Methods for improving cardiac graft function

Abstract

Provided are methods for reducing ischemia-reperfusion injury in a cardiac graft by administering to the graft ex vivo a composition comprising mitochondria-rich extracellular vesicles. Also provided are related methods for improving the function of a cardiac graft and related compositions and methods.

Description

METHODS FOR IMPROVING CARDIAC GRAFT FUNCTIONBACKGROUND

[0001] Despite improved survival rates for heart failure patients over the past three decades, heart failure remains a leading cause of hospital admission in the US with a low 5-year survival rate of about 50%. Cardiac tissue from patients with dilated, hypertrophic, and ischemic cardiomyopathy exhibits mitochondrial structural abnormalities and diminished ATP production. Insufficient energy generation in the damaged tissue results in loss of cardiomyocyte contractility, myocardial pump dysfunction, and ultimately decompensated heart failure. Current therapy includes beta-blockade, diuresis, and renin-angiotensin-aldosterone antagonism. However this pharmacotherapy does not address the mitochondrial abnormalities in heart failure which are directly related to disease progression. In addition to having reduced capacity to generate ATP, the distressed mitochondria become a major source of reactive oxygen species (ROS) and promote cell death through opening of mitochondrial permeability transition pores.

[0002] Transplantation of isolated mitochondria extracted from homogenized healthy cells has been demonstrated to reduce infarct size in murine models of myocardial injury (MI). However, the beneficial effects of this approach are reportedly limited in part due to damage to the isolated mitochondria and failure of the isolated mitochondria to enter target cardiomyocytes.

[0003] Significant challenges remain to be overcome before mitochondrial transplantation therapy can be successfully translated into clinical practice. These challenges include a need for improved methods for mitochondrial isolation that preserve the functional characteristics of the organelles as well as improved methods for delivery of mitochondria to target cells. Current methods for isolating mitochondria include extracting the mitochondria from healthy donor cells by disrupting the cell membrane and separating the mitochondria from other cellular components. Common separation methods include density gradient centrifugation using sucrose or percoll and affinity -based purifications. Current delivery methods include direct injection or the use of a delivery vehicle that may comprise, for example, cell-penetrating peptides or polymers.

[0004] A particular challenges arise with use of Donation after Circulatory Death (DCD) cardiac grafts. DCD grafts provide a much needed alternative source of cardiac grafts to meet the existing need, but the longer ischemic time experienced by DCD grafts contributes toischemia-reperfusion injury and significantly decreases myocardial function and post-transplant graft function. The present invention addresses the need for improved methods of reducing ischemia-reperfusion injury in grafts.BRIEF SUMMARY

[0005] The present disclosure provides methods for reducing ischemia-reperfusion injury and enhancing the function of cardiac grafts by administering mitochondria-rich extracellular vesicles to the grafts ex vivo. Clinical application of the methods described here is expected to improve the prognosis of cardiac transplant patients, including those receiving DCD donor hearts, by enhancing the function and engraftment rate of the donor heart.

[0006] In one aspect, provided is a method of reducing ischemia-reperfusion injury in a cardiac graft comprising administering to the graft ex vivo a composition comprising mitochondria-rich extracellular vesicles. In one aspect, provided is a method for improving the function of a cardiac graft comprising administering to the graft ex vivo a composition comprising mitochondria-rich extracellular vesicles.

[0007] Also provided is a composition comprising mitochondria-rich extracellular vesicles for use in a method of reducing ischemia-reperfusion injury in a cardiac graft by a method including ex vivo administration of the composition to the graft. In one aspect, provided is a composition comprising mitochondria-rich extracellular vesicles for use in a method of improving the function of a cardiac graft by a method including ex vivo administration of the composition to the graft.

