Platelet-derived mitochondria-containing extracellular vesicles for use in the treatment of ocular disorders - Patent Application 20070233633

Abstract

Platelet-derived extracellular vesicles (PEVs) containing mitochondria are used in treating ocular disorders or symptoms of ocular disorders. The PEVs are collected by obtaining blood from one or more donors, adding an anticoagulant and a buffer to the blood to form a mixture, separating the mixture into a supernatant and platelet-rich plasma (PRP), collecting the PRP, stimulating the collected PRP to displace the extracellular vesicles from the platelets in the PRP, and collecting the extracellular vesicles as PEVs.

Description

[Technical field]

[0001] The inventions disclosed and claimed herein generally relate to the use of platelet-derived mitochondria-containing extracellular vesicles (PEVs) for treating ocular disorders via delivery into the intraocular space of the eye. [Background technology]

[0002] explanation Mitochondria are membrane-bound intracellular organelles that contain their own DNA (mtDNA) and their own machinery for synthesizing RNA and proteins. Mitochondria are found in almost all eukaryotic cells and vary in number and location depending on the cell type.

[0003] Mitochondria perform numerous essential roles in eukaryotic cells, such as pyruvate oxidation, the Krebs cycle, and amino acid, fatty acid, and steroid metabolism. The primary function of mitochondria is to generate energy as adenosine triphosphate (ATP) through the electron transport chain and oxidative phosphorylation (the "respiratory chain"). Additional processes involving mitochondria include thermogenesis, calcium ion storage, calcium signaling, programmed cell death (apoptosis), and cell proliferation. Mitochondria are involved in cellular control and signaling events (i.e., Ca, ATP, and ATP-dependent regulation, among others). 2+ It has been disclosed that it plays a role in the regulation of oxidative flux, oxidative stress, and energy-related signaling.

[0004] Thus, mitochondrial dysfunction is a potential contributing factor in multiple diseases, including cardiovascular disease, cancer, Alzheimer's disease, diabetes, vision loss, and frailty. Vision loss can be the result of several eye disorders, such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), Leber's hereditary optic neuropathy (LHON), glaucoma, and diabetic retinopathy. Although this list is not exhaustive, one common aspect among such disorders is mitochondrial dysfunction, which causes the pathology of vision loss or vision impairment. For example, retinal pigment epithelial (RPE) cells are considered to be a key factor in AMD pathology. It is believed that excessive production of reactive oxygen species (ROS) due to mitochondrial dysfunction in RPE cells can cause damage to RPE cells in the retina and ultimately their degeneration. Mitochondrial dysfunction in AMD is thought to result from the long-term accumulation of ROS-induced damage to mitochondrial DNA (mtDNA). Since one of the primary functions of RPE cells is to maintain the health of photoreceptors, degeneration of RPE cells leads to photoreceptor dysfunction and visual variations associated with AMD. Another example is LHON, a known inherited mitochondrial genetic disorder that is manifested by three major mitochondrial DNA (mtDNA) mutations found in 90% of cases. Due to these mtDNA mutations, LHON primarily affects retinal ganglion cells (RGCs), causing their degeneration and resulting in vision loss. Degeneration and eventual loss of RGCs from the inner retina are the cellular hallmarks of this disorder.

[0005] Some have approached the problem of treating such eye disorders by using gene therapy, hoping that damaged or mutated mtDNA will be corrected. However, genotoxic adverse reactions resulting from such approaches may raise safety-related concerns. An alternative approach is to transfer healthy mitochondria into cells affected by such eye disorders. Mitochondrial transfer techniques (collecting mitochondria from an external source and transferring them into the body) have been developed in recent years by several major universities. Although mitochondrial transfer is generally less invasive than other approaches, finding and preparing a source of mitochondria for transfer has long been a challenge. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 9,968,603 [Patent Document 2] U.S. Patent No. 10,537,594 [Non-patent literature]

[0007] [Non-Patent Document 1] Lee et al., Advanced Functional Materials, doi.org / 10.1002 / adfm.202000086 (2020) [Non-Patent Document 2] Hartwig et al., Proteomics, 2009, (9):3209-3214 Summary of the Invention [Problem to be solved by the invention]

