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WO2025090754A1 - Transplantation mitochondriale pour la conservation de tissus à long terme - Google Patents

Transplantation mitochondriale pour la conservation de tissus à long terme Download PDF

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Publication number
WO2025090754A1
WO2025090754A1 PCT/US2024/052800 US2024052800W WO2025090754A1 WO 2025090754 A1 WO2025090754 A1 WO 2025090754A1 US 2024052800 W US2024052800 W US 2024052800W WO 2025090754 A1 WO2025090754 A1 WO 2025090754A1
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tissue
liver
mitochondria
storage
hours
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Mustafa K. UYGUN
Alexandra TCHIR
Basak E. Uygun
Mehmet Toner
Bradley W. ELLIS
Sofia BAPTISTA
Heidi Yeh
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General Hospital Corp
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General Hospital Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes

Definitions

  • the disclosure features a method of prolonging survival of tissue, the method comprising introducing into the tissue one or more exogenous mitochondria.
  • the disclosure features a method of prolonging survival of a cell, the method comprising introducing into the cell one or more exogenous mitochondria.
  • the disclosure features a method of transplanting an isolated tissue into a subject in need of replacement of the tissue, the method comprising: a) introducing, into a cell of the isolated tissue, one or more exogenous mitochondria; b) providing the subject with the tissue resulting from (a) in place of the subject’s endogenous tissue.
  • the disclosure features a method of transplanting an isolated tissue into a subject in need of replacement of the tissue, the method comprising providing the subject with the isolated tissue in place of the subject’s endogenous tissue, wherein prior to the providing, one or more exogenous mitochondria have been introduced into a cell of the isolated tissue.
  • the tissue is a liver, a heart, a kidney, or a lung.
  • the tissue is an isolated tissue.
  • the method comprises introducing the exogenous mitochondria ex vivo.
  • the cell is an endothelial cell. In some embodiments, the cell is a liver cell. In some embodiments, the liver cell is a hepatocyte.
  • the tissue is obtained from a donor after cardiac death (DCD).
  • DCD cardiac death
  • the tissue is characterized by a warm ischemic time (WIT) of greater than 30 minutes. In some embodiments, the tissue is characterized by a WIT of greater than 45 minutes. In some embodiments, the tissue is characterized by a WIT of greater than 60 minutes. In some embodiments, the tissue is characterized by a WIT of from 30 minutes to 72 hours. In some embodiments, the tissue is characterized by a WIT of from 45 minutes to 48 hours. In some embodiments, the tissue is characterized by a WIT of from 60 minutes to 24 hours.
  • WIT warm ischemic time
  • the one or more exogenous mitochondria are obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof.
  • the population of cells is of the same species as the cell into which the one or more mitochondria are introduced.
  • the method further comprises providing the isolated tissue to a subject in need of replacement of the tissue.
  • the tissue is a liver.
  • the subject is diagnosed as having a complication or disease selected from the group consisting of ischemic liver injury, primary non-function (PNF), early allograft dysfunction (EAD), small-for-size syndrome (SFSS), resection for malignant or benign disease, autoimmune hepatitis, cystic fibrosis, cholestatic liver diseases, primary sclerosing cholangitis, primary biliary cirrhosis, inborn errors of metabolism, metabolic diseases, sickle cell hepatopathy, erythropoietic protoporphyria (EPP) hepatopathy, congestive hepatopathy, metabolic dysfunction-associated steatotic liver disease (MAFLD), metabolic dysfunction-associated steatohepatitis (MASH), alcoholic liver disease, hemochromatosis, Alagille syndrome, Wilson's disease, viral hepatitis, alpha-1 antitrypsin deficiency, drug-induced liver injury (DILI), and liver cancer, cancer in the liver, liver met
  • PNF
  • the subject is a human.
  • the exogenous mitochondria are freshly isolated. In some embodiments, the exogenous mitochondria were stored prior to introduction into the tissue or cell. In some embodiments, the exogenous mitochondria are introduced at a dose of 2.5 to 150 pg mitochondria per mL of solution.
  • the disclosure features an ex vivo tissue transplant comprising an isolated tissue that comprises one or more heterologous mitochondria.
  • the tissue is an isolated liver.
  • the tissue transplant is obtained from a DCD.
  • the tissue is characterized by a warm ischemic time (WIT) of greater than 30 minutes.
  • WIT warm ischemic time
  • the tissue is characterized by a WIT of greater than 45 minutes.
  • the tissue is characterized by a WIT of greater than 60 minutes.
  • the tissue is characterized by a WIT of from 30 minutes to 72 hours.
  • the tissue is characterized by a WIT of from 45 minutes to 48 hours.
  • the tissue is characterized by a WIT of from 60 minutes to 24 hours.