[0008] In accordance with any of the foregoing, the method may also include where the composition is administered to the graft within at least about 1 to 2 hours prior to transplantation of the graft into a recipient. The method may also include where the graft has been subjected to ischemic conditions ex vivo prior to administration of the composition. The method may also include where the ischemic conditions comprise storage in an aqueous buffer for a period of time. The method may also include where the period of time is from about 3-20 hours, about 3-5 hours, about 3-10 hours, about 10-20 hours, or about 12-18 hours. The method may also include where the composition is administered to the graft parenterally. The method may also include where the composition is administered via intramyocardial, intracoronary, intravenous, intra-arteriole, or intraventricular injection. The method may also include where the vesicles express one or both of connexin 43 and endothelial marker CD31. The method mayalso include wherein the vesicles comprise functional mitochondria. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0009] FIG. 1A is a schematic representation of an in vitro experiment testing whether human mitochondria rich extracellular vesicles (MEVs) could successfully deliver intact functional human mitochondria to rat cardiac tissue which had been removed from the donor animal and stored in vitro in University of Wisconsin (UW) solution for 17 hours at 4 °C.

[0010] FIG. IB is a bar graph showing dose-dependent expression of human mitochondrial genes in rat heart tissue following injection of either 108or 1010MEVs into the coronary artery of hearts ex vivo.

[0011] FIG. 2A is a schematic representation of an in vitro experiment testing whether MEVs could improve cardiac graft health and function following transplantation where the MEVs are injected into the cardiac tissue following its removal from a donor animal and storage in vitro in University of Wisconsin (UW) solution for 17 hours at 4 °C.

[0012] FIG. 2B is a bar graph showing ATP levels (ng / ml) in untreated control and MEV treated cardiac grafts 3 hours after transplantation.

[0013] FIG. 2C is a bar graph showing troponin-I levels (ng / ml) of recipient rats that received untreated control or MEV treated cardiac grafts. The time point is 3 hours after transplantation.

[0014] FIG. 3 shows representative transmission electron microscopy images of mitochondria in cardiomyocytes. Panel A shows normal structure 30 minutes after procurement. Panels B and C show severe mitochondrial swelling in both MEV-treated (C) and untreated (B) control hearts preserved in University of Wisconsin (UW) solution for 18 hours at 4 °C. Panel D shows further structural changes in the mitochondria untreated (D) control hearts 3 hours after transplantation. Panel E shows that the structural changes were attenuated in MEV-treated hearts.

[0015] FIG. 4A shows representative manganese-enhanced MRI (MEMRI) images acquired 7 days after transplantation

[0016] FIG. 4B is a bar graph showing T1 CNR (left bar in each pair) and T2 CNR (right bar in each pair) for positive control hearts (PC), mitochondrial EV-treated hearts (MEV), and untreated hearts (Control).

[0017] FIG. 5A shows representative Tunel staining images acquired 3 hours after transplantation with donor heart preserved for 18 hours and Tunel positive Ratio. Representative Tunel staining image of control (left panel) and MEV group (right panel). TUNEL-positive cells indicate cell death.

[0018] FIG. 5B is a bar graph showing average Tunel positive cell rate (Tunel & DAPI double positive / DAPI positive) in a randomly selected 6 fields of view from the middle layer of myocardium at the papillary muscle level was significantly lower in the MEV treatment group (p<0.05), suggesting a protective effect of MEV treatment. Values are means ± SD, *P < .05DETAILED DESCRIPTION

[0019] Current constraints on donor heart preservation time often result in organ mismatch or even the discarding of organs, depriving potential recipients of life-saving transplants. Cardiac grafts from Donation after Circulatory Death (DCD) are known to be more challenging due to the longer ischemic time experienced by these grafts. The constraints on donor heart preservation time are primarily due to the need to limit ischemia-reperfusion injury, which compromises the integrity of the mitochondrial function of the cardiac graft. Ischemiareperfusion injury is associated with mitochondrial structural and functional integrities, which significantly decreases myocardial function and post-transplant graft function.

[0020] The present disclosure provides proof of concept evidence that mitochondrial transplantation therapy in the form of mitochondria-rich extracellular vesicles administered to the graft can be used to reduce ischemia-reperfusion injury and enhance function of cardiac grafts, including grafts that have been subjected to a period of ischemia, for example by storage in an appropriate solution for a period of time following removal from the donor animal.