[0008] Each method disclosed herein has several aspects, no single one of which is solely responsible for its desirable properties. Without limiting the scope of the claims, some prominent features will now be briefly discussed. Numerous other embodiments are also contemplated, including embodiments having fewer, additional, and / or different components, steps, features, objects, benefits, and advantages. The components, aspects, and steps may be configured and ordered differently. After reviewing this discussion, and particularly after reading the section entitled "Detailed Description of the Invention," one will understand how the features of the devices and methods disclosed herein provide advantages over other known devices and methods. [Means for solving the problem]

[0009] In some embodiments, a method of treating an ocular disorder or a symptom of an ocular disorder in a patient in need thereof is provided, comprising obtaining platelet-derived extracellular vesicles (PEVs) containing mitochondria, where the PEVs are collected by obtaining blood from one or more donors, adding an anticoagulant and a buffer to the blood to form a mixture, separating the mixture into a supernatant and platelet-rich plasma (PRP), collecting the PRP, stimulating the collected PRP to displace the extracellular vesicles from the platelets in the PRP, and collecting the extracellular vesicles as PEVs. In some embodiments, the method further comprises administering an effective amount of PEVs to the patient's eye, thereby treating the ocular disorder. In some embodiments, the PEVs are collected at a site different from the site where the treatment is performed.

[0010] In some embodiments, the administering step includes delivering the PEV from a syringe configured to deliver the PEV into the eye. In some embodiments, the administering step includes delivering the PEV via a port delivery system that provides a PEV implant for sustained delivery of the PEV into the eye. In some embodiments, the administering step includes delivering an effective amount of the PEV into the vitreous cavity of the eye.

[0011] In some embodiments, the collected PRP contains Ca 2+ In some embodiments, the immune complex comprises heat-aggregated IgG. In some embodiments, the collected PRP is stimulated by freeze-thaw cycles. In some embodiments, the concentration of heat-aggregated IgG is about 0.1 mg / mL to about 2.5 mg / mL, and Ca 2+ The concentration of is about 1 mM to about 25 mM. In some embodiments, the anticoagulant is the anticoagulant acid citrate dextrose (ACD). In some embodiments, the buffer is Tyrode's buffer at about pH 6 to about pH 7. In some embodiments, the separating step is performed by centrifugation. In some embodiments, the blood is stored for 4 days or more. In some embodiments, the blood is stored for up to 1 year.

[0012] In some embodiments, the PEV contacts at least one cell of the eye during and / or after administration of the PEV into the eye. In some embodiments, after the PEV contacts the cell, the PEV is internalized into the cell. In some embodiments, the effective amount corresponds to the amount of internalized PEV, which ranges from about 3 PEVs / cell to about 100 PEVs / cell. In some embodiments, the PEV is frozen during storage. In some embodiments, the frozen PEV is stored in combination with a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

[0013] In some embodiments, the eye disorder is age-related macular degeneration. In some embodiments, the eye disorder is retinitis pigmentosa. In some embodiments, the eye disorder is Leber's hereditary optic neuropathy. In some embodiments, the eye disorder is diabetic retinopathy. In some embodiments, the eye disorder is glaucoma. In some embodiments, the cells comprise retinal pigment epithelial cells. In some embodiments, the cells comprise retinal ganglion cells. In some embodiments, the cells are located in the macula periphery of the retina of the eye.

