] DETAILED DESCRIPTION

[0293] Nuclease

[0294] The nuclease described herein can include any protein or fragment thereof that is capable of cleaving a phosphodiester bond between deoxyribonucleotides. The nuclease described herein can include a nuclease of any origin. In certain embodiments, the nuclease includes a nuclease derived from a human. In certain embodiments, the nuclease is homologous to the cell of interest. In certain embodiments, the nuclease can be codon-optimized.

[0295] In certain embodiments, the nuclease is a non-specific nuclease. For example, the nuclease described herein does not include a Cas enzyme and a zinc finger nuclease. In certain embodiments, the nuclease is an endonuclease. In certain embodiments, the nuclease is a DNA nuclease. For example, the nuclease is a non-specific DNA nuclease. For example, the nuclease is a non-specific DNA endonuclease.

[0296] The nuclease described herein can include a naturally occurring nuclease, or can include an artificially designed nuclease. In certain embodiments, the nuclease described herein does not comprise its naturally occurring localization sequence.

[0297] In certain embodiments, the nuclease comprises a DNase I family member and / or a functional variant thereof. In certain embodiments, the nuclease comprises a human DNase I family member and / or a functional variant thereof. In certain embodiments, the nuclease comprises DNase I and / or a functional variant thereof, DNase1L1 and / or a functional variant thereof, DNase 1L2 and / or a functional variant thereof, and DNase1L3 and / or a functional variant thereof. The functional variant can have, for example, about at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or 80% sequence identity, or at least 90% sequence identity, or at least 95%, 96%, 97%, or 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of the parent.

[0298] In certain embodiments, the nuclease comprises DNase I and / or a functional variant thereof. In certain embodiments, the nuclease comprises the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the nucleic acid molecule encoding the nuclease comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0299] Mitochondrial localization sequence

[0300] The localization of the nuclease to the mitochondria described herein can include localization of the nuclease to the mitochondria in any manner. The localization of the nuclease to the mitochondria described herein includes localization of the nuclease to the outer membrane, the inner membrane of the mitochondria, and / or threading the nuclease through the mitochondrial membrane. In certain embodiments, the localization of the nuclease to the mitochondria includes localization of the nuclease to the outer membrane of the mitochondria.

[0301] In certain embodiments, the nuclease is localized to the mitochondria by a mitochondrial localization sequence. The mitochondrial localization sequences described herein can include any sequence that is capable of localizing a substance to the mitochondria. In certain embodiments, the mitochondrial localization sequence can include any sequence that is capable of localizing a substance to the outer membrane of the mitochondria. The mitochondrial localization sequences described herein can include mitochondrial localization sequences of any origin.

[0302] In certain embodiments, the mitochondrial localization sequence is directly or indirectly linked to the nuclease. For example, a covalent bond (e.g., an amide bond) or a peptide linker can exist between the mitochondrial localization peptide and the nuclease. In certain embodiments, the mitochondrial localization sequence can be located at the N-terminus and / or the C-terminus of the nuclease. In certain embodiments, the mitochondrial localization sequence can be located in the middle of the nuclease, as long as the nuclease is capable of being expressed and localized to the mitochondria.

[0303] The mitochondrial localization sequences described herein can include mitochondrial localization sequences of any origin. In certain embodiments, the mitochondrial localization sequence is homologous to the cell of interest. In certain embodiments, the mitochondrial localization sequence includes a mitochondrial localization sequence derived from a human.

[0304] In certain embodiments, the mitochondrial localization sequence includes a polypeptide sequence. In certain embodiments, the mitochondrial localization sequence is an amino acid sequence. In certain embodiments, the mitochondrial localization sequence can be a naturally occurring sequence that is capable of localizing a substance to the mitochondria. In certain embodiments, the mitochondrial localization sequence includes a mitochondrial localization sequence that localizes the nuclease to various locations in the mitochondria. For example, the mitochondrial localization sequence can include a polypeptide sequence derived from the N-terminal region or the C-terminal region of a mitochondrial protein that is capable of localizing the mitochondrial protein to the mitochondria. For example, the mitochondrial localization sequence can include a polypeptide sequence derived from the N-terminal region or the C-terminal region of a mitochondrial membrane protein that is capable of localizing the mitochondrial membrane protein to the mitochondrial membrane. For example, the mitochondrial localization sequence can include a polypeptide sequence derived from the N-terminal region or the C-terminal region of a mitochondrial outer membrane protein that is capable of localizing the mitochondrial outer membrane protein to the outer membrane of the mitochondria. In certain embodiments, the mitochondrial localization sequence can be an artificially designed sequence that is capable of localizing a substance to the mitochondria. For example, the mitochondrial sequence can include a functional variant sequence of the N-terminal region or the C-terminal region of a mitochondrial protein. In certain embodiments, the nucleic acid molecule encoding the mitochondrial localization sequence can be codon-optimized.

[0305] In some embodiments, the mitochondrial localization sequence comprises a mitochondrial localization sequence derived from cytochrome c oxidase subunit 8A. For example, the mitochondrial localization sequence comprises an amino acid sequence derived from the N-terminus of cytochrome c oxidase subunit 8A. For example, the mitochondrial localization sequence comprises a 29-amino acid sequence derived from the N-terminus of cytochrome c oxidase subunit 8A. The cytochrome c oxidase subunit 8A described herein can comprise a cytochrome c oxidase subunit 8A of any origin. In some embodiments, the cytochrome c oxidase subunit 8A is homologous to the cell of interest. In some embodiments, the cytochrome c oxidase subunit 8A comprises a human cytochrome c oxidase subunit 8A.

[0306] In some embodiments, the mitochondrial localization sequence can comprise the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encoding the mitochondrial localization sequence can comprise the nucleotide sequence set forth in SEQ ID NO: 2.

[0307] Methods of reducing mitochondrial DNA leakage in a cell of interest

[0308] In one aspect, the present application provides a method of reducing mitochondrial DNA leakage in a cell of interest, comprising localizing a nuclease to a mitochondrion.

[0309] In another aspect, the present application provides a modified mitochondrion, wherein a nuclease is localized to the mitochondrion.

[0310] In some embodiments, the method of reducing mitochondrial DNA leakage in a cell of interest described herein comprises genetically engineering a cell of interest to express a nuclease localized to a mitochondrion to reduce mitochondrial DNA leakage in the cell of interest. For example, the method of reducing mitochondrial DNA leakage in a cell of interest described herein comprises causing the cell of interest to express a fusion protein comprising a mitochondrial localization sequence and a nuclease to reduce mitochondrial DNA leakage in the cell of interest. For example, the method of reducing mitochondrial DNA leakage in a cell of interest described herein comprises introducing into the cell of interest a nucleic acid molecule encoding a fusion protein comprising a mitochondrial localization sequence and a nuclease to reduce mitochondrial DNA leakage in the cell of interest. The methods of introducing the nucleic acid molecule into the cell of interest are known in the art.

[0311] In certain embodiments, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein comprise localizing a nuclease to mitochondria and introducing the mitochondria comprising the nuclease into the cell of interest to reduce mitochondrial DNA leakage in the cell of interest. For example, the mitochondria comprising the nuclease is a modified mitochondria described herein. In certain embodiments, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein comprise introducing a modified mitochondria into the cell of interest, wherein the nuclease is localized to the modified mitochondria. For example, the modification comprises localizing the nuclease to the mitochondria. Methods of introducing the modified mitochondria into the cell of interest are known in the art, for example, the modified mitochondria is incubated with the cell of interest, and the modified mitochondria is capable of being taken up by endocytosis.

[0312] In certain embodiments, the modified mitochondria is isolated from a cell that is homologous to the cell of interest. In certain embodiments, the cell from which the modified mitochondria is isolated is from the same species as the cell of interest. For example, the cell from which the modified mitochondria is isolated and the cell of interest are both from a human. In certain embodiments, the cell from which the modified mitochondria is isolated is from the same individual as the cell of interest. For example, the cell from which the modified mitochondria is isolated and the cell of interest are both from the same human.

[0313] In certain embodiments, the modified mitochondria is isolated from a cell that is of the same cell type as the cell of interest. In certain embodiments, the modified mitochondria is isolated from a cell that is of a different cell type than the cell of interest. The cell type can be classified according to characteristics such as structure, function, and / or location in the organism, etc. For example, the modified mitochondria can be isolated from a cell that is of the same function as the cell of interest. For example, when the cell of interest is a cardiomyocyte, the modified mitochondria can be a modified mitochondria isolated from a cardiomyocyte.

[0314] In certain embodiments, to improve the effect of reducing mitochondrial DNA leakage in a cell of interest, a modified mitochondria isolated from a cell that is of a metabolic level that matches the cell of interest can be introduced into the cell of interest. For example, when the cell of interest is a cardiomyocyte, a modified mitochondria isolated from a cell that is of a metabolic level that matches the cardiomyocyte can be introduced into the cardiomyocyte.

[0315] The cell of interest described herein can include a cell in any state, from any source, or of any type. In certain embodiments, the cell of interest comprises a cell line. In certain embodiments, the cell of interest comprises a cultured cell. In certain embodiments, the cell of interest can comprise a healthy cell.

[0316] In some embodiments, the cell of interest comprises a mitochondrially-impaired cell. The mitochondrially-impaired cell comprises a cell with impaired mitochondrial morphology and / or function. The mitochondrial impairment can comprise physiological and / or pathological mitochondrial impairment. For example, the mitochondrially-impaired cell can comprise a healthy cell artificially induced to have impaired mitochondria. In this application, the cell of interest can comprise a cell with impaired mitochondria due to any cause. In some embodiments, the cell of interest comprises a cell with mitochondrial DNA leakage. For example, the cell of interest comprises a cell with abnormal mitochondrial DNA leakage, wherein the abnormal mitochondrial leakage generally refers to an increased amount of mitochondrial DNA leakage relative to a normal cell.

[0317] In some embodiments, the cell of interest comprises a cell in a senescent state. The senescent state can be caused by any cause. For example, the senescence can be physiological senescence. For example, the senescent state can be caused by ischemia and / or hypoxia. For example, the senescent state can be caused by an anti-tumor therapy. For example, the senescent state can be a side effect of an anti-tumor therapy. For example, the senescent cell can comprise a healthy cell artificially induced to exhibit one or more senescence phenotypes. For example, the artificial induction of a cell to exhibit one or more senescence phenotypes can comprise administering a senescence-inducing agent to the cell. For example, the senescence-inducing agent can comprise doxorubicin.

[0318] In some embodiments, the cell of interest comprises an ischemia-reperfusion injury, a hypoxic injury, and / or a cell subjected to a peroxide. For example, the peroxide comprises hydrogen peroxide.

[0319] In some embodiments, the cell of interest is derived from a mammal. In some embodiments, the cell of interest comprises a cell derived from a human or a mouse. In some embodiments, the cell of interest comprises a muscle cell, a neural cell, an endothelial cell, an epithelial cell, an embryonic cell, and / or a tumor cell. In some embodiments, the cell of interest comprises a cardiomyocyte, a skeletal muscle cell, a vascular endothelial cell, a kidney epithelial cell, and / or a neuroblastoma cell.

[0320] The damage to mitochondria and / or the release of mitochondrial DNA in the subject cell described herein can be caused by any cause. In some embodiments, the damage to mitochondria and / or the release of mitochondrial DNA in the subject cell is caused by ischemia-reperfusion. For example, the subject cell is a cardiomyocyte, and the damage to mitochondria and / or the release of mitochondrial DNA in the subject cell can be caused by myocardial ischemia-reperfusion. In some embodiments, the damage to mitochondria and / or the release of mitochondrial DNA in the subject cell is caused by aging. In some embodiments, the damage to mitochondria and / or the release of mitochondrial DNA in the subject cell is caused by hypoxia. In some embodiments, the damage to mitochondria and / or the release of mitochondrial DNA in the subject cell is caused by peroxide stimulation. For example, the peroxide includes hydrogen peroxide.

[0321] The alleviation of the damage to mitochondria and / or the reduction of the release of mitochondrial DNA in the subject cell described herein can be relative to a reference cell. The reference cell can be a cell of the same cell type, under the same culture condition, but not subjected to any treatment to reduce the release of mitochondrial DNA. For example, the reduction of the release of mitochondrial DNA in the subject cell can be less release of mitochondrial DNA in the subject cell after the same induction of the release of mitochondrial DNA, relative to a cell not subjected to any treatment to reduce the release of mitochondrial DNA. In addition, the reduction of the release of mitochondrial DNA in the subject cell can be relative to the cell before the treatment to reduce the release of mitochondrial DNA. For example, in the same cell type, the release of mitochondrial DNA is reduced in the treated cell, relative to the cell before the treatment, by performing the method to reduce the release of mitochondrial DNA.

[0322] The method to reduce the release of mitochondrial DNA in the subject cell described herein includes reducing the amount of the released mitochondrial DNA in the cytoplasm and / or the culture medium, and modulating the reactions in which the released mitochondrial DNA is involved. The amount of the released mitochondrial DNA in the cytoplasm and / or the culture medium can be detected by methods known in the art.

[0323] The methods of reducing mitochondrial DNA leakage in a cell of interest described herein can alleviate mitochondrial damage. The methods of reducing mitochondrial DNA leakage in a cell of interest described herein can alleviate the effects of mitochondrial damage on a cell. The methods of reducing mitochondrial DNA leakage in a cell of interest described herein can include reducing the effects of leaked mitochondrial DNA on a cell. For example, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein can slow the aging of a cell of interest. For example, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein can alleviate one or more aging phenotypes of a cell of interest. For example, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein can alleviate damage to a cell of interest. For example, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein can restore function to a cell of interest. For example, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein can inhibit an inflammatory response in a cell of interest. For example, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein can inhibit a downstream pathway activated by mitochondrial DNA leakage. For example, the downstream pathway activated by mitochondrial DNA leakage includes the cGAS-STING pathway. For example, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein can restore or preserve mitochondrial function in a cell of interest. For example, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein can preserve mitochondrial respiratory capacity.

[0324] In certain embodiments, the methods of reducing mitochondrial DNA leakage in a cell of interest described herein are in vitro and / or ex vivo methods.

[0325] Modified mitochondria

[0326] In another aspect, the present application provides a modified mitochondrion, wherein a nuclease is localized to the mitochondrion. For example, wherein the nuclease comprises a nuclease described herein, the nuclease is localized to the mitochondrion by a means described herein. For example, the nuclease is localized to the mitochondrion by a mitochondrion-localizing peptide sequence described herein.

[0327] The modified mitochondria described herein can be isolated from any cell and / or any tissue. In certain embodiments, the modified mitochondria can be isolated from somatic cells, germ cells, and / or stem cells. In certain embodiments, the modified mitochondria can be isolated from induced pluripotent stem cells. For example, the modified mitochondria can be isolated from cells differentiated from induced pluripotent stem cells. For example, the modified mitochondria can be isolated from various cells differentiated from induced pluripotent stem cells, such as cardiomyocytes. For example, the modified mitochondria can be isolated from muscle cells, neural cells, endothelial cells, epithelial cells, embryonic cells, and / or tumor cells. For example, the modified mitochondria can be isolated from cardiomyocytes and / or skeletal muscle cells.

[0328] The modified mitochondria described herein can be isolated or provided from any source of cells and / or tissues. The modified mitochondria described herein can be isolated from autologous, allogeneic, and / or xenogeneic sources of cells. The modified mitochondria described herein can be isolated from mammalian cells. For example, the modified mitochondria described herein can be isolated from human or mouse cells.

[0329] The modified mitochondria described herein can be isolated or provided from any state of cells and / or tissues. In certain embodiments, the modified mitochondria are isolated from healthy cells. In certain embodiments, the modified mitochondria are isolated from cells that match the metabolic level of the subject to which they will be administered (e.g., the cells of interest).