[0021] Accordingly, the disclosure provides methods for reducing ischemia-reperfusion injury in a cardiac graft subjected to ischemic conditions, the method comprising administering to the graft an effective amount of mitochondria-rich extracellular vesicles within at least about 1 to 2 hours prior to transplantation of the graft into a recipient. In aspects, the graft has been subjected to ischemic conditions for a period of time, for example, about 3-20 hours, about 3-5 hours, about 3-10 hours, about 10-20 hours, or about 12-18 hours.

[0022] Also provided are methods for improving the function and / or engraftment of a cardiac graft, the methods comprising administering to the graft an effective amount of mitochondria- rich extracellular vesicles prior to transplantation of the graft into a recipient. In aspects, themitochondria-rich extracellular vesicles are administered within at least about 1 to 2 hours prior to transplantation of the donor cardiac graft into a recipient. In aspects, the donor graft is a DCD donor cardiac graft.

[0023] The terms “donor” and “recipient” refer to the subject that provides the graft and the subject that receives the graft, respectively. In aspects, the mitochondria-rich extracellular vesicles are heterologous to the recipient. In aspects, the mitochondria-rich extracellular vesicles are autologous to the recipient. In aspects, the donor and recipient are each a mammal. In aspects, the donor and recipient are each human.

[0024] In accordance with aspects of any of the foregoing methods, improved function of a cardiac graft may include one or more of improved left ventricular ejection fraction (LVEF), improved left ventricular end-diastolic volume (LVEDV), improved viability, reduced scar formation, reduced fibrosis and reduced apoptosis in the graft.

[0025] Also provided are methods for treating a subject having myocardial injury, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising a plurality of mitochondria-rich extracellular vesicles as described herein. In aspects, the myocardial injury is a result of myocardial ischemia, cardiac surgery, or circulatory arrest. In aspects, the myocardial injury is myocardial ischemia-reperfusion (IR) injury.

[0026] In accordance with aspects of any of the foregoing methods, the vesicles are administered parenterally. In aspects, the vesicles are administered via intramyocardial, intracoronary, intravenous, intra- arteriole, or intraventricular injection.

[0027] In accordance with aspects of any of the foregoing methods, treating may include one or more of improved left ventricular ejection fraction (LVEF), improved left ventricular end- diastolic volume (LVEDV), improved viability, reduced scar formation, reduced fibrosis and reduced apoptosis in the cardiac tissue.

[0028] In accordance with aspects of any of the foregoing methods, the subject is a mammal. In aspects, the subject is a human.

[0029] Also provided are compositions or pharmaceutical compositions comprising a plurality of mitochondria-rich extracellular vesicles of a donor cell as described herein, for use in the methods described herein. The pharmaceutical compositions may be formulated with one or more pharmaceutically acceptable ingredients including, for example, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants,lubricants, stabilizers, solubilizers, and surfactants. In aspects, the pharmaceutical composition is sterile. In aspects, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier, excipient or diluent. In aspects, the carrier, excipient or diluent is a buffered aqueous solution. In one aspect, the pharmaceutical compositions may be formulated as an aqueous buffered solution having a physiological pH, e.g., a pH in the range of 7-7.4, and isotonic with physiological fluids such as blood, serum, or plasma. In aspects, the pharmaceutical is composition is sterile.

[0030] The mitochondria-rich extracellular vesicles described herein are nanosized lipid membrane-bound vesicles released from donor cells. The term “extracellular vesicle” or “EV” is used herein in accordance with its ordinary and customary meaning in the biomedical arts to refer to nanosized, membrane-bound vesicles released from cells. In organisms, extracellular vesicles may transport cargo, including DNA, RNA, and proteins, between cells as a form of intercellular communication. Different types extracellular vesicles, including microvesicles and exosomes, have been characterized based on their biogenesis or release pathways. The content of extracellular vesicles may include lipids, nucleic acids, proteins, and organelles from donor cells. Microvesicles bud directly from the plasma membrane, are typically 100 nanometers (nm) to 1 micrometer (um) in size, and contain cytoplasmic cargo. Exosomes are formed by a fusion of multivesicular bodies and the plasma membrane in which multivesicular bodies release smaller vesicles (exosomes) whose diameters typically range from 40 to 120 nm.