[0014] The properties of the embodiments of the present disclosure will become apparent upon reference to the following detailed description and drawings, in which like reference numbers correspond to similar, but perhaps not identical, components, and for the sake of brevity, a reference number or feature having a function already described may or may not be described in conjunction with the other figures in which it appears. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows a schematic diagram depicting a method for obtaining a PEV according to some embodiments. [Figure 2A] 2A shows fluorescent images of retinal pigment epithelial cells (RPEC) grown in culture depicting the uptake of PEV into RPEC. FIG. 2A is shown at 20×. [Figure 2B] FIG. 2B shows a fluorescent image of retinal pigment epithelial cells (RPEC) grown in culture, depicting the uptake of PEV into RPEC. FIG. 2B is shown at 40×. [Diagram 3] FIG. 13 shows fluorescent images demonstrating uptake and internalization of PEV into cultured RPEC. [Figure 4] FIG. 1 shows fluorescent images demonstrating in vitro uptake of DsRed-labeled PEV / mitlet by RPEC after 3 and 6 hours. [Diagram 5] FIG. 13 shows fluorescent images depicting internalized PEV-delivered mitochondria in cultured RPEC. [Figure 6A] Figure 6 illustrates the uptake of various effective amounts of PEV into different cell types: Figure 6A illustrates the uptake of PEV into RPEC. [Figure 6B] Figure 6 illustrates the uptake of various effective amounts of PEV into different cell types: Figure 6B illustrates the uptake of PEV into brain endothelial cells (bEND). [Figure 7A]FIG. 1 shows the oxygen consumption rate (OCR) at various stages of oxidative phosphorylation occurring in RPEC mitochondria when at least a portion of mitochondria was transferred from PEV into RPEC in various effective amounts. [Figure 7B] FIG. 1 shows the oxygen consumption rate (OCR) at various stages of oxidative phosphorylation occurring in RPEC mitochondria when at least a portion of mitochondria was transferred from PEV into RPEC in various effective amounts. [Figure 7C] FIG. 1 shows the oxygen consumption rate (OCR) at various stages of oxidative phosphorylation occurring in RPEC mitochondria when at least a portion of mitochondria was transferred from PEV into RPEC in various effective amounts. [Figure 7D] FIG. 1 shows the oxygen consumption rate (OCR) at various stages of oxidative phosphorylation occurring in RPEC mitochondria when at least a portion of mitochondria was transferred from PEV into RPEC in various effective amounts. [Figure 8] FIG. 1 shows the OCR of RPEC receiving various amounts of naked mitochondria in terms of basal respiration. [Figure 9] FIG. 13 shows fluorescence images depicting in vivo internalization of PEV into bone marrow and spleen cells. [Figure 10] FIG. 1 shows a schematic diagram depicting a method of treating an ocular disorder according to some embodiments. [Figure 11] FIG. 1 shows a dot plot representing the PEV population, with DsRed-labeled PEVs accounting for approximately 40% of all CD41+ PEVs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] In the Summary and Detailed Description sections above, and in the claims below, reference is made to specific features of the invention. It is understood that the disclosure of the invention herein includes all possible combinations of such specific features. For example, if a specific feature is disclosed in the context of a particular aspect or embodiment of the invention, or in the context of a particular claim, that feature can also be used in combination with and / or in the context of other specific aspects or embodiments of the invention, and in the invention generally, to the extent possible.

[0017] As shown in Figure 1, some embodiments relate to a method of extracting platelet-derived mitochondria-containing extracellular vesicles (PEVs). In some embodiments, the method includes the steps of: 1) obtaining blood from a donor in step 110; 2) adding an anticoagulant and a buffer to the blood to form a mixture in step 120; 3) separating the mixture into a supernatant and platelet-rich plasma (PRP) in step 130; 4) collecting the platelet-rich plasma (PRP) in step 140; 5) stimulating the collected platelets in step 150; and 6) collecting the PEVs in step 160.

[0018] Finding a source of mitochondria for transplantation is a challenge. As with any donor organ, there is a shortage of mitochondria from young healthy donors. Some diseases or injuries can be cured with autologous mitochondria (e.g., taken from leg muscles within the patient's own body), however, for many other diseases, the quality of the "patient's" mitochondria is declining due to age or mitochondrial DNA (mtDNA) mutations. For such patients, donor mitochondria are the preferred solution. In addition, freshly isolated whole mitochondria die quickly within minutes of isolation and may also provoke an immune response when introduced naked into the bloodstream, reducing their effectiveness as a therapy. Therefore, it would be advantageous to find a convenient and readily available source of mitochondria for immediate donation (while encapsulated in something like a coating, vesicle, or vehicle suspension (or any combination of these) that protects them from the immune system).