[0330] In certain embodiments, the modified mitochondria can be isolated from tissues and / or cells that are cultured in vitro and / or ex vivo. In certain embodiments, the modified mitochondria are isolated from modified cells. Wherein the modified cells have a mitochondrial-targeted nuclease and / or comprise a nucleic acid molecule encoding a mitochondrial-targeted nuclease.

[0331] In certain embodiments, the modified mitochondria described herein can comprise additional modifications in addition to the mitochondrial-localized nuclease. In certain embodiments, the modified mitochondria described herein can comprise mitochondria that are directly and / or indirectly linked to other agents. For example, the modified mitochondria described herein can comprise mitochondria that have a mitochondrial-localized nuclease, and are directly and / or indirectly linked to other agents. In certain embodiments, the agents can be linked to, attached to, embedded in the membrane of, or completely or partially enclosed in the mitochondria. For example, the agents can be directly linked to components of the mitochondrial membrane via a covalent bond, or indirectly linked to components of the mitochondrial membrane via a linker (e.g., a peptide linker) or another covalently bonded agent. The agents can include therapeutic agents, diagnostic agents, and / or imaging agents. Imaging agents are agents that are employed in imaging techniques. The techniques or modalities include, but are not limited to, X-ray, computed tomography (CT), magnetic resonance imaging (MRI), scintigraphy, fluorescence, ultrasound, and the like. The imaging agents can be fluorescent and / or radioactive.

[0332] In certain embodiments, the modified mitochondria described herein are isolated mitochondria. Methods of isolating mitochondria are known in the art. For example, mitochondria can be isolated from cells or tissues by any means known to one of skill in the art. In certain embodiments, a tissue sample or cell sample is collected and then homogenized; after homogenization, the mitochondria are isolated by repeated centrifugation. Alternatively, the cell homogenate can be filtered through a nylon mesh filter to isolate the mitochondria. In certain embodiments, the function of the mitochondria depends on the length of time between isolation and time of use. In certain embodiments, the modified mitochondria are freshly isolated and are viable. The modified mitochondria or compositions comprising the modified mitochondria can be administered to a subject within about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes of the isolation of the mitochondria. In some cases, the modified mitochondria or compositions comprising the modified mitochondria are administered to a subject within about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes of the initiation of the process of isolating the mitochondria. In some cases, the modified mitochondria and / or compositions comprising the modified mitochondria can be stored for a short period of time (e.g., about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes) prior to use. In certain embodiments, the modified mitochondria are not frozen and thawed after isolation from tissue and / or cells.

[0333] The modified mitochondria described herein can alleviate the damage of mitochondria in the subject (e.g., the cell of interest) to which the modified mitochondria is administered. The modified mitochondria described herein can reduce the mitochondrial DNA leakage in the subject (e.g., the cell of interest) to which the modified mitochondria is administered. The reduction of mitochondrial DNA leakage includes reducing the amount of mitochondrial DNA leakage into the cytoplasm and / or the culture medium, and modulating the reactions in which the leaked mitochondrial DNA is involved.

[0334] The modified mitochondria described herein can reduce the impact of leaked mitochondrial DNA on the cell. For example, the modified mitochondria described herein can delay the aging of the cell of interest. For example, the modified mitochondria described herein can delay the aging phenotype of one or more cells of interest. For example, the modified mitochondria described herein can alleviate the damage of the cell of interest. For example, the modified mitochondria described herein can restore the function of the cell of interest. For example, the modified mitochondria described herein can inhibit the inflammatory response. For example, the modified mitochondria described herein can inhibit the downstream pathways activated by mitochondrial DNA leakage. For example, the downstream pathways activated by mitochondrial DNA leakage include the cGAS-STING pathway. For example, the modified mitochondria described herein can restore or retain the function of mitochondria in the cell of interest. For example, the modified mitochondria described herein can retain the respiratory capacity of mitochondria.

[0335] The modified mitochondria described herein can be used to delay aging. For example, wherein the delay of aging comprises reducing the expression level of an aging marker, wherein the aging marker comprises, but is not limited to, p21, p16INK4a, p53, telomere length, and / or a DNA damage marker. For example, wherein the delay of aging comprises delaying cardiac aging, vascular aging, muscle aging, kidney aging, and / or nervous system aging. The modified mitochondria described herein can be used to prevent and / or treat a disease. For example, the disease comprises a disease associated with mitochondrial damage. For example, the disease comprises a disease associated with mitochondrial DNA leakage. For example, the disease comprises an ischemia-related disease. For example, the disease comprises a side effect of an anti-tumor therapy. For example, the side effect of an anti-tumor therapy comprises mitochondrial damage, mitochondrial DNA leakage, and / or inflammation associated with the activation of the cGAS-STING pathway thereby. For example, the anti-tumor therapy comprises chemotherapy and / or radiotherapy. For example, the chemotherapy drug comprises a platinum-based chemotherapy drug and / or an anthracycline-based chemotherapy drug. For example, the anthracycline-based chemotherapy drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin. For example, the platinum-based chemotherapy drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin. For example, the disease comprises a cardiovascular disease. For example, the disease comprises ischemia-reperfusion injury and / or hypoxic injury. The modified mitochondria described herein can be used to downregulate an inflammatory response and / or treat an inflammatory disease. For example, wherein the inflammatory response and / or inflammatory disease comprises or is associated with the activation of the cGAS-STING pathway. For example, the inflammatory response and / or inflammatory disease comprises a kidney inflammatory disease, a liver inflammatory disease, a lung inflammatory disease, a heart inflammatory disease, a spleen-related inflammatory pathology, and / or an inflammatory pathology in smooth muscle tissue. For example, the kidney inflammatory disease comprises acute kidney injury (AKI), chronic kidney disease (CKD), systemic lupus erythematosus (SLE)-related nephritis, IgA nephropathy, interstitial nephritis, and / or diabetic nephropathy. For example, the liver inflammatory disease comprises non-alcoholic steatohepatitis, drug-induced liver injury, viral hepatitis, alcoholic liver disease, liver fibrosis, and / or liver cirrhosis. For example, the lung inflammatory disease comprises acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bacterial or viral infection-related pneumonia, pneumoconiosis, and / or pulmonary fibrosis. For example, the heart inflammatory disease comprises doxorubicin-induced cardiotoxicity, myocardial ischemia-reperfusion injury, myocarditis, and / or chronic heart failure. For example, the spleen-related inflammatory pathology comprises systemic lupus erythematosus (SLE)-related spleen pathology, autoimmune disease-associated spleen immune activation, and / or infection-induced spleen immune activation.For example, the inflammatory disorder in the smooth muscle tissue comprises an inflammatory response in vascular smooth muscle cells, chronic inflammatory disorders in airway smooth muscle, and / or gastrointestinal smooth muscle. For example, the inflammatory disorder in the smooth muscle tissue comprises atherosclerosis and / or arterial injury.

[0336] Fusion protein, nucleic acid molecule, vector, modified cell, method of preparing a modified mitochondrion

[0337] In another aspect, the present application provides a fusion protein comprising a nuclease described herein and a mitochondrial localization sequence described herein.

[0338] In certain embodiments, the mitochondrial localization sequence is directly or indirectly linked to the nuclease. For example, there can be a covalent bond (e.g., an amide bond) or a peptide linker between the mitochondrial localization peptide and the nuclease. In certain embodiments, the mitochondrial localization sequence can be located at the N-terminus and / or C-terminus of the nuclease. In certain embodiments, the mitochondrial localization sequence can be located in the middle of the nuclease, as long as the nuclease can be expressed and localized to mitochondria.

[0339] In certain embodiments, the fusion protein comprises a nuclease sequence set forth in SEQ ID NO: 3 and a mitochondrial localization sequence set forth in SEQ ID NO: 1. In certain embodiments, the nucleic acid molecule encoding the fusion protein comprises a nucleotide sequence encoding a nuclease set forth in SEQ ID NO: 4 and a nucleotide sequence encoding a mitochondrial localization sequence set forth in SEQ ID NO: 2. In certain embodiments, the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the nucleic acid molecule encoding the fusion protein comprises a nucleotide sequence set forth in SEQ ID NO: 6.

[0340] In certain embodiments, the fusion protein is used to alleviate mitochondrial damage in a cell of interest. In certain embodiments, the fusion protein is used to reduce mitochondrial DNA leakage in a cell of interest. In certain embodiments, the fusion protein is used to prepare a modified mitochondrion described herein and / or a modified cell described herein.

[0341] In another aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein described herein.

[0342] In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a nuclease set forth in SEQ ID NO: 4 and a nucleotide sequence encoding a mitochondrial localization sequence set forth in SEQ ID NO: 2. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 6.

[0343] In certain embodiments, the nucleic acid molecule is used to alleviate mitochondrial damage in a cell of interest. In certain embodiments, the nucleic acid molecule is used to reduce mitochondrial DNA leakage in a cell of interest. In certain embodiments, the nucleic acid molecule is used to make a modified mitochondrion described herein and / or a modified cell described herein.

[0344] The nucleic acid molecule described herein can also comprise elements required for expression of the fusion protein. The nucleic acid molecule described herein can be introduced into an organism and / or expressed in any manner. "Introducing" a nucleic acid molecule into a cell includes transforming a cell of the organism with the nucleic acid molecule such that the nucleic acid molecule is capable of functioning in the cell. Means of expressing the nucleic acid molecule described herein include, but are not limited to, plasmid vectors, viral vectors-mediated gene transfer, transposon systems, and the like. Regardless of the method used, as long as the nucleic acid molecule can be effectively introduced into a cell and expressed, it is considered to be in accordance with the means of expressing the nucleic acid molecule described herein.

[0345] In certain embodiments, the nucleic acid molecule comprises expression regulatory elements. For example, the nucleic acid molecule can comprise a promoter and / or an enhancer, wherein the promoter is located upstream of the nucleotide sequence encoding the fusion protein. In certain embodiments, the nucleic acid molecule comprises a polynucleotide signal sequence. For example, the polynucleotide signal sequence is located upstream of the nucleotide sequence encoding the fusion protein.

[0346] The nucleic acid molecule described herein can comprise DNA and / or RNA. The nucleic acid molecule described herein can comprise single-stranded nucleic acid molecules and / or double-stranded nucleic acid molecules.

[0347] In certain embodiments, the nucleic acid molecule is DNA. In certain embodiments, the nucleic acid molecule is constructed in a viral vector to be introduced into an organism and / or expressed. For example, the nucleic acid molecule is constructed in an AAV vector to be introduced into an organism and / or expressed, and the nucleic acid molecule can comprise AAV vector elements. For example, the nucleic acid molecule can further comprise AAV inverted terminal repeats (ITRs), which are located upstream and downstream of the nucleotide sequence encoding the fusion protein. For example, the AAV vector can comprise AAV vectors of various serotypes, for example, the AAV vector can comprise AAV9, and the AAV ITRs comprise ITRs derived from AAV9. For example, the nucleic acid molecule is constructed in a lentiviral vector to be introduced into an organism and / or expressed, and the nucleic acid molecule can comprise lentiviral vector elements. For example, the nucleic acid molecule comprises lentiviral long terminal repeat (LTR) sequences, which are located upstream and downstream of the nucleotide sequence encoding the fusion protein.

[0348] In certain embodiments, the nucleic acid molecule is an RNA. For example, the nucleic acid molecule can be an mRNA. For example, the nucleic acid molecule can further comprise a 5' cap, a 5' untranslated region, and a 3' untranslated region. For example, the nucleic acid molecule can further comprise at least one modified nucleotide.

[0349] The nucleic acid molecule described herein can be an isolated nucleic acid molecule. Methods of isolating nucleic acid molecules are known in the art.

[0350] In another aspect, the present application provides a vector comprising the nucleic acid molecule described herein.

[0351] In certain embodiments, the vector can be a viral vector. For example, the viral vector can comprise a DNA viral vector, an RNA viral vector, and / or a retroviral vector. For example, the viral vector can comprise an integrating viral vector and / or an episomal vector. For example, the vector can comprise an adeno-associated viral vector and / or a lentiviral vector.

[0352] In certain embodiments, the vector can also be a polynucleotide vector, e.g., a plasmid, a cosmid, or a transposon.

[0353] Vectors suitable for use have been widely described and are well known in the art. Those skilled in the art will appreciate that a vector comprising a nucleic acid molecule described herein can further comprise additional sequences and elements that are desirable for replication of the vector in prokaryotic and / or eukaryotic cells. For example, a vector described herein can comprise a prokaryotic replicon, i.e., a nucleotide sequence that has the ability to direct the host's own replication and maintenance in a prokaryotic host cell (e.g., a bacterial host cell). Such replicons are well known in the art. In certain instances, the vector can comprise a shuttle element that makes the vector suitable for replication and integration in both prokaryotes and eukaryotes. In addition, the vector can further comprise a gene that is capable of expressing a detectable marker (e.g., a drug resistance gene). The vector can also have a reporter gene, e.g., a gene that encodes a fluorescent or other detectable protein.

[0354] The vector described herein can be an isolated vector. Methods of isolating vectors are known in the art.

[0355] In another aspect, the present application provides a modified cell comprising the modified mitochondrion described herein, the fusion protein described herein, the nucleic acid molecule described herein, and / or the vector described herein.

[0356] The modified cells described herein can comprise a modified mitochondrion, wherein the nuclease is localized to the modified mitochondrion. The modified cells described herein express a nuclease localized to a mitochondrion. The modified cells described herein comprise a nucleic acid molecule encoding a nuclease localized to a mitochondrion. The modified mitochondrion described herein, the fusion protein described herein, the nucleic acid molecule described herein, and / or the vector described herein can be introduced into the cell to obtain the modified cell. One of skill in the art will understand the conditions necessary to introduce the modified mitochondrion described herein, the fusion protein described herein, the nucleic acid molecule described herein, and / or the vector described herein into a cell, as well as conditions that support or facilitate expression of a gene of interest within a cell. Further, the methods can be in vivo or in vitro methods.

[0357] In certain embodiments, the modified cells can comprise somatic cells, germ cells, and / or stem cells. In certain embodiments, the modified cells can comprise induced pluripotent stem cells (iPSCs). For example, the modified cells can comprise cells differentiated from induced pluripotent stem cells. For example, the modified cells can comprise various cells differentiated from induced pluripotent stem cells, such as cardiomyocytes. For example, the modified cells can comprise muscle cells, neural cells, endothelial cells, epithelial cells, embryonic cells, and / or tumor cells. For example, the modified cells can comprise cardiomyocytes and / or skeletal muscle cells.

[0358] In certain embodiments, the modified cells can comprise prokaryotic cells and eukaryotic cells. In certain embodiments, the modified cells described herein can comprise cells of autologous, allogeneic, and / or xenogeneic origin. In certain embodiments, the modified cells described herein can comprise mammalian cells. For example, the modified cells described herein comprise human or mouse cells.

[0359] The modified cells described herein can comprise cells in any state. For example, the modified cells comprise healthy cells.

[0360] In certain embodiments, the modified cells described herein do not have the potential to develop into a whole organism. In certain embodiments, the modified cells described herein can not include human embryonic stem cells. In certain embodiments, the modified cells described herein can include stem cells isolated or obtained from a human embryo that has not developed in vivo for up to 14 days post-fertilization. In certain embodiments, the modified cells described herein do not include human embryonic stem cells isolated or obtained from a human embryo that has developed in vivo. In certain embodiments, the modified cells described herein do not include stem cells isolated or obtained from a human embryo that has not developed in vivo for more than 14 days post-fertilization.