  • the tissue has been subjected to static cold storage or subzero storage prior to introducing the one or more heterologous mitochondria into the liver.
  • the one or more exogenous mitochondria are obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof.
  • the heterologous mitochondria are freshly isolated.
  • the heterologous mitochondria were stored prior to introduction into the tissue.
  • the heterologous mitochondria are introduced at a dose of 2.5 to 150 pg per mL of solution.
  • FIG. 1A - FIG. 1K show that a mitochondrial transplant improves hepatocyte and liver sinusoidal endothelial cell (LSEC) viability after cold storage.
  • Images of mitochondrial uptake were captured by confocal microscopy (Nikon AXR), donor mitochondria were stained with MitoTrackerRed CMXROS (200 nm), and endogenous mitochondria were dyed with MitoTracker Green (100mM), and excess stain was washed off before imaging.
  • the endogenous mitochondria appear as shading that fills each liver cell whereas the donor mitochondria appear as small, scattered dots within some of the recipient liver cells.
  • FIG. 1 A Cell attachment was substantially increased with mitotherapy (mitochondrial transplantation) in hepatocytes.
  • FIG. 1 B Images of mitochondrial uptake were captured by brightfield microscopy at 20x objective (FIG. 1 B).
  • Hepatocytes, LSECs, stellate, and Kupffer liver cells were exposed to static cold storage (SCS) for 0, 1 , 3, or 5 days.
  • SCS static cold storage
  • Metabolic activity by PrestoBlue (Gokduman et al., Nanomedicine (Lond), 2018;13(11 ):1267-1284) was normalized to positive control. From left to right in the bar graph, for every time point, the cells studied are as follows: Hepatocytes, LSECs, stellate, and Kupffer liver cells (FIG. 1 C).
  • ATP luminescence (Fu et al., Proc Natl Acad Sci U S A., 2013;110(18) :7288-93) for hepatocytes, LSECs, stellate, and Kupffer liver cells exposed to SCS for 0, 1 , 3, or 5 days. From left to right in the bar graph, for every time point, the cells studied are as follows: Hepatocytes, LSECs, stellate, and Kupffer liver cells (FIG. 1 D).
  • FIG. 2A - FIG. 2G show that mitochondria can be transplanted by infusion during machine perfusion and leads to increased oxygen uptake and reduced edema.
  • FIG. 3A - FIG. 3F show results of experiments in which mitochondria were transplanted with the purpose of alleviating warm ischemic injury in liver cells.
  • Hepatocytes were exposed to 2 hours of warm ischemic conditions (FIG. 3A - FIG. 3C).
  • ATP production in cells was compared between the no treatment condition and the mitotherapy condition (FIG. 3A).
  • Metabolic activity was unchanged between hepatocytes of the no treatment condition and the mitotherapy condition (FIG. 3B).
  • FIG. 3C There was a decrease in caspase levels in hepatocytes treated with mitotherapy.
  • LSECs were exposed to 2 hours of warm ischemic conditions (FIG. 3D - FIG. 3F).
  • FIG. 4A - FIG. 4K show that mitochondria improve whole liver viability after warm ischemia. Livers were stored in warm ischemic conditions for 1 .5 hours (FIG. 4A - FIG. 4F). Lactate outflow during perfusion was compared between the vehicle and mitotherapy groups (FIG. 4A). Portal vein resistance was compared between the vehicle and mitotherapy groups (FIG. 4B). A substantial decrease in weight gain was observed in the mitotherapy group (FIG. 4C). An increase in oxygen uptake was observed in the mitotherapy group (FIG. 4D). A decrease in potassium outflow was observed in the mitotherapy group (FIG. 4E).
  • ALT alanine transaminase
  • FIG. 4F Level of alanine transaminase (ALT), a liver injury marker, decreased in the mitotherapy group
  • FIG. 5A - FIG. 5D show mitochondrial isolation protocol design followed by results of isolated mitochondria characterization. Mitochondria were isolated from whole liver through a series of homogenization, centrifugation, and filtration (FIG. 5A). Mitochondrial respiratory health was tested using the gold standard technique, Mitochondrial Stress test (Seahorse, Agilent Technologies) to measure the oxygen consumption rate (OCR, pmol 02/min) (FIG. 5B). As shown in the table in FIG. 5C, the respiratory profile was calculated. Ideal respiratory control ratio is between 4-5, which the mitochondria fell into. Electron microscopy was used to determine the structural integrity of the mitochondria. Both outer and inner membranes were observed as intact (FIG. 5D). FIG.
  • exogenous mitochondria refers to any mitochondria that is not native to a cell, tissue, or organ in the body. Mitochondria can be isolated from a cell, tissue, or organ and then transferred into another cell, tissue, or organ in the same subject or in a different subject, thus, the mitochondria are exogenous in nature as they are not native to the tissue into which they are being transferred.