[0019] Platelets from human blood contain, on average, 4-5 mitochondria, which are released into extracellular vesicles when platelets are activated. Mitochondria-containing extracellular vesicles from platelets are referred to herein as PEVs. These PEVs are typically larger (>400 nM) than other platelet extracts or lysates (30-100 nM), and other sizes may be applicable, although they are less well known.

[0020] PEVs donate their mitochondria to neighboring cells (as shown in Figures 2A-2B, 3, 4, and 5), and it has been shown that different cells that have taken up PEVs can increase the respiratory activity of the cells that absorb them, as shown in Figures 7A-7D, thus regenerating tissues and curing some age-related diseases (as shown in Figures 6A and 6B). PEVs have several advantages for early commercialization: in particular, they can be extracted from donated platelets that are "out of date" and must be discarded; they provide another good, medically useful use for platelets that would otherwise be discarded; they can be collected in most blood banks, which already have all the required skilled personnel, clean handling practices, and equipment, and are already adjacent to hospitals, making a PEV product that could be made available for international use very quickly; PEVs are a variety of platelet infusion and therefore more likely to be included and tested by medical professionals who are already familiar with blood infusion therapy. Furthermore, PEV can be formulated for localized infusion into a variety of internal anatomical regions to treat a variety of clinical disorders using delivery devices already available on the market.

[0021] In some embodiments, a non-limiting example of a delivery device is a syringe comprising a hollow barrel forming at least an interior space, a plunger coupled to and fitted into the hollow barrel, and a needle coupled to the barrel, the needle including a space that is continuous with the interior space of the hollow barrel when the needle is coupled to the barrel. Both the plunger and the needle may be directly or indirectly coupled to the hollow barrel. The syringe is constructed to deliver PEV and / or naked mitochondria intraocularly or intravitreally. The syringe may be constructed to deliver PEV and / or naked mitochondria subretinal. In some embodiments, the syringe is constructed to deliver PEV and / or naked mitochondria subcutaneously. In some embodiments, the syringe is constructed to deliver PEV and / or naked mitochondria intraperitoneally (intraperitoneal injection). In some embodiments, the syringe is constructed to deliver PEV and / or naked mitochondria systemically to a patient.

[0022] In some embodiments, a non-limiting example of a delivery device is a port delivery system, via its intraocular or intravitreal delivery, that provides sustained release of PEVs and / or naked mitochondria (either of which may be optionally combined with other therapeutic agents). Port delivery systems are described in U.S. Patent No. 9,968,603, the disclosure of which is hereby incorporated by reference.

[0023] As another non-limiting example of some embodiments, the ORBIT™ subretinal delivery system has been developed by Gyroscope Therapeutics, Inc., which may be devised for delivery of the PEV. Another delivery system involves providing a PEV on a contact lens, which is then placed in the affected eye. The PEV may also be embedded in a gel-like material and placed in a "microneedle," as described in Lee et al., Advanced Functional Materials, doi.org / 10.1002 / adfm.202000086 (2020), the disclosure of which is hereby incorporated by reference. Many other such delivery systems are known and may be devised for delivering the PEV.

[0024] ] In some embodiments, the blood is derived from a mammalian subject. According to another embodiment, the mammalian subject is a human subject. According to another embodiment, the mammalian subject is selected from the group consisting of a human, a horse, a dog, a cat, a mouse, a rat, a cow, and a sheep. Each possibility represents a separate embodiment of the present invention. According to another embodiment, the PEV of the present invention is derived from a mammalian cell. According to another embodiment, the mammalian cell is a human cell. According to another embodiment, the PEV is derived from a cell in culture. According to another embodiment, the PEV is derived from a tissue.