[0361] In certain embodiments, the modified cells described herein can be used to amplify, replicate, package, and / or purify the modified mitochondria described herein, the fusion proteins described herein, the nucleic acid molecules described herein, and / or the vectors described herein. For example, when the cells are used to package the viral vectors described herein, the modified cells can also be transfected with one or more plasmids or infected with one or more viruses that provide the necessary helper molecules for packaging. In certain embodiments, the modified cells can stably express one or more helper molecules from the genome. Those of skill in the art are capable of selecting appropriate host cells for amplification, replication, packaging, and / or purification of the vectors described herein.

[0362] In certain embodiments, the modified cells described herein can be used to make the modified mitochondria described herein. In certain embodiments, the modified cells described herein can be used to express the fusion proteins described herein, the nucleic acid molecules described herein, and / or the vectors described herein.

[0363] In certain embodiments, the modified cells can be isolated cells. Methods of isolating cells are known in the art.

[0364] The fusion proteins, nucleic acid molecules, vectors, and / or modified cells described herein can be used to alleviate mitochondrial damage in a cell of interest. The fusion proteins, nucleic acid molecules, vectors, and / or modified cells described herein can be used to reduce mitochondrial DNA leakage in a cell of interest. In certain embodiments, the fusion proteins, nucleic acid molecules, vectors described herein are used to make the modified mitochondria described herein and / or the modified cells described herein.

[0365] The fusion proteins, nucleic acid molecules, vectors, and / or modified cells described herein can be used to delay aging. For example, wherein the delay of aging comprises a delay of cardiac aging, vascular aging, muscle aging, kidney aging, and / or nervous system aging. For example, wherein the delay of aging comprises a reduction in the expression level of an aging marker, wherein the aging marker comprises, but is not limited to, p21, p16INK4a, p53, telomere length, and / or a DNA damage marker. The fusion proteins, nucleic acid molecules, vectors, and / or modified cells described herein can be used to prevent and / or treat a disease. For example, the disease comprises a disease associated with mitochondrial damage. For example, the disease comprises a disease associated with mitochondrial DNA leakage. For example, the disease comprises an ischemia-related disease. For example, the disease comprises a side effect of an anti-tumor therapy, wherein the side effect of an anti-tumor therapy comprises, but is not limited to, mitochondrial damage. For example, wherein the mitochondrial damage comprises, but is not limited to, mitochondrial DNA leakage. For example, the anti-tumor therapy comprises chemotherapy and / or radiotherapy. For example, wherein the chemotherapeutic drug comprises a platinum-based chemotherapeutic drug and / or an anthracycline-based chemotherapeutic drug. For example, wherein the anthracycline-based chemotherapeutic drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin. For example, wherein the platinum-based chemotherapeutic drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin. For example, the disease comprises a cardiovascular disease. For example, the disease comprises ischemia-reperfusion injury and / or hypoxic injury. The fusion proteins, nucleic acid molecules, vectors, and / or modified cells described herein can be used to downregulate an inflammatory response and / or treat an inflammatory disease. For example, wherein the inflammatory response and / or inflammatory disease comprises or is associated with cGAS-STING pathway activation. For example, wherein the inflammatory disease is selected from a kidney inflammatory disease, a liver inflammatory disease, a lung inflammatory disease, a heart inflammatory disease, a spleen-related inflammatory disorder, and an inflammatory disorder in smooth muscle tissue.

[0366] For example, wherein the inflammatory disease of the kidney comprises acute kidney injury (AKI), chronic kidney disease (CKD), systemic lupus erythematosus associated nephritis, IgA nephropathy, interstitial nephritis, and / or diabetic nephropathy. For example, wherein the inflammatory disease of the liver comprises nonalcoholic steatohepatitis, drug-induced liver injury, viral hepatitis, alcoholic liver disease, liver fibrosis, and / or cirrhosis. For example, wherein the inflammatory disease of the lung comprises acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bacterial or viral infection associated pneumonia, pneumoconiosis, and / or pulmonary fibrosis. For example, wherein the inflammatory disease of the heart comprises adriamycin-induced cardiotoxicity, myocardial ischemia-reperfusion injury, myocarditis, and / or chronic heart failure. For example, wherein the spleen associated inflammatory disorder comprises systemic lupus erythematosus (SLE) associated splenic disorder, autoimmune disease associated splenic immune activation, and / or infection induced splenic immune activation. For example, wherein the inflammatory disorder in smooth muscle tissue comprises inflammatory response in vascular smooth muscle cells, chronic inflammatory disorder in airway smooth muscle, and / or gastrointestinal smooth muscle. For example, wherein the inflammatory disorder in smooth muscle tissue comprises atherosclerosis and / or arterial injury.

[0367] In another aspect, the present application provides a method of preparing a modified mitochondrion, comprising preparing a modified cell described herein and isolating mitochondria produced by the modified cell.

[0368] The method of preparing a modified mitochondrion described herein can comprise using a nucleic acid molecule described herein and / or a vector described herein, preparing a modified cell described herein, and isolating mitochondria produced by the modified cell.

[0369] In certain embodiments, the method of preparing a modified mitochondrion comprises isolating the modified mitochondrion. In certain embodiments, the method of preparing a modified mitochondrion further comprises detecting the modified mitochondrion. For example, the detecting comprises detecting whether the nuclease is localized to the mitochondrion, detecting the morphology of the mitochondrion, and / or detecting the function of the mitochondrion.

[0370] Pharmaceutical compositions

[0371] The present application also provides a pharmaceutical composition comprising a modified mitochondrion described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, and / or a modified cell described herein.

[0372] The pharmaceutical compositions described herein can also include a pharmaceutically acceptable carrier. For example, the pharmaceutically acceptable carrier can include sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles can include water, ethanol, polyols (such as propylene glycol, glycerol, polyethylene glycol and the like), nontoxic

[0373] The pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (such as inhalation), sublingual, transdermal (such as topical), transmucosal, and rectal administration.

[0374] The pharmaceutical compositions described herein can be formulated in any form, such as a liquid, cream, ointment, salve, oil, emulsion, liposome formulation, etc. Methods of preparing compositions are well known in the art.

[0375] The pharmaceutical compositions described herein can be used to reduce mitochondrial DNA leakage in a cell of interest. The pharmaceutical compositions described herein can be used to delay aging. For example, the delay of aging includes a delay of cardiac aging, vascular aging, muscle aging, kidney aging, and / or nervous system aging. The pharmaceutical compositions described herein can be used to prevent and / or treat a disease. For example, the disease includes a disease associated with mitochondrial DNA leakage. For example, the disease includes an ischemia-related disease. For example, the disease includes a side effect of an anti-tumor therapy. For example, the anti-tumor therapy includes chemotherapy and / or radiotherapy. For example, the chemotherapy drug includes a platinum-based chemotherapy drug and / or an anthracycline chemotherapy drug. For example, the anthracycline chemotherapy drug includes doxorubicin, epirubicin, and / or deoxydoxorubicin. For example, the platinum-based chemotherapy drug includes cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin. For example, the disease includes a cardiovascular disease. For example, the disease includes ischemia-reperfusion injury and / or hypoxic injury.

[0376] In certain embodiments, the pharmaceutical composition comprises a nucleic acid molecule described herein and a carrier for delivering the nucleic acid molecule. For example, the nucleic acid molecule comprises an mRNA, and the carrier for delivering the nucleic acid molecule includes, but is not limited to, a lipid nanoparticle (LNP).

[0377] Uses and methods of treatment

[0378] In another aspect, the present application provides a use of the modified mitochondria described herein, the fusion protein described herein, the nucleic acid molecule described herein, the vector described herein, and / or the modified cell described herein in the preparation of a reagent.

[0379] In certain embodiments, the reagent is used for delaying aging. In certain embodiments, the reagent is used for preventing and / or treating a disease associated with mitochondrial DNA leakage. In certain embodiments, the reagent is used for preventing and / or treating an ischemia-related disease. In certain embodiments, the reagent is used for alleviating side effects of an anti-tumor therapy. In certain embodiments, the anti-tumor therapy comprises side effects including mitochondrial leakage. In certain embodiments, the reagent is used for treating ischemia-reperfusion injury and / or hypoxic injury. In certain embodiments, the reagent is used for down-regulating an inflammatory response and / or treating an inflammatory disease. For example, wherein the inflammatory response and / or inflammatory disease comprises or is associated with cGAS-STING pathway activation. In certain embodiments, the reagent comprises the pharmaceutical composition described herein.

[0380] In another aspect, the present application provides a method of delaying aging, comprising administering to a subject an effective amount of the modified mitochondria described herein, the fusion protein described herein, the nucleic acid molecule described herein, the vector described herein, the modified cell described herein, and / or the pharmaceutical composition described herein.

[0381] In another aspect, the present application provides a method of preventing and / or treating a disease, comprising administering to a subject an effective amount of the modified mitochondria described herein, the fusion protein described herein, the nucleic acid molecule described herein, the vector described herein, the modified cell described herein, and / or the pharmaceutical composition described herein.

[0382] In another aspect, the present application provides a use of the modified mitochondria described herein, the fusion protein described herein, the nucleic acid molecule described herein, the vector described herein, the modified cell described herein, and / or the pharmaceutical composition described herein in delaying aging.

[0383] In another aspect, the present application provides a use of the modified mitochondria described herein, the fusion protein described herein, the nucleic acid molecule described herein, the vector described herein, the modified cell described herein, and / or the pharmaceutical composition described herein in preventing and / or treating a disease.

[0384] In certain embodiments, the delaying of aging comprises reducing the expression level of a marker of aging. For example, wherein the marker of aging comprises, but is not limited to, p21, pl6INK4a, p53, telomere length, and / or a DNA damage marker. For example, wherein the delaying of aging comprises delaying cardiac aging, vascular aging, muscle aging, kidney aging, and / or nervous system aging.

[0385] In certain embodiments, the disease comprises a disease associated with mitochondrial damage. In certain embodiments, the disease comprises a disease associated with mitochondrial DNA leakage. In certain embodiments, the disease comprises an ischemia-related disease. In certain embodiments, the disease comprises a side effect of an anti-tumor therapy. In certain embodiments, the anti-tumor therapy comprises a side effect that comprises mitochondrial leakage. In certain embodiments, the side effect of the anti-tumor therapy comprises, but is not limited to, mitochondrial damage, mitochondrial DNA leakage, and / or inflammation associated with cGAS-STING pathway activation triggered thereby. For example, the anti-tumor therapy comprises chemotherapy and / or radiation therapy. For example, the chemotherapy drug comprises a platinum-based chemotherapy drug and / or an anthracycline-based chemotherapy drug. For example, the anthracycline-based chemotherapy drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin. For example, the platinum-based chemotherapy drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin. In certain embodiments, the disease comprises a cardiovascular disease. In certain embodiments, the disease comprises ischemia-reperfusion injury and / or hypoxic injury. In certain embodiments, the disease comprises an inflammatory response and / or an inflammatory disease. For example, the inflammatory response and / or the inflammatory disease comprises a cGAS-STING pathway activation-mediated or -associated inflammatory response and / or inflammatory disease. For example, the inflammatory response and / or the inflammatory disease comprises a kidney inflammatory disease, a liver inflammatory disease, a lung inflammatory disease, a heart inflammatory disease, a spleen-associated inflammatory condition, and / or an inflammatory condition in smooth muscle tissue. For example, the kidney inflammatory disease comprises acute kidney injury (AKI), chronic kidney disease (CKD), systemic lupus erythematosus-associated nephritis, IgA nephropathy, interstitial nephritis, and / or diabetic nephropathy. For example, the liver inflammatory disease comprises non-alcoholic steatohepatitis, drug-induced liver injury, viral hepatitis, alcoholic liver disease, liver fibrosis, and / or liver cirrhosis. For example, the lung inflammatory disease comprises acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bacterial or viral infection-associated pneumonia, pneumoconiosis, and / or pulmonary fibrosis. For example, the heart inflammatory disease comprises doxorubicin-induced cardiotoxicity, myocardial ischemia-reperfusion injury, myocarditis, and / or chronic heart failure. For example, the spleen-associated inflammatory condition comprises systemic lupus erythematosus (SLE)-associated spleen condition, autoimmune disease-companion spleen immune activation, and / or infection-induced spleen immune activation. For example, the inflammatory condition in smooth muscle tissue comprises an inflammatory response in vascular smooth muscle cells, chronic inflammatory conditions in airway smooth muscle, and / or gastrointestinal smooth muscle. For example, the inflammatory condition in smooth muscle tissue comprises atherosclerosis and / or arterial injury.

[0386] The modified mitochondria described herein, the fusion proteins described herein, the nucleic acid molecules described herein, the vectors described herein, the modified cells described herein, and / or the pharmaceutical compositions described herein can be administered in a therapeutically effective amount. For example, a therapeutically effective amount can include an amount effective to prevent or ameliorate one or more symptoms of a disease or condition or the progression of the disease or condition when administered to a subject. For example, a therapeutically effective amount can include an amount of a binding compound sufficient to result in an improvement in symptoms, e.g., an amount that treats, cures, prevents or ameliorates the relevant medical condition or that enhances the therapeutic, healing, prophylactic or ameliorative effect(s) of another treatment. For example, when referring to an individual active ingredient administered alone, a therapeutically effective dose refers only to that ingredient. For example, when administered in combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, sequentially or simultaneously. The therapeutically effective amount can vary depending on the type of disorder, the route of administration, the duration of treatment, the size of the area to be treated, and / or the location of the treatment site, among other factors.

[0387] In the present application, the delaying of aging includes delaying the time at which an aging state occurs (e.g., relative to a normal aging progression), mitigating and / or reducing an aging state, preventing an aging state, and / or reversing an aging state. For example, the delaying of aging includes ameliorating one or more aging phenotypes.

[0388] In the present application, the prevention and / or treatment includes not only preventing and / or treating a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing the onset of one or more symptoms associated with a disease, reducing and / or alleviating one or more symptoms associated with a disease, reducing the severity and / or duration of a disease and / or any symptoms associated therewith, and / or preventing further increases in the severity of a disease and / or any symptoms associated therewith, preventing, reducing, or reversing any physiological damage caused by a disease, and generally any pharmacological effect that is beneficial to the patient being treated. For example, the modified mitochondria described herein, the fusion proteins described herein, the nucleic acid molecules described herein, the vectors described herein, the modified cells described herein, and / or the pharmaceutical compositions described herein need not achieve a complete cure or eradicate any symptom or manifestation of a disease. As recognized in the relevant art, a drug used as a therapeutic can reduce the severity of a given disease state, but need not eliminate every manifestation of the disease to be considered a useful therapeutic. Similarly, a prophylactically administered therapeutic constitutes a useful prophylactic need not be completely effective in preventing the onset of a disorder. It is sufficient to simply reduce the impact of a disease in a subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or to reduce the likelihood of the disease occurring or worsening.

[0389] In the present application, the subject can include a mammal. For example, the subject can include a human or a mouse.

[0390] In the present application, the subject can be in an old age stage. In the present application, the subject can include a subject suffering from a disease associated with mitochondrial DNA leakage. In the present application, the subject can include a subject suffering from an ischemic disease. In the present application, the subject can include a subject receiving an effect of a side effect of an anti-tumor treatment. In the present application, the subject can include a subject suffering from a cardiovascular disease. In the present application, the subject can include a subject having ischemia-reperfusion injury and / or hypoxic injury.

[0391] Without wishing to be bound by any theory, the following examples are merely intended to illustrate the various technical solutions of the present application, and are not used to limit the scope of the present application.