  • the exogenous mitochondria can be from a human source.
  • the exogenous mitochondria can be from a non-human mammalian source (e.g., porcine, monkey, bovine etc.).
  • the exogenous mitochondria can be obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof.
  • the exogenous mitochondria can be obtained from skeletal muscles (e.g., rectus abdominis muscle, pectoralis major muscle, gastrocnemius muscle), fibroblasts, platelets, liver, or cardiac muscles.
  • the exogenous mitochondria can be mitochondria secreted extracellularly by many cell types, including mesenchymal stem cells (MSCs), astrocytes, neural stem cells, induced pluripotent stem cells (iPSCs), platelets, adipocytes, hepatocytes, cardiomyocytes, endothelial progenitor cells, osteoblasts, and various cell lines.
  • MSCs mesenchymal stem cells
  • astrocytes astrocytes
  • neural stem cells induced pluripotent stem cells (iPSCs)
  • iPSCs induced pluripotent stem cells
  • platelets adipocytes
  • hepatocytes hepatocytes
  • cardiomyocytes endothelial progenitor cells
  • osteoblasts and various cell lines.
  • heterologous refers to any biological product (for example, protein, organelle, ceil, tissue, or organ) that is derived from a different individual.
  • the mitochondria are heterologous mitochondria.
  • the heterologous mitochondria can be from a non-human mammalian source (e.g., porcine, monkey, bovine etc.) and can be transplanted into the cell, tissue, or organ of a human or a non-human mammal other than porcine.
  • isolated tissue refers to a tissue or an organ that has been removed or isolated from the body of a subject and is present outside the body.
  • a tissue or an organ for example, heart, liver, kidney, lung, eye, muscle, brain tissue, skin etc.
  • the tissue or organ is an isolated tissue or isolated organ.
  • isolated liver refers to a liver that has been removed or isolated from the body of a subject and is present outside the body.
  • a liver can be procured from one individual and then transplanted into another individual. Once procured and before transplant, the liver is an isolated liver.
  • the term “prolonging survival” refers to increasing the length of time an isolated cell, tissue, or organ, which has been transplanted with exogenous mitochondria at any point between procurement and post-transplant into the recipient, will survive relative to an isolated cell, tissue, or organ that has not been transplanted with exogenous mitochondria.
  • the cell is an endothelial cell.
  • the cell is a liver cell.
  • the liver cell is a hepatocyte.
  • the tissue or organ is heart, liver, kidney, lung, eye, muscle, brain tissue, or skin.
  • subzero storage refers to storage at a temperature below 0 °C.
  • Subzero storage at a very low temperature e.g., -196 °C (the temperature of liquid nitrogen)
  • subzero storage may encompass supercooling, partial freezing cryopreservation, and vitrification techniques known to those skilled in the art.
  • xenotransplantation refers to transplantation of a cell, tissue, or organ from one species to another.
  • the disclosure is directed towards advancing the therapeutic potential of mitochondria for tissue or organ (e.g., heart, liver, kidney, lung, eye, muscle, brain tissue, or skin) transplantation using both in vitro and in vivo models of liver injury, improving the utilization of extended warm ischemia tissues or organs, and extending the limits of tissue or organ preservation.
  • tissue or organ e.g., heart, liver, kidney, lung, eye, muscle, brain tissue, or skin
  • End stage organ failures contribute to several deaths annually in the United States and in many cases, there is no treatment other than transplant.
  • end stage liver disease contributes to around 77,000 deaths annually in the United States (Asrani et al., J Hepatol., 2019; 70(1 ):151 -171 ).
  • To enhance the availability of tissues or organs for transplantation many transplant centers are increasingly turning to extended criteria donor organs.
  • One significant, yet underutilized group is deceased cardiac donors, also known as donors after cardiac death (DCD).
  • DCD cardiac death
  • the warm ischemic time or the period during which blood flow is halted, can cause damage to these tissues or organs.
  • the current clinical maximum for warm ischemic time is >30 minutes for liver donation (Paterno et al., Liver Transpl., 2019;25(9):1342-1352).
  • the main course of the ischemia-reperfusion cascade is facilitated by mitochondrial injury, but there is a significant gap in our knowledge about how to treat the injury to mitochondria, preventing the use of a large donor organ pool (Horvath et al., Int J Mol Sci . , 2021 ;22(6)) .
  • livers Beyond sourcing tissues or organs, maintaining and distributing tissues or organs is also limited.
  • the current method for storing and transporting organs is to place them in storage bags with preservation fluid on ice in a cooler.
  • the time limit for cold storage of livers has been 12 hours.
  • Extended criteria livers have a higher level of injury, which further limits how much preservation damage they can sustain (Maggi et al., Transplant Proc., 2014;46(7):2295-9).