[0025] According to another embodiment, the PEV is derived from a cell or tissue selected from the group consisting of human placenta, human placental cells grown in culture, and human blood cells. According to another embodiment, the PEV of the present invention is derived from a cell or tissue selected from the group consisting of placenta, placental cells grown in culture, and blood cells. According to another embodiment, the naked mitochondria can be isolated from a cell or tissue selected from the group consisting of donor liver, bone marrow, placenta, human placental cells, or any other tissue. According to another embodiment, the naked mitochondria can be isolated from a cell grown in culture or a tissue grown in culture selected from the group consisting of donor liver, bone marrow, placenta, human placental cells, or any other tissue.

[0026] As used herein, the phrase "naked mitochondria" refers to mitochondria isolated from cells or tissues. In some embodiments, the cells are cells grown in culture. In some embodiments, the tissue is tissue grown in culture. The naked mitochondria can be suspended in a freezing buffer, a hydrogel, a pharma- ceutically acceptable liquid medium capable of supporting naked mitochondria, or a buffer solution containing sugars. In some embodiments, the hydrogel is biocompatible, biodegradable, and capable of supporting naked mitochondria. In some embodiments, the hydrogel can be thermosensitive, which includes temperature-dependent hydrophilicity and hydrophobicity. In some embodiments, the hydrogel is biocompatible, biodegradable, capable of supporting naked mitochondria, and thermosensitive (including hydrogels with temperature-dependent hydrophilicity and hydrophobicity).

[0027] As used herein, the phrases "cells grown in culture" or "tissues grown in culture" refer to a number of cells or tissues, respectively, grown in a liquid, semi-solid, or solid medium outside the organism from which the cells or tissues originate. According to some embodiments, the cells grown in culture are cells grown in a bioreactor. According to a non-limiting example, cells can be grown in a bioreactor, followed by isolation of PEVs from the cells. According to another non-limiting example, cells can be grown in a bioreactor, followed by isolation of mitochondria from the cells. In some embodiments, the mitochondria isolated from cells grown in a bioreactor are naked mitochondria.

[0028] In some embodiments, the blood is from a mouse. Mouse blood is used to test the feasibility of the PEV extraction method. In some embodiments, the blood is from a human donor.

[0029] Once blood is obtained in step 110, an anticoagulant and buffer are added in step 120 to prevent the blood from thickening and solidifying. In some embodiments, the anticoagulant is ACD (20%). In some embodiments, the buffer is 40% Tyrode's buffer having a pH of about 6 to about 7, preferably pH 6.5.

[0030] After adding an anticoagulant and a buffer to the blood in step 120, the mixture is then separated into a supernatant and platelet-rich plasma (PRP) in step 130. In some embodiments, the separation is by centrifugation. Plasma is the liquid portion of whole blood. Plasma is composed primarily of water and proteins, and provides the medium for red blood cells, white blood cells, and platelets to circulate throughout the body. Platelets are the blood cells that provide blood clots and other necessary growth and healing functions. After centrifugation of the mixture, the blood cells form a pellet that accumulates at the bottom of the tube. The pellet is called platelet-rich plasma (PRP) and contains concentrated platelets. The pellet is then collected in step 140.

[0031] A buffer is then added to the collected PRP in step 140 to resuspend the platelets. The platelets are then activated or stimulated in step 150. There are many ways to activate platelets. Any of several substances can be used for this purpose, including carbon radioisotopes, prostaglandins, serotonin, adenosine triphosphate, collagen, l-lactate dehydrogenase, thrombin, magnesium, adenosine, calcium, and heat-aggregated antibodies. In some embodiments, the platelets are activated by freeze-thaw cycles. As used herein, the term "freeze-thaw cycles" refers to freezing the mitochondria of the present invention at a temperature below 0°C, maintaining the mitochondria at a temperature below 0°C for a defined period of time, and thawing the mitochondria at room temperature or body temperature or any temperature above 0°C. The term "room temperature" as used herein refers to a temperature between 18°C ​​and 25°C. The term "body temperature" as used herein refers to a temperature between 35.5°C and 37.5°C, preferably 37°C.