[0392] Examples

[0393] The present application is further illustrated by the following specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, for which specific conditions are not specified, are carried out according to the conventional conditions in the art, for example, the conditions described in Sambrook and Rusself et al., Molecular Cloning: A Laboratory Manual (3rd edition) (2001), CSHL Press, or the conditions suggested by the manufacturer. Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available.

[0394] Table 1 Experimental materials and reagents used in the examples

[0395] Example 1 Design and construction of lentiviral plasmid overexpressing mtDNase I

[0396] 1.1 Construction of lentiviral overexpression plasmid vector

[0397] Design upstream and downstream specific amplification primers, introduce restriction enzyme sites by PCR, synthesize DNase I gene CDS region without leader peptide sequence (DNA sequence shown in SEQ ID NO: 4), and introduce mitochondrial localization sequence (DNA sequence shown in SEQ ID NO: 2), and load into lentiviral overexpression plasmid vector (as shown in FIG. 1A).

[0398] 1.2 Packaging and concentration purification of lentivirus

[0399] The three-plasmid virus packaging system was used, and the composition was pspax2, pMD2G, pLVX-IRES-ZsGreenl. The ZsGreenl expression frame on the plasmid can express green fluorescent protein (GFP), and the three plasmids were extracted by high-purity endotoxin-free extraction, co-transfected into 293T cells, replaced with complete culture medium 6h after transfection, and the cell supernatant rich in lentivirus particles was collected 24 and 48h after culture. The virus supernatant was concentrated using Lenti-X Concentrator concentration reagent. The slow virus Lenti-mtDNase I overexpressing mtDNase I (as shown in FIG. IB) was successfully packaged, and the mitochondrial targeting sequence was introduced before the DNase I CDS region. The mitochondrial localization DNase I (mtDNase I) was expressed.

[0400] Example 2 DNase I co-staining with TOM20 in cells

[0401] This example shows that DNase I is successfully localized to mitochondria.

[0402] This example studies whether DNase I is successfully expressed on mitochondria after the slow virus Lenti-mtDNase I in Example 1 infects human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). DNase I is successfully expressed on mitochondria by using immunofluorescence to label mitochondrial marker TOM20 and DNase I expression localization in cells. The experimental steps are as follows:

[0403] 1. After treatment, the cells were washed twice with pre-cooled PBS, and then 4% paraformaldehyde was added for room temperature fixation for 10 min.

[0404] 2. Remove the paraformaldehyde and wash with pre-cooled PBS for 3 times, 5 min each time.

[0405] 3. Add PBS containing 0.3% Triton X-100 and 20% FBS (Blocking buffer) and block at room temperature for 60 min.

[0406] 3. Dilute DNase I and TOM20 antibodies in Blocking buffer solution at a ratio of 1:300 as primary antibodies. After removing the blocking solution in step 3, add the primary antibodies and incubate overnight at 4°C.

[0407] 4. Remove the primary antibodies and add Blocking buffer, wash 3 times, 5 min each time.

[0408] 5. Dilute the fluorescent secondary antibody in Blocking buffer solution at a ratio of 1:400, add the secondary antibody, and incubate at room temperature in the dark for 1 hour.

[0409] 6. Remove the secondary antibody, add blocking buffer, and wash 3 times, 5 minutes each time.

[0410] 7. Add 100 ng / mL DAPI solution and incubate at room temperature in the dark for 10 min.

[0411] 8. Remove DAPI, add PBS, wash 3 times, 5 min each time, add anti-fluorescence quenching solution, place at 4℃ in the dark or take pictures directly under a laser confocal fluorescence microscope.

[0412] The experimental results are shown in Figure 2. Compared with cells not infected with Lenti-mtDNase I, cells infected with Lenti-mtDNase I showed more DNase I and co-localized with the mitochondrial marker TOM20, indicating that DNase I was successfully expressed on mitochondria.

[0413] Example 3: qPCR detection of mitochondrial DNA in cytoplasm

[0414] This embodiment illustrates that localizing DNase I in mitochondria can reduce mitochondrial DNA leakage.

[0415] This example investigated whether mtDNase I could reduce mitochondrial DNA in the cytoplasm after hypoxia-reperfusion or doxorubicin treatment. The mitochondrial DNA content in the cytoplasm of human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) infected with the lentivirus Lenti-mtDNase I obtained in Example 1 was detected using mitochondrial DNA-specific qPCR. The experimental steps are as follows:

[0416] 1. The cells were divided into two equal volumes. Total mtDNA control: Resuspended in 500 μL of 50 mM NaOH, boiled for 30 min to dissolve the DNA, and then neutralized with 50 μL of 1 M Tris-HCl (pH 8). Cytoplasmic fraction: Resuspended in 500 μL of buffer consisting of 150 mM NaCl, 50 mM HEPES (pH 7.4), and 25 mg / ml digitalis glycoside. After mixing, the mixture was centrifuged at 1000g for 10 min to form spheroids of intact cells. The supernatant containing cytoplasm was then transferred to a new tube.

[0417] 2. Centrifuge at 17000g for 10 minutes to remove remaining cell debris, and perform quantitative PCR on total DNA and cytoplasmic DNA using mitochondrial DNA-specific primers.

[0418] Table 2 Quantitative PCR primer sequences

[0419] The experimental results are shown in Figure 3. After hypoxia reperfusion or doxorubicin treatment, the expression of mitochondrial DNA in the cytoplasm increased, while in the cells treated with mtDNase I, the increase in the expression of mitochondrial DNA caused by hypoxia reperfusion or doxorubicin treatment was lower, indicating that after overexpression of mtDNase I, the leakage of mitochondrial DNA was reduced.

[0420] Example 4 Western blot detection of cGAS-STING pathway in cells

[0421] This example shows that positioning DNase I in mitochondria can reduce the level of downstream pathways activated by mitochondrial DNA, such as the cGAS-STING pathway.

[0422] This example studies whether infection with the lentivirus Lenti-mtDNase I constructed in Example 1 can prevent the activation of the downstream cGAS-STING pathway triggered by mitochondrial DNA leakage in hiPSC-CMs. Western blot is used to detect cGAS-STING pathway proteins in cells, including cGAS / STING / phosphorylated TBK1 / phosphorylated IRF3 expression. The experimental steps are as follows:

[0423] 1. Protein sample acquisition: Add RIPA to lyse the cells, incubate on ice for 10 minutes, then centrifuge at 13,000g at 4°C for 15 minutes, and take the supernatant as the sample.

[0424] 2. Electrophoresis: Prepare an electrophoresis gel and perform SDS-PAGE.

[0425] 3. Transfer:

[0426] 3.1 After electrophoresis, cut the gel strip to the appropriate size, and equilibrate with transfer buffer for 5 minutes x 3 times.

[0427] 3.2 Membrane treatment: Cut filter paper and PVDF membrane of the same size as the gel strip in advance, and immerse in transfer buffer after methanol activation for 15 seconds.

[0428] 3.3 Transfer: Place the anode carbon plate, filter paper, PVDF membrane, gel, filter paper, and cathode carbon plate in the transfer device from bottom to top in the order of precise alignment, and remove air bubbles at each step. Connect the power supply, set the constant current to 300mA, and transfer for 1.5 hours. After the transfer is complete, disconnect the power supply and remove the membrane, and cut the membrane strip to be tested for immunoblotting.

[0429] 4. Immunoblotting:

[0430] 4.1 Put the film strip to be tested into 2.5% milk for blocking for 1 hour, remove the blocking solution, add diluted primary antibody in milk, and incubate at 4°C overnight.

[0431] 4.2 After washing with TBST for 15 minutes for 3 times, add secondary antibody, and incubate at room temperature for 1 hour.

[0432] 4.3 After washing with TBST for 15 minutes for 3 times, add developing solution for development.

[0433] The experimental results are shown in FIG. 4. Compared with the control group, the expression amount of cGAS-STING pathway protein in the hiPSC-CMs after hypoxia-reperfusion treatment increased, and the increase in protein expression caused by hypoxia-reperfusion treatment in the cells after the addition of mtDNase I decreased, indicating that overexpression of mtDNase I can prevent the activation of the downstream cGAS-STING pathway triggered by mitochondrial DNA leakage.

[0434] Example 5 Live cell staining of hiPSC-CMs overexpressing mtDNase I

[0435] This example shows that positioning DNase I in mitochondria can reduce the inflammatory response triggered by mitochondrial DNA leakage and reduce damage.

[0436] This example studies the effect of the slow virus Lenti-mtDNase I constructed in Example 1 on mitochondrial superoxide and mitochondrial membrane potential in cells after injury after infecting human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). MitoSOX is used to detect superoxide in mitochondria, and TMRM is used to detect mitochondrial membrane potential. After labeling the cells, take pictures for statistics. The experimental steps are as follows:

[0437] 1. After infecting HiPSC-CMs with a carrier or Lenti-mtDNase I, perform normoxic or hypoxia-reperfusion treatment.

[0438] 2. Use the cell-permeable fluorescent probe MitoSOX Red to measure the production of mitochondrial ROS in hiPSC-CMs. Add 5 μM MitoSOX Red to the hiPSC-CM culture solution, incubate at 37°C for 30 minutes, and then wash with PBS. For mitochondrial membrane potential measurement, use the Image-iT TM TMRM kit, add 10 μM TMRM to the hiPSC-CM culture solution, incubate at 37°C for 30 minutes, and then wash with PBS.

[0439] 3. Hoechst 33342 was used to stain the nucleus, and after 5 minutes, PBS was used for washing. Fresh culture solution was added, and the Operetta CLS high content imaging system from Perkin Elmer was used to capture and quantify the fluorescence intensity.

[0440] The experimental results are shown in Figure 5. Compared with the normal cells in the control group, the mitochondrial superoxide of the hiPSC-CMs increased and the membrane potential decreased after the hypoxia-reperfusion treatment (Figure 5A and Figure 5B), while the mitochondrial superoxide of the cells overexpressing mtDNase I was less and the membrane potential was high after the hypoxia-reperfusion treatment (Figure 5A and Figure 5B), which indicated that mtDNase I could reduce the inflammatory response caused by mitochondrial DNA leakage to alleviate the damage.

[0441] Example 6: Detection of the senescence phenotype of hiPSC-CMs overexpressing mtDNase I

[0442] This example shows that locating DNase I in mitochondria can delay the aging of cells.

[0443] This example studies the effect of the lentivirus Lenti-mtDNase I constructed in Example 1 on the aging of cells after infecting human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The cell aging marker p21 is labeled by immunofluorescence, and the cardiomyocyte marker cTnT is used to label the cardiomyocytes. The experimental steps are as follows:

[0444] 1. After treatment, the cells were gently washed twice with pre-cooled PBS, and then 4% paraformaldehyde was added for fixation at room temperature for 10 min.

[0445] 2. The paraformaldehyde was removed, and the cells were washed with pre-cooled PBS for 3 times, each for 5 min.

[0446] 3. Blocking buffer containing 0.3% Triton X-100 and 20% FBS was added, and the cells were blocked at room temperature for 60 min.

[0447] 3. The p21 and cTnT antibodies were diluted in the Blocking buffer solution at a ratio of 1:300 and 1:500, respectively, as the primary antibody. After removing the blocking solution in step 3, the primary antibody was added, and the cells were incubated at 4°C overnight.

[0448] 4. The primary antibody was removed, and Blocking buffer was added, and the cells were washed for 3 times, each for 5 min.

[0449] 5. The secondary antibody was diluted in the Blocking buffer solution at a ratio of 1:400, and the secondary antibody was added, and the cells were incubated at room temperature for 1 h in the dark.

[0450] 6. Remove secondary antibody, add Blocking buffer, wash 3 times, 5 min each.

[0451] 7. Add 100 ng / mL DAPI solution, incubate at room temperature for 10 min in the dark.

[0452] 8. Remove DAPI, add PBS, wash 3 times, 5 min each, add anti-fluorescence quenching solution, place at 4°C in the dark or directly take pictures under laser confocal fluorescence microscope.

[0453] The experimental results are shown in Figure 6. Compared with cells not infected with Lenti-mtDNase I, cells infected with Lenti-mtDNase I have a smaller proportion of p21 positive cells after hypoxia reperfusion or doxorubicin treatment, indicating that mtDNase I can reduce aging caused by hypoxia reperfusion or doxorubicin damage.

[0454] Example 7 Mouse ischemia reperfusion surgery, doxorubicin heart damage and echocardiography detection

[0455] This example shows that positioning DNase I in mitochondria can protect the heart and reduce damage to heart function.

[0456] This example studies whether the use of mtDNase1 in mice can reduce the level of heart function in a chronic heart failure model caused by ischemia reperfusion or doxorubicin. The left ventricular ejection fraction and left ventricular fractional shortening of the mice were detected by echocardiography to verify the heart failure index of the mice. The experimental steps are as follows:

[0457] 1. Mouse ischemia reperfusion surgery

[0458] 1.1 Experimental animals: Wild-type C57BL / 6J mice were purchased from China Jiangsu Jucu Yakang Biotechnology Co., Ltd. All animals were raised in the SPF animal room of the Ninth People's Hospital of Shanghai Jiaotong University School of Medicine, with room temperature set at 25°C, 12-hour regular cycle of day and night, conventional feed, and free access to water and food. (Ethical statement: The feeding, experimental operation, and post-experimental disposal of all experimental animals in this experiment comply with the 1996 version of the Guide for the Care and Use of Laboratory Animals in the United States, and all experimental protocols comply with the requirements of the Animal Ethics Committee of Shanghai Jiaotong University School of Medicine and the Ninth People's Hospital of Shanghai Jiaotong University School of Medicine.)

[0459] 1.2 After the mouse is anesthetized, the mouse is fixed on the operating table in a supine position, the mouse's chest hair is shaved and disinfected to ensure the sterility of the surgical area. Make a 1 cm long incision along the edge of the pectoralis major muscle between the 3rd and 4th intercostal spaces on the left side of the sternum, bluntly separate the subcutaneous tissue, pectoralis major muscle and anterior serratus muscle to expose the heart.

[0460] 1.3 Myocardial ischemia: The heart was squeezed quickly through the 3rd, 4th intercostal space with forceps. The left anterior descending coronary artery (LAD) was ligated with 7-0 silk to cause myocardial ischemia.

[0461] 1.4 Reperfusion: The ligature was tied into a slipknot, and after 40 minutes of ischemia, the ligature was slowly pulled out to restore blood supply to the myocardium, thus achieving reperfusion.

[0462] 1.5 Suture the wound: After the myocardial ischemia and reperfusion operation was completed, the surgical incision was carefully sutured.

[0463] 2, Doxorubicin-induced heart injury model

[0464] 2.1 Experimental animals: Wild-type C57BL / 6J mice were purchased from Jiangsu Jucu Yakang Biotechnology Co., Ltd. in China. All animals were housed in the SPF animal room of the Central Laboratory of the Ninth People's Hospital of Shanghai Jiaotong University School of Medicine, with room temperature set at 25°C, 12-hour regular cycle of day and night, regular feed, and free access to water. (Ethical statement: The feeding, experimental operation, and post-experimental disposal of all experimental animals in this experiment comply with the 1996 version of the Guide for the Care and Use of Laboratory Animals in the United States, and all experimental protocols comply with the requirements of the Animal Ethics Committee of the School of Medicine of Shanghai Jiaotong University and the Ninth People's Hospital of Shanghai Jiaotong University.)

[0465] 2.2 Doxorubicin group: Mice were injected intraperitoneally with 2.5 mg / mL doxorubicin solution, with each injection amount being 0.5 mg / kg, once a week for a total of 4 times, to establish a doxorubicin-induced chronic heart failure model. The control group was injected with the same dose of normal saline, and the body weight change of the mice was recorded during the modeling period.