  • the ability to increase storage time or rescue suboptimal livers through advanced ex vivo treatment may increase the number of livers that are able to be transplanted.
  • Mitochondrial damage during cold storage has been well documented to leading to decreased respiration and higher levels of cell death (Horvath et al., Int J Mol Sci., 2021 ;22(6); Vajdova et al., Hepatology, 2002;36(6):1543-52).
  • Current methods to address mitochondrial damage are limited (Martins et al., Int J Med Sci., 2018;15(3):248-256; Saeb-Parsy et al., Trends Mol Med., 2021 ;27(2):185-198; Xu etal., Front Pharmacol., 2021 ;12:796207) and the critical and lasting damage to native mitochondria may not be recovered by such methods.
  • An innovative approach to treating mitochondrial dysfunction is using live, isolated mitochondrial transplantation. Treatment with exogenous mitochondria may be a more effective route to addressing mitochondrial damage and rescue widespread mitophagy. Mitochondrial transplant may rescue the mitochondrial network after the structural and functional damage induced by cold storage.
  • the isolated cell, tissue, or organ can be transplanted with exogenous mitochondria at any point between procurement and post-transplant into the recipient. Exogenous mitochondria can be transplanted into the isolated cell, tissue, or organ after it has been removed or isolated from the donor. In some embodiments, the isolated cell, tissue, or organ can be transplanted with exogenous mitochondria post-transplant into the recipient.
  • Mitochondrial transfer is a stress-relieving rescue mechanism to improve ATP content, decrease apoptosis, promote repair, and decrease oxidative stress (Hayakawa etal., Nature, 2016;535(7613):551 -555; Islam et al., Nat Med., 2012;18(5):759-65) and has improved ischemic damage in complex systems of hearts, kidneys, and livers in animal models (Blitzer et al., Ann Thorac Surg., 2020;109(3):711 -719; Jabbari et al., Biochim Biophys Acta Mol Basis Dis., 2020;1866(8):165809; Ko et al., J Cell Mol Med., 2020;24(17):10088-10099), and a pediatric clinical trial for cardiogenic shock (Guariento et al., The Journal of Thoracic and Cardiovascular Surgery, 2021 ;162(3):992-1001 ).
  • a tissue or an organ for example, heart, liver, kidney, lung, eye, muscle, brain tissue, skin, etc.
  • the tissue or organ is an isolated tissue or isolated organ.
  • the isolated tissue is obtained from a DCD.
  • mitochondrial injury or other damage can happen during the warm ischemic time (WIT), or the period during which blood flow is halted.
  • WIT warm ischemic time
  • the isolated tissue is characterized by a WIT of greater than 30 minutes.
  • the isolated tissue is characterized by a WIT of greater than 45 minutes.
  • the isolated tissue is characterized by a WIT of greater than 60 minutes.
  • the isolated tissue is characterized by a WIT of from 30 minutes to 72 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 45 minutes to 48 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 60 minutes to 24 hours. In some embodiments, the isolated tissue is characterized by a WIT of greater than 72 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 72 hours to 96 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 96 hours to 120 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 120 hours to 144 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 30 minutes to 144 hours.
  • the isolated tissue can be subjected to static cold storage or subzero storage prior to the introducing of the one or more exogenous mitochondria.
  • the static cold storage or subzero storage comprises storage of the isolated tissue at a temperature of about 4° C to about - 196° C (the temperature of liquid nitrogen).
  • the static cold storage or subzero storage comprises storage of the isolated tissue at a temperature of about 4° C to about - 80° C.
  • the static cold storage or subzero storage comprises storage of the isolated tissue at a temperature of about 4° C to about - 20° C.
  • the static cold storage or subzero storage further comprises storage of the isolated tissue at the temperature for at least 24 hours.
  • the static cold storage or subzero storage comprises storage of the isolated tissue at the temperature for at least 48 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the isolated tissue at the temperature for at least 72 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the isolated tissue at the temperature for at least 96 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the isolated tissue at the temperature for at least 144 hours. In some embodiments, the static cold storage or subzero storage further comprises storage of the isolated tissue at the temperature for from about 24 hours to about 144 hours.
  • the subzero storage further comprises storage of the isolated tissue at - 196° C (e.g., using liquid nitrogen) and may encompass supercooling, partial freezing cryopreservation or vitrification. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 1 month. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 3 months. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 6 months. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 9 months.
  • the isolated tissue at - 196° C e.g., using liquid nitrogen
  • the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 1 month. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 3 months. In some embodiments, the subzero
  • the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 1 year. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 2 years. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 3 years. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for about 1 month to about 3 years or more.
  • the currently disclosed study was performed to investigate the promise of mitochondrial transplant as a targeted option for mitochondria-driven pathologies.