[0032] In another embodiment, the freeze-thaw cycled mitochondria are frozen at a temperature of at least -70°C. In another embodiment, the freeze-thaw cycled mitochondria are frozen at a temperature of at least -20°C. In another embodiment, the freeze-thaw cycled mitochondria are frozen at a temperature of at least -4°C. In another embodiment, the freeze-thaw cycled mitochondria are frozen at a temperature of at least 0°C. According to another embodiment, the freezing of mitochondria is gradual. According to some embodiments, the freezing of mitochondria is by flash freezing. As used herein, the term "flash freezing" refers to rapid freezing of mitochondria by exposing them to extremely low temperatures.

[0033] In another embodiment, the freeze-thaw cycled mitochondria are frozen for at least 30 minutes before thawing. According to another embodiment, the freeze-thaw cycle comprises freezing the partially purified functional mitochondria for at least 30, 60, 90, 120, 180, 210 minutes before thawing. Each possibility represents a separate embodiment of the present invention. According to another embodiment, the freeze-thaw cycled mitochondria are frozen for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 48, 72, 96, 120 hours before thawing. Each freezing time represents a separate embodiment of the present invention. According to another embodiment, the freeze-thaw cycled mitochondria are frozen for at least 4, 5, 6, 7, 30, 60, 120, 365 days before thawing. Each freezing time represents a separate embodiment of the present invention. According to another embodiment, the freeze-thaw cycle comprises freezing the partially purified functional mitochondria for at least 1, 2, 3 weeks before thawing. Each possibility represents a separate embodiment of the present invention. According to alternative embodiments, the freeze-thaw cycle comprises freezing the partially purified, functional mitochondria for at least 1, 2, 3, 4, 5, 6 months prior to thawing, with each possibility representing a separate embodiment of the present invention.

[0034] According to another embodiment, the mitochondria subjected to freeze-thaw cycles are frozen in a freezing buffer. According to another embodiment, the mitochondria subjected to freeze-thaw cycles are frozen in an isolation buffer. As used herein, the term "isolation buffer" refers to a buffer in which the mitochondria of the present invention are partially purified. In a non-limiting example, the isolation buffer comprises 200 mM sucrose, 10 mM Tris-MOPS, and 1 mM EGTA. According to some embodiments, BSA (bovine serum albumin) is added to the isolation buffer during partial purification. According to some embodiments, 0.2% BSA is added to the isolation buffer during partial purification. According to some embodiments, HSA (human serum albumin) is added to the isolation buffer during partial purification. According to some embodiments, 0.2% HAS is added to the isolation buffer during partial purification. According to other embodiments, HSA or BSA is removed from the mitochondria of the present invention after partial purification. Each possibility represents a separate embodiment of the present invention. Without being bound by any mechanism or theory, freezing mitochondria in isolation buffer saves time and the isolation process by eliminating the need to replace the isolation buffer with a freezing buffer prior to freezing or to replace the freezing buffer upon thawing.

[0035] According to another embodiment, the freezing buffer comprises a cryoprotectant. According to some embodiments, the cryoprotectant is a sugar, an oligosaccharide, or a polysaccharide. Each possibility represents a separate embodiment of the present invention. According to another embodiment, the sugar concentration in the freezing buffer is a sugar concentration sufficient to act to maintain mitochondrial function. According to another embodiment, the isolation buffer comprises a sugar. According to another embodiment, the sugar concentration in the isolation buffer is a sugar concentration sufficient to act to maintain mitochondrial function. According to another embodiment, the sugar is sucrose. According to another embodiment, the sugar is a sugar other than trehalose. Without being bound by any theory or mechanism, mitochondria frozen in a freezing or isolation buffer comprising sucrose exhibit comparable or higher oxygen consumption rates after thawing compared to control mitochondria that were not subjected to a freeze-thaw cycle or were frozen in a freezing or isolation buffer that does not contain sucrose.

[0036] According to some embodiments, when a saccharide is added to the mitochondrial composition of the present invention at a sufficient concentration, such addition acts to maintain mitochondrial function. According to another embodiment, the saccharide concentration sufficient to act to maintain mitochondrial function is a concentration of 100 mM to 400 mM, preferably 100 mM to 250 mM, and most preferably 200 mM to 250 mM. Each possibility represents a separate embodiment of the present invention. According to another embodiment, the saccharide according to the present invention is sucrose. According to some embodiments, the saccharide of the present invention is a saccharide other than trehalose. According to some embodiments, the saccharide of the present invention is a saccharide other than mannitol.