[0466] 2.3 After the last doxorubicin injection, the mice were continued to be fed for two weeks, and the drug was completely absorbed to exert its effect.

[0467] 3, Echocardiography to detect changes in mouse cardiac contractility

[0468] 3.1 The fur on the precordial region of the mouse was removed with depilatory cream, and the mouse was placed in an anesthesia box containing 2% isoflurane for 3 minutes of induction anesthesia.

[0469] 3.2 The anesthetized mouse was fixed supine on the operating table surface of a 37°C constant temperature heating plate, connected to an electrocardiograph to keep the heart rhythm stable, maintained anesthesia with an isoflurane mask, and applied an appropriate amount of ultrasonic gel.

[0470] 3.3 When the heart rate of the mouse is maintained at 350-450 bp / s, the ultrasound probe is cut off towards the head of the mouse, and is rotated counterclockwise by 30-45 degrees. M-mode cardiac ultrasound images are taken at the level of the mitral valve chord papillary muscle, and the heart rate of the mouse is measured and recorded, and the left ventricular fractional shortening (FS) and left ventricular ejection fraction (EF) are calculated.

[0471] 4. Construction of AAV9-mtDNase I and injection scheme of mice

[0472] 4.1 Construction of pAAV9-mtDNase I plasmid: The DNA sequence of mtDNase I is synthesized and purified, and is connected to the pAAV-GOI plasmid. After transformation, the pAAV9-mtDNase I plasmid is extracted, and the sequence is verified to be complete and free of mutations.

[0473] 4.2 Construction of AAV9-mtDNase I virus

[0474] 1) Cell preparation

[0475] The cryopreserved 293T cells are taken out of the liquid nitrogen, quickly placed in a 37°C water bath for recovery, and after centrifugation, fresh culture medium is added to the cells, which are cultured at 37°C and 5% CO2, and the cells are passaged every 2-3 days. After the cells grow normally, they are transferred to a 10cm culture dish for adherent culture.

[0476] 2) Plasmid transfection

[0477] When the cell density reaches about 80-90% confluence, transfection can be performed. The pAAV9-mtDNase I, pAAV-RC and helper plasmid (pHelper) are configured according to a certain ratio according to the three-plasmid transfection system, and transfection and culture are performed according to 1000 μL system per dish.

[0478] 3) Cell culture

[0479] After one day of culture, the transfection of cells is observed under a microscope. Generally, the transfection efficiency should be above 80%, and a low transfection rate will lead to a decrease in virus yield.

[0480] 4) Virus collection and impurity removal

[0481] After 72 hours of culture, the virus supernatant is collected. The virus culture supernatant is centrifuged at low temperature to remove cell debris, and an appropriate amount of nuclease is added to remove free nucleic acids at 37°C, and then PEG8000 / NaCl solution is added for overnight precipitation.

[0482] 5) Virus purification and concentration

[0483] The precipitated virus was resuspended with PBS and subjected to density gradient centrifugation in an ultracentrifuge. After centrifugation, the solution corresponding to the virus layer was extracted, and dialysis bags were used for further dialysis at 4°C overnight. The next day, the dialysate was filtered through a 0.22 um filter and concentrated through a concentration tube to achieve the purpose of impurity removal and concentration.

[0484] 4.3 AAV9-mtDNase I virus titer detection: The DNA copy number of rAAV virions, i.e., the number of rAAV VG (Viral genomes), was detected, which was converted into the content of rAAV per unit volume, i.e., the titer of rAAV vg / mL (Viral genomes / mL). QC standard: titer ≥ 1.0E+12 vg / mL

[0485] 4.4 AAV9-mtDNase I virus mouse administration scheme: The obtained virus titer was about 5E+12 vg / mL, and 4E+12 VG was administered by tail vein injection before modeling. After administration, the mice were raised separately, and the growth state and feeding state of the mice were observed every other day. MI and doxorubicin modeling were performed one week after administration.

[0486] The experimental results are shown in Figure 7: After myocardial ischemia-reperfusion and doxorubicin injury, the left ventricular ejection fraction (Figure 7A) and the fractional shortening (Figure 7B) of the heart decreased, and the ejection fraction and fractional shortening of the heart infected with AAV9-mtDNase I increased compared with the control group, indicating that AAV9-mtDNase I can protect the heart and reduce the damage to the heart function after ischemia-reperfusion or doxorubicin action.

[0487] Example 8 HE and MASSON staining of mouse myocardial tissue sections to observe the morphological changes of myocardial tissue

[0488] This example shows that locating DNase I in mitochondria can reduce the fibrosis of myocardial tissue.

[0489] This example studies whether the use of mtDNase I overexpression mice, MASSON staining of heart sections of chronic heart failure models caused by ischemia-reperfusion or doxorubicin, detects the level of cardiac fibrosis in mice. The experimental steps are as follows:

[0490] 1. Paraffin section preparation:

[0491] 1) Tissue dehydration: Place the heart tissue fixed with 4% paraformaldehyde into the embedding box, wash under running water for 10 minutes, then place the embedding box in 75% ethanol-80% ethanol-95% ethanol I-95% ethanol II-anhydrous ethanol I-anhydrous ethanol II, respectively, dehydrate at room temperature, soak for 1 hour each time.

[0492] 2) Tissue transparency: Put the dehydrated tissue into anhydrous ethanol: xylene = 1 : 1 - xylene I - xylene II in turn, and gradually transparent at room temperature, each grade soaking for 20 minutes.

[0493] 3) Tissue wax immersion: Put the transparent tissue into xylene: paraffin = 1 : 1 - paraffin I - paraffin II - paraffin III in turn, and gradually immersed at 58°C, each grade soaking for 30 minutes.

[0494]

[0495] 4) Tissue embedding: First pour a little embedding paraffin into the embedding box, then quickly move the tissue block into the embedding box with forceps, pour paraffin, and take out after complete solidification.

[0496] 5) Sectioning and mounting: Put the wax block on the microtome, adjust the microtome so that the wax block cutting surface is parallel to the cutting knife edge, then trim the wax block to the desired part, and then cut into continuous wax strips, divide the wax strips, and separate the wax slices on 42°C warm water to make the wax slices unfold. Take the unfolded wax slices with a glass slide, adjust the position of the wax slices to the center of the glass slide, and dry naturally.

[0497] 2, HE staining and MASSON staining:

[0498] 1) De-waxing: Take the prepared myocardial tissue paraffin sections, 5 pieces per group, and put them into a 60°C oven for 20 minutes, then put them into xylene I - xylene II in turn, and gradually de-wax at room temperature, each grade soaking for 5 minutes.

[0499] 2) Hydration: Put the de-waxed tissue sections into anhydrous ethanol - 95% ethanol - 80% ethanol in turn, and gradually hydrate at room temperature, each grade soaking for 5 minutes. Then wash with PBS solution for 2 times, each time for 3 minutes.

[0500] 3) MASSON staining: Put the tissue sections into hematoxylin solution for 10 minutes, take them out and rinse under running water for 10 minutes, then differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse under running water for a few minutes, then blue in MASSON blue solution for 3-5 minutes, rinse under running water for a few minutes, then immerse in Lichun red dye for 5-10 minutes. After washing with prepared weak acid working solution (ddH2O: weak acid solution = 1 : 1) for 1 minute, wash with 1% phosphomolybdic acid for 1-2 minutes, and wash with weak acid working solution for 1 minute. Without water washing, directly put into aniline blue staining solution for 1-2 minutes, and wash with weak acid working solution for 1 minute.

[0501] 4) Dehydration and mounting: Put the stained sections into 70% ethanol - 80% ethanol - 95% ethanol - anhydrous ethanol - xylene I - xylene II in turn, and gradually dehydrate at room temperature, each grade soaking for 5 minutes, then dry in a fume hood and mount with neutral resin.

[0502] ​3. Observation: Observed under upright light microscope and stored photos, analyzed the degree of myocardial fibrosis with Image J.

[0503] Results are shown in Figure 8. MASSON staining results showed that ischemia-reperfusion or doxorubicin induced chronic heart failure in mice resulted in severe fibrosis in the heart, and mtDNase I treatment could alleviate the cardiac fibrosis phenotype caused by ischemia-reperfusion or doxorubicin.

[0504] Example 9 Transmission electron microscopy detection of mitochondrial state in myocardial tissue of mice

[0505] This example shows that positioning DNase I in mitochondria can alleviate mitochondrial damage and myocardial damage.

[0506] This example investigates whether the use of mtDNase I overexpressing mice can alleviate the morphology of cardiac mitochondria and sarcomeres in a chronic heart failure model induced by ischemia-reperfusion or doxorubicin. The experimental steps are as follows:

[0507] 1. Sample fixation:

[0508] 1) Cut the myocardial tissue into 1 mm3volume and place it in 2.5% glutaraldehyde fixing solution at 4°C for 2 hours.

[0509] 2) Discard the fixing solution and wash with 0.1M PB buffer for 3 times, 3 minutes each time.

[0510] 3) Fix with 1% osmium acid fixing solution at 4°C for 2 hours.

[0511] 4) Discard the osmium acid and wash with 0.1M PB buffer for 3 times, 3 minutes each time.

[0512] 2. Gradient dehydration: Place the sample in 30% ethanol-50% ethanol-70% ethanol-85% ethanol-95% ethanol-anhydrous ethanol I-anhydrous ethanol II successively at room temperature, dehydrate step by step, soak for 15 minutes each time, and then place in alcohol-acetone 1:1 mixture-acetone I-acetone II successively, dehydrate for 20 minutes each time.

[0513] 3. Infiltration: Place the sample in different proportions of acetone resin mixture (3:1-1:1-1:3) successively, infiltrate for 1 hour each time. Then place in pure resin for infiltration overnight.

[0514] 4. Embedding: Transfer the infiltrated sample to the embedding plate and polymerize in the oven at 60°C for 48 hours.

[0515] 5. Sectioning: Perform ultrathin sectioning of the sample with an ultramicrotome.

[0516] 6. Staining: Stain with lead citrate for 10 minutes, and then wash with ddH2O without carbon dioxide for 3 times.

[0517] 7. Observation: After the ultra-thin sections are dried, the sections are placed into a HITACHI H-7650 transmission electron microscope to observe the ultrastructure of the myocardial cells.

[0518] The experimental results are shown in FIG. 9. Electron microscopy results show that the mitochondria of the ischemia-reperfusion mice are enlarged and the mitochondrial cristae are damaged. The mitochondria of the adriamycin-induced chronic heart failure mice are vacuolated and the sarcomere is broken. The mtDNase 1 treatment can alleviate the damage to the mitochondria and sarcomere caused by ischemia-reperfusion or adriamycin.

[0519] Example 10 Isolated perfusion of mouse hearts and isolation and purification of adult mouse myocardial cells

[0520] This example shows that positioning the DNase I in the mitochondria can improve myocardial function.

[0521] This example investigates whether the use of mtDNase I overexpressing mice can improve the systolic and diastolic function of the Langendorff purified myocardial cells of an adriamycin-induced chronic heart failure model. The experimental steps are as follows:

[0522] 1. Reagent preparation

[0523] 1) CIB (cardiac perfusion buffer): Weigh the components in the following table, dissolve in 900 mL ultrapure water, adjust the pH to 7.4, then make up to 1 L, filter with a 0.22 μm filter, and store at 4°C.

[0524] Table 3: CIB (cardiac perfusion buffer)

[0525] 2) MEM Base Solution: Weigh all the components in the following table, dissolve in 900 mL ultrapure water, adjust the pH to 7.4, then make up to 1 L, filter with a 0.22 μm filter, and store at 4°C.

[0526] Table 4: MEM Base Solution

[0527] 3) BSA solution: The formula is shown in the following table. After complete dissolution, place in a 37°C water bath for use.

[0528] Table 5: BSA solution

[0529] 4) Digestive enzyme mixture: Weigh all the components in the following table, dissolve in CIB, and place in a 37°C water bath for use.

[0530] Table 6: Digestive enzyme mixture

[0531] 2. Mouse heart perfusion ex vivo:

[0532] 1) The experimental mouse was injected intraperitoneally with heparin (1:500 U) 0.3 mL, and placed for 15 minutes to be heparinized in the whole body.

[0533] 2) CIB was perfused into the Langendroff perfusion system, so that the whole system was filled with CIB, and then 30 mL of digestive enzyme was perfused into the Langendroff perfusion system.

[0534] 3) The mouse was sacrificed by cervical dislocation method, and the heart was quickly removed. The aortic root of the heart was suspended to the perfusion needle head, and the perfusion needle was installed to the perfusion system.

[0535] 4) Open the Langendroff perfusion system, set the flow rate to 3 mL / min for perfusion, discard the first 3 minutes of perfusion liquid, and collect the subsequent perfusion liquid into a 50 mL centrifuge tube.

[0536] 3. Myocardial tissue digestion:

[0537] 1) The perfused heart was removed, at this time the heart had become soft and loose, and was cut into pieces and placed in the collected perfusion liquid.

[0538] 2) The centrifuge tube was placed in a 37°C shaking bed at a speed of 100 rpm for 5 minutes.

[0539] 3) The digestive solution was gently blown and the myocardial tissue was completely dispersed, and then filtered using a 100 μm filter.

[0540] 4) The digestive enzyme was neutralized using BSA Solution and centrifuged at 500 rpm for 5 minutes.

[0541] 5) Discard the supernatant, and the collected cells were frozen in liquid nitrogen or resuspended for subsequent experiments.

[0542] 4. Inopotix myocardial cell contraction and ion concentration synchronous measurement system detects myocardial cell contraction and diastolic function

[0543] 1) The myocardial cells after perfusion and digestion were resuspended using 10 mL of BSA Solution.

[0544] 2) Gradient calcium was performed using 10 mL of liquid system, and 1.25 μL-8.75 μL-30 μL-50 μL of 100 mm CaCl2 solution was added, with an interval of 2 minutes each time.

[0545] 3) Centrifuged at 500 rpm for 5 minutes, removed the supernatant, and added DMEM complete medium (DMEM + 10% FBS) for resuspension.

[0546] 4) The cell suspension was seeded in the middle of the laminin pre-coated confocal dishes at 200 μl per dish.

[0547] 5) The cells were incubated in a 37 °C cell incubator for 30 minutes.

[0548] 6) The supernatant was aspirated, 1.5 mL DMEM complete medium was added, and the Ionoptix MyoCam system was used to measure the contraction and relaxation function of the cardiomyocytes.

[0549] The results are shown in Figure 10. The Ionoptix results show that the doxorubicin-induced cardiomyocyte contractile dysfunction is significantly improved by mtDNase I treatment.

[0550] Example 11. Targeting DNase I to mitochondria can be used to treat a variety of diseases

[0551] This example investigates the therapeutic effect of targeting DNase I to mitochondria for different disease models. This example shows that targeting DNase I to mitochondria, either by overexpressing DNase I containing mitochondrial localization sequences or by administering mitochondria with DNase I, has a certain therapeutic or alleviating effect on a variety of diseases, can delay aging, for example, has a therapeutic effect on cardiovascular diseases, ischemic diseases, hypoxia-reperfusion injury, hypoxic injury.

[0552] Targeting DNase I to mitochondria in different disease cell models, or introducing mitochondria expressing DNase I on the outer membrane into different disease cell models (cardiovascular disease-oxLDL+HUVEC cells, ischemia-related disease-Ischemia+HUVEC cells, ischemia-reperfusion injury-I / R+HUVEC cells, hypoxic injury-hypoxia+HUVEC cells), and observing the effect of treatment / symptom / indicator alleviation. Using a doxorubicin-injected mouse model (causing systemic aging), evaluating the degree of aging and inflammation in each organ by expressing DNase I targeted to mitochondria, and injecting mitochondria with DNase I.