  • the objective of this study was to advance the therapeutic potential of mitochondria for tissue or organ (e.g., liver, heart, kidney, lung, eye, muscle, brain tissue, or skin) transplantation using both in vitro and in vivo models of liver injury, to improve the utilization of extended warm ischemia organs, and to extend the limits of tissue or organ preservation.
  • tissue or organ e.g., liver, heart, kidney, lung, eye, muscle, brain tissue, or skin
  • mitochondrial transplantation can also be useful for heart, kidney, and lung transplantation. Further, successful mitochondrial transplantation would inform the ischemia reperfusion field, and help develop mitochondrial therapy applications to a wide range of conditions from aging to muscular dystrophy, blindness (Ng and Turnbull; J Neurol., 2016;263:179-191 ), and acute conditions such as stroke (Hayakawa et al., Nature, 2016;535(7613):551 -555), and traumatic brain injury (Kong et al., Mil Med Res., 2022;9:2), among others.
  • Mitochondria can be isolated from a cell, tissue, or organ and then transferred into another cell, tissue, or organ in the same subject or in a different subject, thus, the mitochondria are exogenous in nature as they are not native to the tissue into which they are being transferred.
  • the exogenous mitochondria can be from a human source.
  • the exogenous mitochondria can be from a non-human mammalian source (e.g., porcine, monkey, bovine etc.).
  • the exogenous mitochondria can be obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof.
  • the exogenous mitochondria can be obtained from skeletal muscles (e.g., rectus abdominis muscle, pectoralis major muscle, gastrocnemius muscle), fibroblasts, platelets, liver, or cardiac muscles.
  • the exogenous mitochondria can be mitochondria secreted extracellularly by many cell types, including mesenchymal stem cells (MSCs), astrocytes, neural stem cells, platelets, adipocytes, hepatocytes, cardiomyocytes, endothelial progenitor cells, osteoblasts, and various cell lines.
  • MSCs mesenchymal stem cells
  • astrocytes astrocytes
  • neural stem cells e.g., platelets, adipocytes, hepatocytes, cardiomyocytes, endothelial progenitor cells, osteoblasts, and various cell lines.
  • the source of the mitochondria is the same as that of the source of the organ that will be transplanted. In some embodiments, the source of the mitochondria is independent of the source of the organ that will be transplanted. In some embodiments, the source of the mitochondria is independent of the source of the liver that will be transplanted. In some embodiments, the mitochondria can be isolated from the organ donor. In some embodiments, the mitochondria can be isolated from the organ donor’s muscle. In some embodiments, the mitochondria can be isolated from the organ donor’s liver. In some embodiments, the mitochondria can be isolated from the recipient. In some embodiments, the mitochondria can be isolated from the recipient’s liver. In some embodiments, the mitochondria can be isolated from the recipient’s muscle.
  • the mitochondria can be isolated from an individual who is neither the donor nor the recipient. In some embodiments, the mitochondria can be isolated from a different species and transplanted into a human’s isolated issue. In some embodiments, the mitochondria can be isolated from a pig or a monkey. In some embodiments, healthy mitochondria can be isolated from damaged tissue. In some embodiments, healthy mitochondria can be isolated from healthy tissue. In some embodiments, the mitochondria can be freshly isolated before transplant. In some embodiments, the mitochondria can be isolated and stored before transplant.
  • Example 1 Investigation of the impact of mitochondrial transplantation on long-term liver preservation The aim of this study was to investigate the impact of mitochondrial transplantation on long-term liver preservation.
  • Mitochondria were isolated from 200-300 mg of Lewis rat liver tissue and resuspended in isolation solution [210 mannitol, 70 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA at pH 7.3 KOH, in DI water] (Preble et al., Journal of Visualized Experiments : JoVE, 2014(91 ):51682). The liver was sectioned into 50 mg portions and minced with a razor while in isolation solution.
  • each portion was homogenized in 5 mL of fresh isolation solution using a gentle MACS dissociator in C tubes (Miltenyi Biotec) and combined in 50 mL conicals with a final concentration of 5% BSA, added after homogenization to avoid froth. Next, this homogenate was centrifuged for 10 minutes at 800 g. Supernatant was strained through subsequent 40 urn, 40 urn, and 10 urn filters in 50 mL conicals. Next, the solution was spun down at 5,500 g for 10 minutes in 15 mL conicals.
  • the fluffy part of the pellet was removed by careful mixing using a 1 mL pipet approximately 1 cm above the pellet, for 5 cycles, before aspiration of waste and replacement with 5 mL of fresh solution. A final centrifugation was performed at 5,500 g for 5 minutes.
  • the crude mitochondrial pellet was resuspended in 1 mL of mitochondrial isolation solution. Total mitochondrial protein content was determined using a simple Bradford assay.