[0031] The mitochondria-rich extracellular vesicles described herein may be collected from cell culture medium of donor cells, for example by differential centrifugation (e.g., 10,000xg for 30 min). In aspects, the extracellular vesicles may be isolated from donor cell culture medium by a method comprising one or more of differential centrifugation, ultrafiltration, density gradient / cushion centrifugation, and immunoaffinity-based capture. In aspects, the cell culture medium is a conditioned medium. The term conditioned medium refers to medium in which donor cells have been cultured for a period of time, for example a period of from 1 to 14 days.

[0032] In aspects, the donor cells are peripheral blood mononucleocyte cells (PBMCs). PBMCs may be extracted from whole blood using art recognized methods, for example a Ficoll gradient centrifugation method which separates blood into a top layer of plasma, followed by a layer of PBMCs and a bottom fraction including neutrophils, eosinophils and erythrocytes.

[0033] In aspects, the donor cells are induced pluripotent stem cells (iPSC) derived from a PBMC fraction of donor blood. Pluripotency may be induced in the PBMC in accordance with art-recognized methods which include exposing cells cultured in vitro to specific factors that induce pluripotency, for example as described in Takahashi et al., Cell. (2006) 126 (4): 663-76. In aspects, pluripotency may be induced by exposing the PBMC to Klf4-Oct3 / 4-Sox2 (KOS) and L-Myc.

[0034] In aspects, the donor cells are iPSC that have been differentiated in vitro into cardiomyocytes. In an aspect, the iPSC are differentiated into cardiomyocytes by culturing the iPSC in a base medium, such as Roswell Park Memorial Institute (RPMI) 1640, in the absence of insulin and supplemented with a combination of factors including a B-27™ Supplement, recombinant human albumin, L-asorbic acid 2-phosphate, lactate, the GSK3 inhibitor, CH1R99021, and the Wnt inhibitor, Wnt-C59. In aspects, the methods include treatment with CHIRR99021 for 2 or 3 days followed by treatment with Wnt-C59 for 2 days.

[0035] Accordingly, in aspects the mitochondria-rich extracellular vesicles are vesicles of cardiomyocytes or vesicles of iPSC. In aspects, the vesicles express one or more cardiomyocyte marker proteins such as Connexin 43 and endothelial marker CD31.

[0036] The present inventors previously developed a mitochondrial transplantation therapy using extracellular vesicles secreted from autologous induced pluripotent stem cell (iPSC)- derived cardiomyocytes, as described in W02020232301.

[0037] Here, using a heterotopic rat heart transplant model, the present disclosure demonstrates that human mitochondria-rich extracellular vesicles (MEVs) injected directly into a cardiac graft just prior to transplantation but following 17 hours of storage can substantially reduce myocardial injury and improve graft function.

[0038] An initial experiment was performed to test whether human mitochondria-rich extracellular vesicles (MEVs) could successfully deliver intact functional human mitochondria to murine cardiac tissue where the heart had been removed from the donor animal and stored in vitro in University of Wisconsin (UW) solution for 17 hours at 4 °C. FIG. 1A shows a schematic representation of the experiment. The heart is removed from the donor animal and stored in vitro in University of Wisconsin (UW) solution for 17 hours at 4 °C followed by either sham injection for untreated controls or injection with a dose of 108MEVs or 1010MEVs. Cardiac tissue was then assayed for expression of human mitochondrial genes.

[0039] Rat cardiac tissue was assayed by quantitative PCR one hour after MEV injection. As illustrated in FIG. IB, the data show a dose-dependent increase in the expression of human mitochondrial genes for the two doses of MEVs administered.

[0040] Another experiment was conducted to test the effects of MEVs on a cardiac transplant that had been stored after removal from the donor. Similar to the in vitro experiment discussed above, donor heart was removed and stored in vitro in University of Wisconsin (UW) solution for 17 hours at 4 °C followed by either sham injection for untreated controls or injection with MEVs. The donor heart was then transplanted in the recipient animal. Cardiac MRI was used to confirm the spontaneous contraction of the heart and blood flow into the right ventricle at three hours post-transplantation. Cardiac tissue was also visually inspected for the presence of human mitochondria by detecting MitoTracker™ (MT) labelled mitochondria according to the manufacturer's instructions (Thermo Fisher Scientific). At 3 hours post-transplantation, MT positive mitochondria were detected throughout the entire heart. A schematic representation of the experiment is provided in FIG. 2A.