[0553] Example 12. Therapeutic advantages of targeting DNase I to mitochondria

[0554] This example investigates the comparison of the therapeutic effect of overexpressing DNase I localized in mitochondria versus overexpressing DNase I localized in cytoplasm. The results show that overexpressing DNase I localized in mitochondria has better therapeutic effect than overexpressing DNase I localized in cytoplasm.

[0555] This example investigates the comparison of the therapeutic effect of mitochondria with DNase I versus mitochondria without DNase I. The results show that mitochondria with DNase I has better therapeutic effect than mitochondria without DNase I.

[0556] Example 13 Localizing nuclease to mitochondria reduces mtDNA leakage and its activated downstream pathways

[0557] This example shows that localizing nuclease to mitochondria, such as DNase I variants, can also reduce mtDNA leakage and the level of downstream pathways activated by mtDNA leakage.

[0558] The level of mtDNA leakage / cGAS-STING pathway activation after ischemia-reperfusion and doxorubicin stimulation was evaluated by fluorescence co-localization staining.

[0559] Example 14 Localizing nuclease to mitochondria using different mitochondrial localization methods

[0560] This example investigates that using mitochondrial localization sequences other than COX8A mitochondrial localization sequence can also successfully localize nuclease to mitochondria, and can reduce mtDNA leakage and the level of downstream pathways activated by mtDNA leakage.

[0561] This example investigates that placing mitochondrial localization sequence at the C-terminus of nuclease can also successfully localize nuclease to mitochondria, and can reduce mtDNA leakage and the level of downstream pathways activated by mtDNA leakage.

[0562] The level of mtDNA leakage / cGAS-STING pathway activation after ischemia-reperfusion and doxorubicin stimulation was evaluated by fluorescence co-localization staining.

[0563] Example 15 Energy level matching relationship between the cell from which mitochondria is generated and the target cell

[0564] This example investigates the energy level matching relationship between the cell from which mitochondria is generated and the target cell.

[0565] Four different cell sources of mitochondria were used: human colon cancer cell line (RKO, R-mito), mouse embryonic fibroblasts (NIH 3T3, F-mito), mouse skeletal muscle (C2C12, M-mito), and mouse neonatal cardiomyocytes (H-mito). Measurements by flow cytometry showed that the number of Mitotracker green (MTG, indicating the number of mitochondria) and mitochondrial membrane potential (TMRM) were very consistent across the four cells. By measuring the number of mitochondria (MTG), membrane potential (TMRM), and oxidative state (MitoSox) of isolated mitochondria, the number of mitochondria per cell was the highest, the mitochondrial membrane potential was the highest, but the mitochondrial superoxide level was average for intact cardiomyocytes compared to other cell types. However, when detected in isolated mitochondria, the differences in mitochondrial membrane potential and oxidative state both disappeared. Next, by examining the contraction and mitochondrial respiratory function of these cardiomyocytes, we found that metabolically matched mitochondria (H-mito) were necessary to improve contractile function. Under DOX stimulation, mitochondrial pretreatment restored basal mitochondrial respiration, but only H-mito treatment restored maximal mitochondrial respiration. These data suggest that mitochondrial transplantation protects the heart by restoring mitochondrial abundance and metabolism in a DOX-induced heart failure model.

[0566] This example demonstrates that, for different cell types, the production of mitochondria from cells with energy levels matched to the target cells can better reduce mtDNA leakage and / or the level of downstream pathways activated by mtDNA.

[0567] Example 16 Effect of localizing DNase I to mitochondria on different cell types

[0568] This example demonstrates that localizing DNase I to mitochondria can reduce mitochondrial DNA leakage and the level of downstream pathways activated by mitochondrial DNA leakage for a variety of cell types.

[0569] This example demonstrates that localizing DNase I to mitochondria can alleviate mtDNA leakage or downstream pathways activated by mtDNA for a variety of cell types induced by doxorubicin, by expressing mitochondria-localized DNase I in HUVEC vascular endothelial cells (for vascular aging), C2C12 skeletal muscle cell lines (muscle aging), HEK293 kidney epithelial cell lines (kidney injury), and SH-SY5Y neural cell lines.

[0570] Example 17 Mitochondria-localized DNase I can be delivered in a variety of ways

[0571] This example studies different delivery methods of the tested mitochondrial localization sequence-DNase I nucleic acid molecule, including mRNA, plasmid transfection, establishing overexpression cell lines, and then directly delivering mitochondria with mtDNase I after extraction, all of which can achieve the expression of nucleic acid localization in mitochondria, and can reduce mitochondrial DNA leakage and the level of downstream pathways activated by mitochondrial DNA leakage.

[0572] Example 18 Mitochondrial localization of DNase I degrades DNA in vitro

[0573] This example demonstrates that DNase I is localized in mitochondria and still retains the activity of degrading DNA after extraction of mitochondria. This example studies the function of mitochondrial localized DNase I (mtDNase) in degrading DNA in vitro.

[0574] The experimental steps are as follows:

[0575] 1. Mitochondrial preparation: mitochondria were extracted from cell lines expressing mtDNase I (extraction method and buffer used can refer to the published mitochondrial extraction method Frezza et al. 2007 doi: 10.1038 / nprot.2006.478); a small amount of extracted mitochondria was stained with MitoTracker Green FM Dye (M46750) and then quantified by flow cytometry.

[0576] 2. DNA preparation: this example uses conventionally cultured C2C12 (mouse muscle cell line) as a DNA sample for testing; the extraction method is a routine operation in the field, and this example uses Beijing TransGen Biotech DNA extraction kit-EasyPure Genomic DNA Kit (EE101) for DNA extraction; the extracted DNA is quantitatively detected by Thermo Scientific Nanodrop One Spectrophotometer (13-400-525).

[0577] 3. mtDNase DNA degradation efficiency test: 1 ug of C2C12 DNA was mixed with different amounts of mtDNase (4000, 40,000, 100,000); MgCl2 was added to each mixed sample to ensure DNase I in vitro function according to the instructions of DNase I, RNase-free (EN0525) kit produced by Thermo Scientific, and the DNase I provided by the kit was used as a positive control and pure DNA as a negative control for DNA degradation experiment; the prepared sample was incubated in a 37 degree Celsius oven for 30 minutes, and then Southern Blot was performed.

[0578] 4. Southern Blot or DNA gel is a commonly used molecular experimental method in the field, and the experimental conditions of this embodiment are described as follows: 1% agarose gel was prepared; DNAGel Loading Dye (6X) (Thermo Scientific R0611) was added to the incubated sample; the sample was added to the cooled and buffer-soaked agarose gel; after connecting the power supply, the running gel box was started; first run at 60V for 20 minutes, then adjust the voltage to 100V for 2 hours; move the run gel to the developer for development and take pictures.

[0579] The experimental results are shown in Figure 11, and the results show that mitochondria containing DNase I can degrade DNA in vitro.

[0580] Example 19. Localizing DNase I to mitochondria can effectively reduce the cGAS-STING pathway caused by doxorubicin

[0581] This embodiment shows that expressing DNase I localized to mitochondria in cells and transplanting DNase I with mitochondrial localization can reduce the inflammatory response caused by DNA leakage and reduce damage.

[0582] The experimental steps are as follows:

[0583] 1. Wild-type HL1 cell lines and HL1 cell lines expressing mitochondria-localized DNase I were seeded in different wells of a 96-well plate at a ratio of 2:1 (for specific distribution, see Figure 12);

[0584] 2. Each day, 100 uL of fresh culture medium (DMEM / F12 + 10% FBS + 1x Pen-Strep) was changed for each well;

[0585] 3. Two hours before the start of the experiment, mitochondria with DNase I were added to half of the wild-type HL1 cells;

[0586] 4. Replace the culture medium in the culture dish with the culture medium containing 1 uM doxorubicin at different time points. In this example, the cells were placed in doxorubicin for 0 minutes, 60 minutes, 120 minutes, 240 minutes, and 360 minutes, respectively;

[0587] 5. After 360 minutes of experiment, use room temperature PBS to clear twice, then add 4% paraformaldehyde at room temperature for 10 minutes for cell fixation;

[0588] 6. Remove the paraformaldehyde and add pre-cooled PBS for washing 3 times, each time for 5 minutes;

[0589] 7. Add PBS containing 0.3% Triton X-100 and 20% FBS (Blocking buffer) for blocking at room temperature for 60 minutes;

[0590] 8. Dilute the STING (Proteintech 19851-1-AP) and γH2AX (CST 9718S) antibodies in the Blocking buffer solution at a ratio of 1:200 and 1:500, respectively, as the primary antibody. After removing the blocking solution in step 3, add the primary antibody and incubate overnight at 4°C;

[0591] 9. Remove the primary antibody and add Blocking buffer for washing 3 times, each time for 5 minutes;

[0592] 10. Dilute the fluorescent secondary antibody in the Blocking buffer solution at a ratio of 1:400, add the secondary antibody, and incubate at room temperature for 1 hour in the dark;

[0593] 11. Remove the secondary antibody and add Blocking buffer for washing 3 times, each time for 5 minutes;

[0594] 12. Add 100 ng / mL DAPI solution, incubate at room temperature for 10 minutes in the dark;

[0595] 13. Remove DAPI, add PBS for washing 3 times, each time for 5 minutes, add a fluorescence quenching solution, and place at 4°C in the dark or directly take microphotographs on the Operetta CLS High-Content Analysis System (Revvty HH16000020);

[0596] 14. After photographing, use the fluorescence analysis software provided by Operetta to analyze the cell fluorescence intensity and cell nucleus base to obtain the experimental conclusion.

[0597] The experimental results are shown in FIGS. 13A and 13B. Transplanting the extracted mtDNase I mitochondria into wild-type cells (+mtDNase mito) and the cells themselves expressing DNase I localized to mitochondria can effectively reduce the expression of Sting and the positive rate of γH2AX caused by doxorubicin damage. Although the difference in the positive rate of γH2AX in each group in the histogram is relatively small, the main reason is that this indicator is expressed as the proportion of positive cells to the total number of cells, which is affected by the difference in the total number of cells in the sample.

[0598] Example 20 Localizing DNase I to mitochondria reduces inflammation caused by mitochondrial DNA leakage and alleviates damage in a kidney cell line

[0599] This example demonstrates that mitochondria with mtDNase I extracted from a myocardial cell line can also reduce inflammation caused by DNA leakage and alleviate damage in other organs after transplantation.

[0600] The experimental steps are as follows: the cells are seeded into a six-well plate for culture; the culture medium (MEM+10%FBS+1%Pen-Strep) is replaced daily; mitochondria with mtDNase I are transplanted into half of the cells; the culture medium is replaced 2 hours after transplantation, and one half of the cells are replaced with culture medium containing 1.5 uM cisplatin (see FIG. 14 for the specific experimental design); and the cell state is observed after 24 hours of incubation in the incubator.

[0601] The experimental results are shown in FIG. 14. The results show that the group of cells that were added with cisplatin but not transplanted with mitochondria had the most visible cell apoptosis and morphological abnormalities (lower left), and the transplantation of mitochondria with mtDNase I effectively alleviated cell damage.

[0602] Example 21 Protective effect of mitochondria with mtDNase I in an AKI model

[0603] This example demonstrates that acute kidney injury caused by cisplatin in vivo can be effectively prevented by transplanting mitochondria with DNase I. The cisplatin-induced acute kidney injury (AKI) mouse model is a commonly used animal model for studying the mechanisms of kidney damage and related treatment strategies. This model induces damage to renal tubular epithelial cells, inflammation, and decreased kidney function in a short period of time, and can stably reproduce a pathological process similar to clinical AKI, and is widely used to evaluate the effects of antioxidant, anti-inflammatory, or cell protection candidate drugs in AKI. Therefore, this example uses this model to verify the protective effect of mitochondria with mtDNase I under AKI conditions in vivo.

[0604] The experimental steps are as follows:

[0605] 1. Cisplatin preparation: Weigh out the appropriate amount of cisplatin powder; dissolve using DMSO; prepare for injection, use fresh drug whenever possible.

[0606] 2. Mitochondria extraction: Culture the cell line expressing mitochondrial DNase I in advance according to the required amount; extract mitochondria from the cell line expressing mtDNase I (the extraction method and the buffer used can refer to the published mitochondrial extraction method Frezza et al. 2007 doi: 10.1038 / nprot.2006.478); a small amount of extracted mitochondria is stained with MitoTracker Green FM Dye (M46750) and then quantified by flow cytometry.

[0607] 3. Mitochondria injection: This example performs kidney positioning injection, at the beginning of the experiment, the mouse is placed in an anesthesia box containing 2% isoflurane for anesthesia; the anesthetized mouse is fixed supine on the 37°C constant temperature heating plate operation table, connected with the electrocardiogram device to keep the heart rhythm stable, the isoflurane mask maintains anesthesia, and the appropriate amount of ultrasonic gel is applied; under the guidance of ultrasound, the injection needle is pushed into the abdomen to perform assisted kidney positioning injection; each mouse is injected with 200,000 / 50uL of mitochondria with DNase I in the left kidney.

[0608] 4. Cisplatin injection: After the mouse receives positioning mitochondria injection for more than 2 hours, 15-20ug / kg of cisplatin liquid is injected according to the body weight of the mouse, and 15ug / kg dose is used in this example; 48 hours after cisplatin injection, measurement and sampling are performed.

[0609] The experimental results are shown in Figures 15A-15C, which show that the transplantation of mitochondria by positioning injection plays a protective role, and the protection of mitochondria with DNase I is better than that of wild-type mitochondria, which inhibits the kidney swelling caused by the classic AKI modeling method of cisplatin injection, and the weight loss caused by renal failure. In addition, Figure 15C shows that compared with the injection of wild-type mitochondria and mitochondria with mtDNase I, mitochondria with mtDNase I effectively inhibit the weight loss caused by chemotherapy drugs (cisplatin), and unmodified mitochondria cannot effectively inhibit this phenotype.

[0610] Example 22 Protection of live mice by transplantation of mitochondria with mtDNase I against doxorubicin

[0611] This example compares the protective effect of mitochondria with DNase I on an acute doxorubicin mouse model (high dose drug injection). Doxorubicin is a commonly used anthracycline chemotherapy drug known for its strong cytotoxicity, which can induce mitochondrial dysfunction, oxidative stress, and apoptosis. In animal experiments, high-dose injection of doxorubicin can quickly induce an acute organ injury model, especially in tissues such as the kidney and heart, which exhibit significant mitochondrial damage and inflammatory response. It is commonly used to study drug toxicity mechanisms and related protection strategies. Therefore, this example uses an acute injury model induced by doxorubicin to verify the relief and protection of mitochondria with mtDNase I on drug-induced tissue damage.

[0612] The experimental steps are as follows:

[0613] 1. Doxorubicin preparation: weigh an appropriate amount of doxorubicin powder; dissolve with DMSO; calculate the injection amount according to the mouse body weight.

[0614] 2. Mitochondria extraction: culture the cell line expressing mitochondria-localized DNase I in advance according to the required amount; extract mitochondria from the cell line expressing mtDNase I (the extraction method and buffer used can refer to the published mitochondria extraction method Frezza et al. 2007 doi:10.1038 / nprot.2006.478); a small amount of extracted mitochondria is stained with MitoTracker Green FM Dye (M46750) and quantified by flow cytometry.