  • Oxygen consumption ratio (OCR) and respiratory complex activity of donor mitochondria were analyzed using a Seahorse XF Mini Analyzer (Divakaruni et al., Current Protocols in Toxicology, 2014;60(1 ):25.2.1 - 25.2.16). Transmission electron microscopy was used to evaluate the ultrastructure of mitochondria as additional quality control (Franko et al., PLoS One., 2013;8(12):e82392).
  • hepatocytes Primary hepatocytes, liver sinusoidal endothelial cells (LSECs), stellate, and Kupffer cells were isolated from Lewis rats. Cells were kept at 37°C, 5% CO2 for 24 hours after seeding. Then, media was replaced with University of Wisconsin (UW) solution for SCS and cells were kept at 4°C for 24 hours. Cells were recovered with William’s E medium (WE) for hepatocytes, endothelial growth medium-2 (EGM2) for LSECs, and DMEM supplemented with 10% fetal bovine serum (FBS) for stellate and Kupffer cells and kept in 37°C, 5% CO2 for 24 hours before assays were performed.
  • WE William’s E medium
  • ECM2 endothelial growth medium-2
  • FBS fetal bovine serum
  • mitochondria were diluted to a concentration of 300 pg/mL in the cell’s respective media and co-incubated with the cells for 90 minutes followed by several wash steps with PBS and a final cell respective media (Caicedo et al., 2015).
  • the vehicle control was mitochondria isolation solution added in same volume as mitochondria.
  • Mitochondria were incubated with 100 nM MitoTracker CMXRos fluorescent dye for 10 minutes at room temperature. Then, mitochondria were spun out of the solution at 5,500 g for 10 minutes before being resuspended in the cell’s respective media. Mitochondria were transplanted as described above. To visualize cell uptake, we performed fluorescent microscopy at 20x objective.
  • the ex vivo perfusion protocol has been previously described (Tessier et al., Nat Commun., 2022;13(1 ):4008) in brief. Livers were procured from rats and the portal vein was cannulated. Directly after procurement, the liver was flushed with cold University of Wisconsin (UW) solution and placed in a culture dish in cold UW. On ice, the liver was transferred to a 4°C refrigerator for 72 hours covered in UW. After storage, the liver was then transferred to the perfusion basin that was warmed to 37°C with a WE- based media. To remove the UW solution, the livers were flushed with 30 mL of room temperature WE medium before attachment to the perfusion system.
  • UW cold University of Wisconsin
  • the portal vein cannula previously secured in place, was attached to the inflow tubing and perfusion was initiated. A mechanical rotor pump was used to circulate the perfusing fluid.
  • donor mitochondria were injected directly into the portal vein cannula after 30 minutes of machine perfusion at a concentration of 1 x 10 9 mitochondria per gram wet weight of tissue, an amount chosen based on previous groups evaluations (Orfany et al., J Vase Surg., 2020;71 (3):1014-1026).
  • Livers were procured and flushed with room temperature PBS. They were placed in small plastic bags filled with PBS before being placed in a 37°C water bath for 1 .5 or 2 hours. Mitochondria were delivered as a post-warm ischemia (post-WI) treatment through the portal vein, as described above. The perfusion was run for 4 hours and followed by end-point assessments.
  • post-WI post-warm ischemia
  • Perfusate was collected every 30 minutes to be measured through blood gas analysis (Blood Gas Analyzer 500, Siemens) and for alanine transaminase/aspartate aminotransferase (ALT/AST) (Piccolo Xpress, Abaxis). Weight change was tracked before and after machine perfusion to monitor edema. Pressure through the portal vein was measured using a pressure monitor in line with the portal vein, which was later used to calculate resistance through the liver. At the end of perfusion, liver tissue biopsies were snap frozen for NMR to evaluate metabolite content and tissue biopsies were collected in formalin for histopathological assessment (H&E, Masson’s trichrome, TUNEL).
  • Mitochondria were delivered in 1 mL of vehicle followed by two hours of machine perfusion at 37°C.
  • the positive control group was flushed with 30 mL of 200 nm MitoTracker Red CMXRos which was allowed to incubate for 20 minutes before a 30 mL flush of saline.
  • the negative control group was flushed with 30 mL of saline.
  • the fluorescence intensity of donor mitochondria was imaged by the SPECTRAL AMI imaging system (Spectral Instruments) by the Mouse Imaging Program at MGH. Donor mitochondria were stained by 100 nm MitoTracker Red CMXRos.