[0041] Graft tissue was also tested for ATP at 3 hours post-transplantation. As illustrated in FIG. 2B, MEV-treated grafts had significantly (p=0.0095) higher ATP levels than untreated control grafts.

[0042] Recipient serum troponin I levels were also assayed at 3 hours post-transplantation and found to be significantly lower (p<0.0001) in recipients that had received the MEV-treated grafts, as illustrated in FIG. 2C.

[0043] When viewed by transmission electron microscopy, structural changes to the mitochondria were visible under ischemic conditions (storage in vitro in University of Wisconsin (UW) solution for 18 hours at 4 °C) for both untreated controls and MEV-treated cardiac tissue. However, at 3 hours post-transplantation, while the mitochondria of untreated control tissue continues to show structural alterations, the structural changes of the mitochondria in MEV-treated hearts are substantially reduced. Representative images are shown in FIG. 3.

[0044] FIG. 4A shows representative manganese-enhanced MRI (MEMRI) images acquired 7 days after transplantation and Contrast-to-Noise Ratio (CNR). The contrast effect was analyzed by calculating the difference in CNR between plain MRI T1 -weighted images and manganese- enhanced MRI T1 -weighted images. Noise (N) was measured as the standard deviation of the air signal (Air), background signal (Sbackground) was the mean signal intensity of skeletal musclenear the heart (M), and the target signal (Starget) was the mean signal intensity of six sections at the papillary muscle level of the donor heart. Thus, CNR was measured as

[0045] The mean signal intensity of each section was determined using the largest possible circle that did not exceed the boundaries of each section. In the Positive Control (PC) group, donor hearts from rats were preserved for less than 1 hour before transplantation. In the MEV and Control groups, donor hearts were preserved for 18 hours before transplantation.Manganese is taken up by viable cardiomyocytes; therefore, Tl CNR correlates with myocardial cell viability. Conversely, high signal intensity on T2-weighted images suggests tissue edema, meaning that T2 CNR correlates with the degree of myocardial injury.

[0046] Quantification of the CNR in Tl - and T2-weighted images is represented by the bar graph of FIG. 4B. The Tl CNR was significantly higher in the MEV group (n=4) compared to the control group (n=4). For T2-weighted images, MEV group (n=5) tended to have lower CNR compared to the control group (n=4).

[0047] These results demonstrate that MEV-treated hearts exhibited significantly higher Tl CNR, indicating better myocardial viability.

[0048] FIG. 5A illustrates Tunel staining results obtained 3 hours after transplantation, revealing that the MEV treatment group had a significantly lower rate of cell death compared to the control group. The data are quantified in the bar graph shown in FIG. 5B.

[0049] These data further support the therapeutic potential of MEV in preserving myocardial function and minimizing injury.

[0050] In summary, cardiomyocytes exposed to ischemia exhibit altered mitochondria, including alterations in structure evidenced by swelling, as well as alterations in function, evidenced by changes in gene and protein expression. Reperfusion of such altered mitochondria induces substantial additional damage leading to cell death in a process of ischemic reperfusion injury. Importantly, the data presented here show that MEV treatment attenuates this ischemic reperfusion injury.

[0051] While the invention has been described by means of specific embodiments and applications thereof, modifications and variations could be made thereto by those skilled in the art without departing from the scope set forth in the claims.

[0052] The present invention is set forth in various levels of detail. In certain instances, details not necessary for one of ordinary skill in the art to understand the invention may have been omitted.

[0053] Section headings are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0054] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. An “embodiment” may refer to an illustrative representation of a method or article in which a disclosed concept or feature may be provided or embodied, or a representation of a manner in which a concept or feature may be provided or embodied. Such illustrated embodiments are to be understood as examples (unless otherwise stated), and other manners of embodying the described concepts or features, such as may be understood by one of ordinary skill in the art upon learning the concepts or features from the present disclosure, are within the scope of the disclosure. Accordingly, disclosed embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the claimed subject matter being indicated by the appended claims, and not limited to the foregoing description or particular embodiments or arrangements described or illustrated herein. It is intended that the present subject matter covers such modifications and variations as come within the scope of the appended claims and their equivalents.