[0615] 3. Mitochondria injection: intraperitoneal injection for systemic administration in this example, the mitochondria-containing suspension is loaded into a 1 mL syringe (Biyun Tian FS801); the mouse is grasped using the dorsal fixation method, with its head slightly tilted back and abdomen upwards, and the left hind leg is fixed with fingers; the injection site (right thigh root, lower abdomen midline 0.5 cm position) is disinfected with 75% alcohol, the syringe needle is inserted into the skin, and then advanced 2-3 mm subcutaneously, with the needle at a 45-degree angle to the skin; the needle is withdrawn, and if there is no blood or liquid, the slow injection can begin; after injection, the needle is rotated gently and the syringe is slowly pulled out to prevent liquid leakage; each mouse is injected with 1,000,000 / 100 uL of mitochondria with DNase I.

[0616] 4. Doxorubicin injection: after the mouse receives the mitochondria injection for more than 2 hours, 8-15 mg / kg of doxorubicin liquid is injected according to the mouse body weight, and 12.5 mg / kg dose is used in this example; sample collection is performed 2 hours after doxorubicin injection.

[0617] The experimental results, as shown in FIGS. 16A-16C, show that the protection of mtDNase is superior to wild-type mitochondria, and in multiple organs (including liver, kidney, lung, heart, spleen, and smooth muscle) not only inhibits the up-regulation of cGAS-STING due to DNA leakage, but also avoids the P21 up-regulation that occurs with wild-type mitochondria transplantation.

[0618] The foregoing detailed description has been presented for purposes of illustration and description. Various modifications and changes can be made to the implementation detailed in this disclosure, in light of the above detailed description, without departing from the scope of the disclosure. It is intended that the scope of the application be limited not by this detailed description, but rather by the claims appended hereto.

Claims

1. A method of reducing mitochondrial DNA leakage in a cell of interest, comprising localizing a nuclease to mitochondria.

2. The method of claim 1, wherein the nuclease comprises a human nuclease.

3. The method of any one of claims 1-2, wherein the nuclease comprises a DNAse.

4. The method of claim 3, wherein the DNAse comprises a member of the DNase I family and / or a functional variant thereof.

5. The method of any one of claims 3-4, wherein the DNAse comprises DNase I and / or a functional variant thereof.

6. The method of any one of claims 3-5, wherein the DNAse comprises the amino acid sequence set forth in SEQ ID NO:

3.

7. The method of any one of claims 3-6, wherein a nucleic acid molecule encoding the DNAse comprises the nucleotide sequence set forth in SEQ ID NO:

4.

8. The method of any one of claims 1-7, wherein the nuclease is localized to the mitochondria by a mitochondrial localization sequence.

9. The method of claim 8, wherein the mitochondrial localization sequence is directly or indirectly linked to the nuclease.

10. The method of any one of claims 8-9, wherein the mitochondrial localization sequence is at the N-terminus or C-terminus of the nuclease.

11. The method of any one of claims 8-10, wherein the mitochondrial localization sequence comprises a polypeptide sequence.

12. The method of any one of claims 8-11, wherein the mitochondrial localization sequence comprises a polypeptide sequence derived from an N-terminal region or C-terminal region of a mitochondrial protein.

13. The method of any one of claims 8-12, wherein the mitochondrial localization sequence comprises a polypeptide sequence derived from an N-terminal region or C-terminal region of a mitochondrial membrane protein.

14. The method of any one of claims 8-13, wherein the mitochondrial localization sequence comprises a mitochondrial localization sequence derived from cytochrome c oxidase subunit 8A.

15. The method of claim 14, wherein the cytochrome c oxidase subunit 8A comprises human cytochrome c oxidase subunit 8A.

16. The method of any one of claims 8-15, wherein the mitochondrial localization sequence comprises the amino acid sequence set forth in SEQ ID NO:

1.

17. The method of any one of claims 8-16, wherein a nucleic acid molecule encoding the mitochondrial localization sequence comprises the nucleotide sequence set forth in SEQ ID NO:

2.

18. The method of any one of claims 1-17, comprising causing the cell of interest to express a fusion protein comprising a mitochondrial localization sequence and the nuclease and / or to express a nucleic acid molecule encoding the fusion protein.

19. The method of claim 18, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

5.

20. The method of any one of claims 18-19, wherein the nucleic acid molecule encoding the fusion protein comprises the nucleotide sequence set forth in SEQ ID NO:

6.

21. The method of any one of claims 1-20, comprising introducing an isolated modified mitochondrion into a cell of interest, wherein the nuclease is localized to the modified mitochondrion.

22. The method of claim 21, wherein the modified mitochondrion is isolated from a cell and / or tissue.

23. The method of any one of claims 21-22, wherein the cell from which the modified mitochondrion is isolated comprises a somatic cell, a germ cell, and / or a stem cell.

24. The method of any one of claims 21-23, wherein the modified mitochondrion is isolated from an induced pluripotent stem cell.

25. The method of any one of claims 21-24, wherein the modified mitochondrion is isolated from a muscle cell, a neural cell, an endothelial cell, an epithelial cell, an embryonic cell, and / or a tumor cell.

26. The method of any one of claims 21-25, wherein the modified mitochondrion is isolated from a cardiomyocyte and / or a skeletal muscle cell.

27. The method of any one of claims 21-26, wherein the modified mitochondrion is isolated from an induced pluripotent stem cell differentiated cardiomyocyte.

28. The method of any one of claims 21-27, wherein the modified mitochondrion is isolated from a mammalian cell.

29. The method of any one of claims 21-28, wherein the modified mitochondrion is isolated from a human or mouse cell.

30. The method of any one of claims 21-29, wherein the modified mitochondrion is isolated from a healthy cell.

31. The method of any one of claims 21-30, wherein the modified mitochondrion is isolated from a cell that is syngeneic to the cell of interest.

32. The method of any one of claims 21-31, wherein the modified mitochondrion is isolated from a cell that is allogeneic to the cell of interest.

33. The method of any one of claims 21-32, wherein the modified mitochondrion is isolated from a cell that is of the same cell type as the cell of interest.

34. The method of any one of claims 21-33, wherein the modified mitochondrion is isolated from a cell that is metabolically compatible with the cell of interest.

35. The method of any one of claims 1-34, wherein the cell of interest comprises a cell with impaired mitochondria.

36. The method of any one of claims 1-35, wherein the cell of interest comprises a cell with activation of the cGAS-STING pathway.

37. The method of any one of claims 1-36, wherein the cell of interest comprises a cell in a state of senescence.

38. The method of any one of claims 1-37, wherein the cell of interest comprises a cell with ischemia-reperfusion injury, hypoxic injury, and / or exposure to an oxidant.

39. The method of any one of claims 1-38, wherein the cell of interest is derived from a mammal.

40. The method of any one of claims 1-39, wherein the cell of interest is derived from a human or a mouse.

41. The method of any one of claims 1-40, wherein the cell of interest comprises a muscle cell, a neural cell, an endothelial cell, an epithelial cell, an embryonic cell, and / or a tumor cell.

42. The method of any one of claims 1-41, wherein the cell of interest comprises a cardiomyocyte, a skeletal muscle cell, a vascular endothelial cell, a kidney epithelial cell, and / or a neuroblastoma cell.

43. The method of any one of claims 1-42, wherein the mitochondrial DNA leakage in the cell of interest is caused by ischemia reperfusion.

44. The method of any one of claims 1-43, wherein the mitochondrial DNA leakage in the cell of interest is caused by aging.

45. The method of any one of claims 1-44, wherein the mitochondrial DNA leakage in the cell of interest is caused by an anti-tumor therapy.

46. The method of claim 45, wherein the anti-tumor therapy comprises a drug or a therapeutic means capable of inducing mitochondrial damage, tDNA leakage, or activation of the cGAS-STING pathway.

47. The method of claim 46, wherein the anti-tumor therapy comprises chemotherapy and / or radiation therapy.

48. The method of any one of claims 46-47, wherein the chemotherapeutic drug comprises a platinum-based chemotherapeutic drug and / or an anthracycline-based chemotherapeutic drug.

49. The method of claim 48, wherein the anthracycline-based chemotherapeutic drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin.

50. The method of claim 48, wherein the platinum-based chemotherapeutic drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin.

51. The method of any one of claims 1-50, wherein the mitochondrial DNA leakage in the cell of interest is caused by hypoxia.

52. The method of any one of claims 1-51, wherein the mitochondrial DNA leakage in the cell of interest is caused by peroxide stimulation.

53. The method of claim 52, wherein the peroxide comprises H2O2.

54. The method of any one of claims 1-53, wherein the method is capable of delaying aging of the cell of interest.

55. The method of any one of claims 1-54, wherein the method is capable of alleviating damage of the cell of interest.

56. The method of any one of claims 1-55, wherein the method is capable of suppressing inflammatory responses in the cell of interest.

57. The method of any one of claims 1-56, wherein the method is capable of suppressing downstream pathways activated by mitochondrial DNA leakage.

58. The method of claim 57, wherein the downstream pathway comprises the cGAS-STING pathway.

59. The method of any one of claims 1-58, wherein the method is capable of preserving the respiratory capacity of mitochondria.

60. The method of any one of claims 1-59, wherein the method is in vitro and / or ex vivo.

61. A modified mitochondrion, wherein, the mitochondrion.

62. The mitochondrion of claim 61, wherein the nuclease is localized to the outer membrane of the mitochondrion.

63. The mitochondrion of any one of claims 61-62, wherein the nuclease comprises a human nuclease.

64. The mitochondrion of any one of claims 61-63, wherein the nuclease comprises a DNAse.

65. The mitochondrion of claim 64, wherein the DNAse comprises a member of the DNase I family and / or a functional variant thereof.

66. The mitochondrion of any one of claims 64-65, wherein the DNAse comprises DNase I and / or a functional variant thereof.

67. The mitochondrion of any one of claims 64-66, wherein the DNAse comprises the amino acid sequence set forth in SEQ ID NO:

3.

68. The mitochondrion of any one of claims 64-67, wherein a nucleic acid molecule encoding the DNAse comprises the nucleotide sequence set forth in SEQ ID NO:

4.

69. The mitochondrion of any one of claims 61-68, wherein the nuclease is localized to the mitochondrion by a mitochondrial localization sequence.

70. The mitochondrion of claim 69, wherein the mitochondrial localization sequence is directly or indirectly linked to the nuclease.

71. The mitochondrion of any one of claims 69-70, wherein the mitochondrial localization sequence is at the N-terminus or C-terminus of the nuclease.

72. The mitochondrion of any one of claims 69-71, wherein the mitochondrial localization sequence comprises a polypeptide sequence.

73. The mitochondrion of any one of claims 69-72, wherein the mitochondrial localization sequence comprises a polypeptide sequence derived from an N-terminal region or C-terminal region of a mitochondrial protein.

74. The mitochondrion of any one of claims 69-73, wherein the mitochondrial localization sequence comprises a polypeptide sequence derived from an N-terminal region or C-terminal region of a mitochondrial membrane protein.

75. The mitochondrion of any one of claims 69-74, wherein the mitochondrial localization sequence comprises a mitochondrial localization sequence derived from cytochrome c oxidase subunit 8A (COX8A).

76. The mitochondrion of claim 75, wherein the cytochrome c oxidase subunit 8A comprises human cytochrome c oxidase subunit 8A.

77. The mitochondrion of any one of claims 69-76, wherein the mitochondrial localization sequence comprises the amino acid sequence set forth in SEQ ID NO:

1.

78. The mitochondrion of any one of claims 69-77, wherein a nucleic acid molecule encoding the mitochondrial localization sequence comprises the nucleotide sequence set forth in SEQ ID NO:

2.

79. The mitochondrion of any one of claims 61-78, wherein the mitochondrion is isolated from a cell or a tissue.

80. The mitochondrion of any one of claims 61-79, wherein the mitochondrion is isolated from a somatic cell, a germ cell, and / or a stem cell.

81. The mitochondrion of any one of claims 61-80, wherein the mitochondrion is isolated from an induced pluripotent stem cell.

82. The mitochondrion of any one of claims 61-81, wherein the mitochondrion is isolated from a muscle cell, a neural cell, an endothelial cell, an epithelial cell, an embryonic cell, and / or a tumor cell.

83. The mitochondrion of any one of claims 61-82, wherein the mitochondrion is isolated from a cardiac cell and / or a skeletal muscle cell.

84. The mitochondrion of any one of claims 61-83, wherein the mitochondrion is isolated from an induced pluripotent stem cell differentiated cardiac cell.

85. The mitochondrion of any one of claims 61-84, wherein the mitochondrion is isolated from a mammalian cell.

86. The mitochondrion of any one of claims 61-85, wherein the mitochondrion is isolated from a human or a mouse cell.

87. The mitochondrion of any one of claims 61-86, wherein the modified mitochondrion is isolated from a healthy cell.

88. A fusion protein comprising a mitochondrion localization sequence and a nuclease.

89. The fusion protein of claim 88, wherein the nuclease comprises a human nuclease.

90. The fusion protein of any one of claims 88-89, wherein the nuclease comprises a DNAse.

91. The fusion protein of claim 90, wherein the DNAse comprises a member of the DNase I family and / or a functional variant thereof.

92. The fusion protein of any one of claims 90-91, wherein the DNAse comprises DNase I and / or a functional variant thereof.

93. The fusion protein of any one of claims 90-92, wherein the DNAse comprises the amino acid sequence set forth in SEQ ID NO:

3.

94. The fusion protein of any one of claims 91-93, wherein the nucleic acid molecule encoding the DNAse comprises the nucleotide sequence set forth in SEQ ID NO:

4.

95. The fusion protein of any one of claims 90-94, wherein the nuclease is localized to the mitochondrion by a mitochondrion localization sequence.

96. The fusion protein of claim 95, wherein the mitochondrion localization sequence is directly or indirectly linked to the nuclease.

97. The fusion protein of any one of claims 95-96, wherein the mitochondrion localization sequence is N-terminal to the nuclease.

98. The fusion protein of any one of claims 95-97, wherein the mitochondrion localization sequence comprises a polypeptide sequence.

99. The fusion protein of any one of claims 95-98, wherein the mitochondrion localization sequence comprises a polypeptide sequence derived from an N-terminal region or a C-terminal region of a mitochondrion protein.

100. The fusion protein of any one of claims 95-99, wherein the mitochondrial localization sequence comprises a polypeptide sequence derived from an N-terminal region or a C-terminal region of a mitochondrial membrane protein.

101. The fusion protein of any one of claims 95-100, wherein the mitochondrial localization sequence comprises a mitochondrial localization sequence derived from cytochrome c oxidase subunit 8A (COX8A).

102. The fusion protein of claim 101, wherein the cytochrome c oxidase subunit 8A comprises a human cytochrome oxidase subunit 8A.

103. The fusion protein of any one of claims 95-102, wherein the mitochondrial localization sequence comprises the amino acid sequence set forth in SEQ ID NO:

1.

104. The fusion protein of any one of claims 95-103, wherein the nucleic acid molecule encoding the mitochondrial localization sequence comprises the nucleotide sequence set forth in SEQ ID NO:

2.

105. The fusion protein of any one of claims 88-104, comprising the amino acid sequence set forth in SEQ ID NO:

5.

106. The fusion protein of any one of claims 88-105, a nucleotide sequence encoding which comprises the nucleotide sequence set forth in SEQ ID NO:

6.

107. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 88-106.

108. The nucleic acid molecule of claim 107, comprising the nucleotide sequence set forth in SEQ ID NO:

6.

109. The nucleic acid molecule of any one of claims 107-108, further comprising an expression regulatory element.

110. The nucleic acid molecule of claim 109, wherein the expression regulatory element comprises a promoter and / or an enhancer, wherein the promoter is positioned upstream of the nucleotide sequence encoding the fusion protein.

111. The nucleic acid molecule of any one of claims 109-110, further comprising a polynucleotide signal sequence positioned downstream of the nucleotide sequence encoding the fusion protein.