  • Mitotherapy improves metabolic function in primary liver cells
  • Donor mitochondria were stained with MitoTrackerRed CMXROS for detection, with staining demonstrating successful uptake (FIG. 1 A). It has been previously demonstrated that after 24 hours of hypothermic storage in UW, rat hepatocytes display high viability one hour after rewarming, however, viability drops significantly during the first day of long-term culture with significant cell death at 3 days (Usta et al., PLoS One., 2013;8(7):e69334). The damage from hypothermic storage was quantified in additional liver cell types, with findings of universally decreased metabolic activity, ATP levels, and increased caspase expression, though each cell type had a varied injury profile (FIG. 1 C - FIG. 1 E).
  • Hepatocytes were targeted as they are the majority cell type in livers and have clear damage with increasing cold storage. At 24 hours post-rewarming, hepatocyte viability dropped past 50% below fresh, healthy controls (FIG. 1 C - FIG. 1 E). Mitochondrial transplant substantially improved cell metabolic rate following SCS (FIG. 1 F - FIG. 1 H) and gross morphology (FIG. 1 B) compared to the negative control. Surprisingly, the cells were able to recover enough to match the metabolic rate of the positive control, which were kept in standard culture conditions throughout the experiment. Mitochondrial transplantation improved cell survival and attachment. A marked improvement in cell attachment was observed with mitochondrial transplant, demonstrated by increased density of cells (FIG. 1 B).
  • LSECs Liver sinusoidal endothelial cells
  • FIG. 1A - FIG. 1K show that a mitochondrial transplant improves hepatocyte and LSEC viability after cold storage.
  • Images of mitochondrial uptake were captured by confocal microscopy (Nikon AXR), donor mitochondria were stained with MitoTrackerRed CMXROS (200 nm), and endogenous mitochondria were dyed with MitoTracker Green (1 OOmM), and excess stain was washed off before imaging.
  • the endogenous mitochondria appear as shading that fills each liver cell whereas the donor mitochondria appear as small, scattered dots within some of the recipient liver cells.
  • FIG. 1 A Cell attachment was substantially increased with mitotherapy in hepatocytes.
  • FIG. 1 B Images of mitochondrial uptake were captured by brightfield microscopy at 20x objective (FIG. 1 B).
  • Hepatocytes, LSECs, stellate, and Kupffer liver cells were exposed to static cold storage (SCS) for 0, 1 , 3, or 5 days.
  • SCS static cold storage
  • Metabolic activity by PrestoBlue (Gokduman et al., Nanomedicine (Lond), 2018;13(11 ):1267-1284) was normalized to positive control. From left to right in the bar graph, for every time point, the cells studied are as follows: Hepatocytes, LSECs, stellate, and Kupffer liver cells (FIG. 1 C).
  • ATP luminescence (Fu et al., Proc Natl Acad Sci U S A., 2013;110(18):7288-93) for hepatocytes, LSECs, stellate, and Kupffer liver cells exposed to SCS for 0, 1 , 3, or 5 days. From left to right in the bar graph, for every time point, the cells studied are as follows: Hepatocytes, LSECs, stellate, and Kupffer liver cells (FIG. 1 D).
  • Mitotherapy alleviates cold storage injury in rat livers during ex vivo machine perfusion
  • FIG. 2A - FIG. 2G show that mitochondria can be transplanted by infusion during machine perfusion and leads to increased oxygen uptake and reduced edema.
  • Mitotherapy improves metabolic function in primary liver cells
  • FIG. 3B While metabolic activity (FIG. 3B) and ATP production (FIG. 3A) in hepatocytes were similar regardless of mitotherapy treatment, markers of cell death decreased in hepatocytes that received mitotherapy after two hours of warm ischemia (FIG. 3C). In contrast, while LSECs exhibited a slight increase in ATP levels (FIG. 3D), their cell death activity remained unchanged (FIG. 3F).
  • FIG. 3A - FIG. 3F show results of experiments in which mitochondria were transplanted with the purpose of alleviating warm ischemic injury in liver cells.
  • Hepatocytes were exposed to 2 hours of warm ischemic conditions (FIG. 3A - FIG. 3C).
  • ATP production in cells was compared between the no treatment condition and the mitotherapy condition (FIG. 3A).
  • Metabolic activity was unchanged between hepatocytes of the no treatment condition and the mitotherapy condition (FIG. 3B).
  • FIG. 3C There was a decrease in caspase levels in hepatocytes treated with mitotherapy.
  • LSECs were exposed to 2 hours of warm ischemic conditions (FIG. 3D - FIG. 3F).
  • Mitotherapy alleviates warm ischemic injury in rat livers during ex vivo machine perfusion
  • FIG. 4A - FIG. 4K show that mitochondria improve whole liver viability after warm ischemia. Livers were stored in warm ischemic conditions for 1 .5 hours (FIG. 4A - FIG. 4F). Lactate outflow during perfusion was compared between the vehicle and mitotherapy groups (FIG. 4A). Portal vein resistance was compared between the vehicle and mitotherapy groups (FIG. 4B). A substantial decrease in weight gain was observed in the mitotherapy group (FIG. 4C). An increase in oxygen uptake was observed in the mitotherapy group (FIG. 4D). A decrease in potassium outflow was observed in the mitotherapy group (FIG. 4E).