[0055] Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0056] The phrases “at least one”, “one or more”, and “and / or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0057] The term “about” when used before a numerical designation, e.g, temperature, time, amount, concentration, and such other, including a range, indicates approximations which mayvary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” means that the value may vary by + / - 10%.

[0058] The term “comprises / comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel character! stic(s)” of the claimed invention.

[0059] As used herein, a “subject” refers to a mammal, including non-human primates and humans. In aspects, the subject is a human, a non-human primate, swine, sheep, cow, equine, canine, feline or rodent, including a mouse, rat, a guinea pig or other rodent.

[0060] The terms “treatment” or “treating” as used herein pertain generally to describe the management and care of a subject, including human and animal subjects (e.g., in veterinary applications), to achieve a therapeutic effect in relation to a disease or disorder, for example, to alleviate, ameliorate, or mitigate one or more symptoms or complications of the disease or disorder, to slow the progression of the disease or disorder, including a reduction in the rate of progression or a halt in the progression, and cure of the condition. In the context of the present invention, treating may include improving mitochondrial function. In aspects, improved mitochondrial function may include increased intracellular ATP levels, and / or increased expression of PGC-Ia. In aspects, treating advanced heart failure may include reducing or ameliorating one or more symptoms of advanced heart failure including exercise intolerance, unintentional weight loss, refractory volume overload, recurrent ventricular arrhythmias, hypotension and inadequate perfusion for example manifested as low pulse pressure.

[0061] The term “effective amount” or “therapeutically effective amount” or “pharmaceutically effective amount” refers to an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example in the context of treating a subject with a disease, disorder, or condition characterized by a mitochondrial abnormality, an effective amount is an amount that, for example, provides some alleviation, amelioration, mitigation and / or decrease in one or more symptoms of the disease, disorder, or condition experienced by a subject.

[0062] Effective amounts may vary according to factors such as the disease state, age, sex and / or weight of the subject. The amount of a given therapeutic agent that will correspond to a therapeutically effective amount will vary depending upon factors including the pharmaceutical formulation, the route of administration, the type or stage of condition, disease or disorder, and subject-specific factors that may include sex, age, race, age, weight, comorbidities, etc. Methods for determining an effective dose are known in the art.

[0063] In aspects, the route of administration is an intravenous or parenteral route. Parenteral administration includes, e.g, intramyocardial, intracoronary, intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.

Claims

CLAIMSWhat is claimed is:

1. A method of reducing ischemia-reperfusion injury in a cardiac graft comprising administering to the graft ex vivo a composition comprising mitochondria-rich extracellular vesicles.

2. A method for improving the function of a cardiac graft comprising administering to the graft ex vivo a composition comprising mitochondria-rich extracellular vesicles.

3. The method of claim 1 or 2, wherein the composition is administered to the graft within at least about 1 to 2 hours prior to transplantation of the graft into a recipient.

4. The method of claim 1, wherein the graft has been subjected to ischemic conditions ex vivo prior to administration of the composition.

5. The method of claim 4, wherein the ischemic conditions comprise storage in an aqueous buffer for a period of time.

6. The method of claim 5, wherein the period of time is from about 3-20 hours, or from about 3-5 hours.

7. The method of any one of claims 1 to 6, wherein the composition is administered to the graft parenterally.

8. The method of claim 7, wherein the composition is administered via intramyocardial, intracoronary, intravenous, intra-arteriole, or intraventricular injection.

9. The method of any one of claims 1 to 8, wherein the vesicles express one or both of connexin 43 and endothelial marker CD31.

10. The method of any one of claims 1 to 9, wherein the vesicles comprise functional mitochondria.

11. A composition comprising mitochondria-rich extracellular vesicles for use in a method of reducing ischemia-reperfusion injury in a cardiac graft by a method comprising ex vivo administration of the composition to the graft.

12. A composition comprising mitochondria-rich extracellular vesicles for use in a method of improving the function of a cardiac graft by a method comprising ex vivo administration of the composition to the graft.

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