112. The nucleic acid molecule of any one of claims 107-111, comprising DNA and / or RNA.

113. The nucleic acid molecule of any one of claims 107-112, comprising mRNA.

114. The nucleic acid molecule of claim 113, further comprising a 5’ cap, a 5’ untranslated region, and a 3’ untranslated region.

115. The nucleic acid molecule of any one of claims 113-114, comprising at least one modified nucleotide.

116. The nucleic acid molecule of any one of claims 113-115, further comprising AAV inverted terminal repeat (ITR) sequences positioned upstream and downstream of the nucleotide sequence encoding the fusion protein.

117. The nucleic acid molecule of claim 116, wherein the AAV ITRs are derived from AAV9.

118. The nucleic acid molecule of any one of claims 107-112, further comprising a lentiviral long terminal repeat (LTR) located upstream and downstream of the nucleotide sequence encoding the fusion protein.

119. A vector comprising the nucleic acid molecule of any one of claims 107-118.

120. The vector of claim 119, which is a viral vector or a polynucleotide vector.

121. The vector of any one of claims 119-120, which is a plasmid.

122. The vector of any one of claims 119-120, which is a viral vector, and the viral vector includes an AAV vector and a lentiviral vector.

123. The vector of claim 122, wherein the AAV vector is an AAV9 vector.

124. A modified cell comprising the mitochondrion of any one of claims 61-87, the fusion protein of any one of claims 88-106, the nucleic acid molecule of any one of claims 107-118, and / or the vector of any one of claims 119-123.

125. The cell of claim 124, which includes a somatic cell, a germ cell, and / or a stem cell.

126. The cell of any one of claims 124-125, which includes an induced pluripotent stem cell.

127. The cell of any one of claims 124-126, which includes a muscle cell, a neural cell, an endothelial cell, an epithelial cell, an embryonic cell, and / or a tumor cell.

128. The cell of any one of claims 124-127, wherein the cell includes a cardiomyocyte and / or a skeletal muscle cell.

129. The cell of any one of claims 124-128, which includes a cardiomyocyte differentiated from an induced pluripotent stem cell.

130. The cell of any one of claims 124-129, which includes a mammalian cell.

131. The cell of any one of claims 124-130, which includes a human or a mouse cell.

132. The cell of any one of claims 124-131, which includes a healthy cell.

133. A method of making a modified mitochondrion, comprising making the modified cell of any one of claims 124-132 and isolating a mitochondrion produced by the modified cell.

134. A pharmaceutical composition comprising the mitochondrion of any one of claims 61-87, the fusion protein of any one of claims 88-106, the nucleic acid molecule of any one of claims 107-118, the vector of any one of claims 119-123, and / or the cell of any one of claims 124-132.

135. The pharmaceutical composition of claim 134, further comprising a pharmaceutically acceptable carrier.

136. The pharmaceutical composition of any one of claims 134-135, comprising the nucleic acid molecule of any one of claims 103-114 and a carrier for delivering the nucleic acid molecule.

137. The pharmaceutical composition of claim 134, wherein the nucleic acid molecule comprises an mRNA and the carrier for delivering the nucleic acid molecule comprises a lipid nanoparticle (LNP).

138. Use of the mitochondrion of any one of claims 61-87, the fusion protein of any one of claims 88-106, the nucleic acid molecule of any one of claims 107-118, the vector of any one of claims 119-123, and / or the cell of any one of claims 124-132 in the manufacture of a medicament.

139. The use of claim 138, wherein the medicament is for delaying aging.

140. The use of claim 139, wherein the delaying of aging comprises delaying cardiac aging, vascular aging, muscle aging, kidney aging, and / or nervous system aging.

141. The use of any one of claims 139-140, wherein the delaying of aging comprises reducing the expression level of an aging marker.

142. The use of claim 141, wherein the aging marker comprises p21, p16INK4a, p53, telomere length, and / or a DNA damage marker.

143. The use of any one of claims 138-142, wherein the medicament is for preventing and / or treating a disease associated with mitochondrial damage.

144. The use of claim 143, wherein the mitochondrial damage comprises mitochondrial DNA leakage.

145. The use of any one of claims 138-144, wherein the medicament is for preventing and / or treating an ischemia-related disease.

146. The use of any one of claims 138-145, wherein the medicament is for alleviating side effects of an anti-tumor therapy.

147. The use of claim 146, wherein the side effects of the anti-tumor therapy comprise mitochondrial damage, mitochondrial DNA leakage, and / or inflammation associated with activation of the cGAS-STING pathway triggered thereby.

148. The use of any one of claims 146-147, wherein the anti-tumor therapy comprises chemotherapy and / or radiotherapy.

149. The use of any one of claim 148, wherein the chemotherapeutic drug comprises a platinum-based chemotherapeutic drug and / or an anthracycline-based chemotherapeutic drug.

150. The use of claim 149, wherein the anthracycline-based chemotherapeutic drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin.

151. The use of claim 149, wherein the platinum-based chemotherapeutic drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin.

152. The use of any one of claims 138-151, wherein the medicament is for treating a cardiovascular disease.

153. The use of any one of claims 138-152, wherein the agent is used to treat ischemia reperfusion injury and / or hypoxic injury.

154. The use of any one of claims 138-153, wherein the agent is used to downregulate an inflammatory response and / or treat an inflammatory disease.

155. The use of claim 154, wherein the inflammatory response and / or inflammatory disease comprises an inflammatory response and / or inflammatory disease mediated by or associated with cGAS-STING pathway activation.

156. The use of any one of claims 154-155, wherein the inflammatory disease is selected from the group consisting of a kidney inflammatory disease, a liver inflammatory disease, a lung inflammatory disease, a heart inflammatory disease, a spleen-related inflammatory disorder, and an inflammatory disorder in smooth muscle tissue.

157. The use of claim 156, wherein the kidney inflammatory disease comprises acute kidney injury (AKI), chronic kidney disease (CKD), systemic lupus erythematosus associated nephritis, IgA nephropathy, interstitial nephritis, and / or diabetic nephropathy.

158. The use of claim 156, wherein the liver inflammatory disease comprises nonalcoholic steatohepatitis, drug-induced liver injury, viral hepatitis, alcoholic liver disease, liver fibrosis, and / or cirrhosis.

159. The use of claim 156, wherein the lung inflammatory disease comprises acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bacterial or viral infection associated pneumonia, pneumoconiosis, and / or pulmonary fibrosis.

160. The use of claim 156, wherein the heart inflammatory disease comprises doxorubicin-induced cardiotoxicity, myocardial ischemia reperfusion injury, myocarditis, and / or chronic heart failure.

161. The use of claim 156, wherein the spleen-related inflammatory disorder comprises systemic lupus erythematosus (SLE)-associated spleen disorder, autoimmune disease- associated spleen immune activation, and / or infection-induced spleen immune activation.

162. The use of claim 156, wherein the inflammatory disorder in smooth muscle tissue comprises an inflammatory response in vascular smooth muscle cells, chronic inflammatory disorder in airway smooth muscle, and / or gastrointestinal tract smooth muscle.

163. The use of claim 162, wherein the inflammatory disorder in smooth muscle tissue comprises atherosclerosis and / or arterial injury.

164. A method of delaying aging, comprising administering to a subject an effective amount of the mitochondrion of any one of claims 61-87, the fusion protein of any one of claims 88-106, the nucleic acid molecule of any one of claims 107-118, the vector of any one of claims 119-123, the cell of any one of claims 124-132, and / or the pharmaceutical composition of any one of claims 134-137.

165. The method of claim 164, wherein the delaying aging comprises delaying cardiac aging, vascular aging, muscle aging, kidney aging, and / or nervous system aging.

166. The method of any one of claims 164-165, wherein the delaying aging comprises reducing expression level of an aging marker.

167. The method of claim 166, wherein the aging marker comprises p21, pl6INK4a, p53, telomere length, and / or a DNA damage marker.

168. A method of preventing and / or treating a disease, comprising administering to a subject an effective amount of the mitochondria of any one of claims 61-87, the fusion protein of any one of claims 88-106, the nucleic acid molecule of any one of claims 107-118, the vector of any one of claims 119-123, the cell of any one of claims 124-132, and / or the pharmaceutical composition of any one of claims 134-137.

169. The method of claim 168, wherein the disease comprises a disease associated with mitochondrial damage.

170. The method of claim 169, wherein the mitochondrial damage comprises mitochondrial DNA leakage.

171. The method of any one of claims 168-170, wherein the disease comprises an ischemia-related disease.

172. The method of any one of claims 168-171, wherein the disease comprises a side effect of an anti-tumor therapy.

173. The method of claim 172, wherein the side effect of an anti-tumor therapy comprises mitochondrial damage, mitochondrial DNA leakage, and / or an inflammation reaction associated with the activation of cGAS-STING pathway thereby.

174. The method of any one of claims 172-173, wherein the anti-tumor therapy comprises chemotherapy and / or radiotherapy.

175. The method of claim 174, wherein the chemotherapeutic drug comprises a platinum-based chemotherapeutic drug and / or an anthracycline-based chemotherapeutic drug.

176. The method of claim 175, wherein the anthracycline-based chemotherapeutic drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin.

177. The method of claim 174, wherein the platinum-based chemotherapeutic drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin.

178. The method of any one of claims 168-177, wherein the disease comprises a cardiovascular disease.

179. The method of any one of claims 168-178, wherein the disease comprises ischemia-reperfusion injury and / or hypoxic injury.

180. The method of any one of claims 168-179, wherein the disease comprises an inflammation reaction and / or an inflammatory disease.

181. The method of claim 180, wherein the inflammatory response and / or inflammatory disease comprises an inflammatory response and / or inflammatory disease mediated by or associated with cGAS-STING pathway activation.

182. The method of claim 181, wherein the inflammatory disease is selected from the group consisting of a kidney inflammatory disease, a liver inflammatory disease, a lung inflammatory disease, a heart inflammatory disease, a spleen-related inflammatory disorder, and an inflammatory disorder in smooth muscle tissue.

183. The method of claim 182, wherein the kidney inflammatory disease comprises acute kidney injury (AKI), chronic kidney disease (CKD), systemic lupus erythematosus- associated nephritis, IgA nephropathy, interstitial nephritis, and / or diabetic nephropathy.

184. The method of claim 182, wherein the liver inflammatory disease comprises nonalcoholic steatohepatitis, drug-induced liver injury, viral hepatitis, alcoholic liver disease, liver fibrosis, and / or cirrhosis.

185. The method of claim 182, wherein the lung inflammatory disease comprises acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bacterial or viral infection-associated pneumonia, pneumoconiosis, and / or pulmonary fibrosis.

186. The method of claim 182, wherein the heart inflammatory disease comprises doxorubicin-induced cardiotoxicity, myocardial ischemia-reperfusion injury, myocarditis, and / or chronic heart failure.

187. The method of claim 182, wherein the spleen-related inflammatory disorder comprises systemic lupus erythematosus (SLE)-associated spleen disorder, autoimmune disease- associated spleen immune activation, and / or infection-induced spleen immune activation.

188. The method of claim 182, wherein the inflammatory disorder in smooth muscle tissue comprises an inflammatory response in vascular smooth muscle cells, chronic inflammatory disorder in airway smooth muscle, and / or gastrointestinal tract smooth muscle.

189. The method of claim 188, wherein the inflammatory disorder in smooth muscle tissue comprises atherosclerosis and / or arterial injury.

190. Use of the mitochondrion of any one of claims 61-87, the fusion protein of any one of claims 88-106, the nucleic acid molecule of any one of claims 107-118, the vector of any one of claims 119-123, the cell of any one of claims 124-132, and / or the pharmaceutical composition of any one of claims 134-137 in delaying aging.

191. The use of claim 190, wherein the delaying of aging comprises heart aging, vascular aging, muscle aging, kidney aging, and / or nervous system aging.

192. The use of any one of claims 190-191, wherein the delaying of aging comprises reducing the expression level of an aging marker.

193. The use of claim 192, wherein the senescence markers comprise p21, p16INK4a, p53, telomere length, and / or DNA damage markers.

194. Use of the mitochondria of any one of claims 61-87, the fusion protein of any one of claims 88-106, the nucleic acid molecule of any one of claims 107-118, the vector of any one of claims 119-123, the cell of any one of claims 124-132, and / or the pharmaceutical composition of any one of claims 134-137 in the treatment and / or prevention of a disease.

195. The use of claim 194, wherein the disease comprises a disease associated with mitochondrial damage.

196. The use of claim 195, wherein the mitochondrial damage comprises mitochondrial DNA leakage.

197. The use of any one of claims 194-196, wherein the disease comprises an ischemia-related disease.

198. The use of any one of claims 194-197, wherein the disease comprises a side effect of an anti-tumor therapy.

199. The use of claim 198, wherein the side effect of an anti-tumor therapy comprises mitochondrial damage, mitochondrial DNA leakage, and / or an inflammation reaction associated with the activation of the cGAS-STING pathway thereby.

200. The use of any one of claims 194-199, wherein the anti-tumor therapy comprises chemotherapy and / or radiotherapy.

201. The use of claim 200, wherein the chemotherapeutic drug comprises a platinum-based chemotherapeutic drug and / or an anthracycline-based chemotherapeutic drug.

202. The use of claim 201, wherein the anthracycline-based chemotherapeutic drug comprises doxorubicin, epirubicin, and / or deoxydoxorubicin.

203. The use of claim 201, wherein the platinum-based chemotherapeutic drug comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, metaplatin, and / or apraplatin.

204. The use of any one of claims 194-203, wherein the disease comprises a cardiovascular disease.

205. The use of any one of claims 194-204, wherein the disease comprises ischemia-reperfusion injury and / or hypoxic injury.

206. The use of any one of claims 194-205, wherein the disease comprises an inflammation reaction and / or an inflammatory disease.

207. The use of claim 206, wherein the inflammation reaction and / or the inflammatory disease comprises an inflammation reaction and / or an inflammatory disease mediated by or associated with the activation of the cGAS-STING pathway.

208. The use of any one of claim 207, wherein the inflammatory disease is selected from the group consisting of a kidney inflammatory disease, a liver inflammatory disease, a lung inflammatory disease, a heart inflammatory disease, a spleen-related inflammatory lesion, and an inflammatory lesion in smooth muscle tissue.

209. The use of claim 207, wherein the inflammatory disease of the kidney comprises acute kidney injury (AKI), chronic kidney disease (CKD), systemic lupus erythematosus- associated nephritis, IgA nephropathy, interstitial nephritis, and / or diabetic nephropathy.

210. The use of claim 207, wherein the inflammatory disease of the liver comprises nonalcoholic steatohepatitis, drug-induced liver injury, viral hepatitis, alcoholic liver disease, liver fibrosis, and / or cirrhosis.

211. The use of claim 207, wherein the inflammatory disease of the lung comprises acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bacterial or viral infection- associated pneumonia, pneumoconiosis, and / or pulmonary fibrosis.

212. The use of claim 207, wherein the inflammatory disease of the heart comprises doxorubicin-induced cardiotoxicity, myocardial ischemia-reperfusion injury, myocarditis, and / or chronic heart failure.

213. The use of claim 207, wherein the spleen-associated inflammatory disorder comprises systemic lupus erythematosus (SLE)-associated splenic disorder, autoimmune disease- associated splenic immune activation, and / or infection-induced splenic immune activation.

214. The use of claim 207, wherein the inflammatory disorder in smooth muscle tissue comprises inflammatory responses in vascular smooth muscle cells, chronic inflammatory disorders in airway smooth muscle, and / or gastrointestinal smooth muscle.

215. The use of claim 214, wherein the inflammatory disorder in smooth muscle tissue comprises atherosclerosis and / or arterial injury.

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