  • ALT alanine transaminase
  • FIG. 4F Level of alanine transaminase (ALT), a liver injury marker, decreased in the mitotherapy group
  • Isolated mitochondria from whole Lewis rat livers were evaluated via respirometry and electron microscopy (FIG. 5A - FIG. 5D).
  • Donor mitochondria were stained with MitoTrackerRed CMXROS for detection, with staining demonstrating successful uptake (FIG. 1 A) and dose-dependence of donor mitochondria incorporation into the cells (FIG. 6A).
  • FIG. 5A - FIG. 5D show mitochondrial isolation protocol design followed by results of isolated mitochondria characterization. Mitochondria were isolated from whole liver through a series of homogenization, centrifugation, and filtration (FIG. 5A). Mitochondrial respiratory health was tested using the gold standard technique, Mitochondrial Stress test (Seahorse, Agilent Technologies) to measure the oxygen consumption rate (OCR, pmol 02/min) (FIG. 5B). As shown in the table in FIG. 5C, the respiratory profile was calculated. Ideal respiratory control ratio is between 4-5, which the mitochondria fell into. Electron microscopy was used to determine the structural integrity of the mitochondria. Both outer and inner membranes were observed as intact (FIG. 5D).
  • a method of prolonging survival of tissue comprising introducing into the tissue one or more exogenous mitochondria.
  • a method of prolonging survival of a cell comprising introducing into the cell one or more exogenous mitochondria.
  • a method of transplanting an isolated tissue into a subject in need of replacement of the tissue comprising: a) introducing, into a cell of the isolated tissue, one or more exogenous mitochondria; b) providing the subject with the tissue resulting from (a) in place of the subject’s endogenous tissue.
  • a method of transplanting an isolated tissue into a subject in need of replacement of the tissue comprising providing the subject with the isolated tissue in place of the subject’s endogenous tissue, wherein prior to the providing, one or more exogenous mitochondria have been introduced into a cell of the isolated tissue. 5. The method of any one of embodiments 1 , 3, and 4, wherein the tissue is a liver, a heart, a kidney, or a lung.
  • liver cell is a hepatocyte.
  • ischemic liver injury primary non-function (PNF), early allograft dysfunction (EAD), small-for-size syndrome (SFSS), resection for malignant or benign disease, autoimmune hepatitis, cystic fibrosis, cholestatic liver diseases, primary sclerosing cholangitis, primary biliary cirrhosis, inborn errors of metabolism, metabolic diseases, sickle cell hepatopathy, erythropoietic protoporphyria (EPP) hepatopathy, congestive hepatopathy, metabolic dysfunction-associated steatotic liver disease (MAFLD), metabolic dysfunction-associated steatohepatitis (MASH), alcoholic liver disease, hemochromatosis, Alagille syndrome, Wilson's disease, viral hepatitis, alpha-1 antitrypsin deficiency, drug-induced liver injury (DILI),
  • PNF primary non-function
  • EAD early allograft dysfunction
  • SFSS small-for-size syndrome
  • An ex vivo tissue transplant comprising an isolated tissue that comprises one or more heterologous mitochondria.

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Abstract

L'invention concerne des procédés pour améliorer la conservation et l'utilisation de tissus ou d'organes par transplantation mitochondriale.
PCT/US2024/052800 2023-10-24 2024-10-24 Transplantation mitochondriale pour la conservation de tissus à long terme Pending WO2025090754A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180057610A1 (en) * 2016-01-15 2018-03-01 Children's Medical Center Corporation Therapeutic Use of Mitochondria and Combined Mitochondrial Agent
US20190070211A1 (en) * 2016-02-26 2019-03-07 Beth Israel Deaconess Medical Center, Inc. Niacinamide (nam) in ischemic tissue injury
WO2023003418A1 (fr) * 2021-07-23 2023-01-26 주식회사 파이안바이오테크놀로지 Composition destinée à la conservation de cellules, de tissus ou d'organes, comprenant des mitochondries isolées, et son utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180057610A1 (en) * 2016-01-15 2018-03-01 Children's Medical Center Corporation Therapeutic Use of Mitochondria and Combined Mitochondrial Agent
US20190070211A1 (en) * 2016-02-26 2019-03-07 Beth Israel Deaconess Medical Center, Inc. Niacinamide (nam) in ischemic tissue injury
WO2023003418A1 (fr) * 2021-07-23 2023-01-26 주식회사 파이안바이오테크놀로지 Composition destinée à la conservation de cellules, de tissus ou d'organes, comprenant des mitochondries isolées, et son utilisation

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