[0037] According to another embodiment, the sugar concentration in the composition of the present invention is 100 mM to 150 mM. According to another embodiment, the sugar concentration in the composition of the present invention is 150 mM to 200 mM. According to another embodiment, the sugar concentration in the composition of the present invention is 100 mM to 200 mM. According to another embodiment, the sugar concentration in the composition of the present invention is 100 mM to 400 mM. According to another embodiment, the sugar concentration in the composition of the present invention is 150 mM to 400 mM. According to another embodiment, the sugar concentration in the composition of the present invention is 200 mM to 400 mM. According to another embodiment, the sugar concentration in the composition of the present invention is at least 100 mM. According to another embodiment, the sugar concentration in the composition of the present invention is at least 200 mM. Without being bound by any theory or mechanism of action, a sugar concentration of less than 100 mM may not be sufficient to maintain mitochondrial function.

[0038] In some embodiments, the stimulatory agent is heat-aggregated IgG.

[0039] The stimulated platelets are centrifuged to remove any residual platelets or cells. The supernatant containing the PEVs is then collected in step 160.

[0040] According to some embodiments, the PEV is derived from a subject in need thereof. According to another embodiment, the PEV is derived from a subject different from the subject in need thereof. According to another embodiment, the PEV is derived from the same subject to which the PEV is administered. According to another embodiment, the PEV is derived from a subject different from the subject to which the PEV is administered. According to another embodiment, the PEV of the present invention is derived from a source selected from autologous, allogeneic, and xenogeneic. Each possibility represents a separate embodiment of the present invention. As used herein, mitochondria of autologous origin refer to mitochondria derived from the same subject to be treated. As used herein, mitochondria of allogeneic origin refer to mitochondria derived from a subject of the same species but different from the subject to be treated. As used herein, mitochondria of xenogeneic origin refer to mitochondria derived from a subject of different species, different from the subject to be treated. According to another embodiment, the PEV of the present invention is derived from a donor. According to some embodiments, the donor is an allogeneic donor. According to some embodiments, the donor is an autologous donor.

[0041] The term "subject in need thereof" as used herein refers to a subject suffering from or at risk of suffering from a condition that would benefit from enhanced mitochondrial function. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the "subject in need thereof" is a subject suffering from a condition that would benefit from pro-apoptotic activity. In a non-limiting example, the condition that would benefit from pro-apoptotic activity is cancer. According to another embodiment, the subject in need thereof is a mammal. According to another embodiment, the subject in need thereof is a human. According to another embodiment, the subject in need thereof is selected from the group consisting of a human, a horse, a dog, a cat, a mouse, a rat, a cow, and a sheep.

[0042] Some embodiments relate to a method for transducing platelet-derived mitochondria-containing extracellular vesicles (PEVs) into cells, the method comprising 1) extracting PEVs from blood, and 2) incubating the PEVs with cells for a time sufficient to transduce the PEVs into the cells. Incubating the cells can be in vitro or in vivo, the latter being depicted in FIG.

[0043] In certain in vivo embodiments, incubating the PEV with the cells comprises injecting the PEV into the blood, cerebrospinal fluid, pleural fluid, pericardial fluid, peritoneal / ascites fluid, synovial fluid, saliva, or any other bodily fluid of the subject. This can be accomplished in a variety of ways, including using a suitable catheter, such as an intra-arterial or intrathecal catheter. The PEV can also be introduced into a particular organ or tissue of the subject, such as the eye or retina of the subject. To this end, the PEV can be delivered via intravitreal, intravenous, or intra-arterial injection.

[0044] According to another embodiment, as shown in FIG. 10, a method of treating an ocular disorder or a symptom of an ocular disorder in a patient in need thereof is provided, the method comprising obtaining platelet-derived extracellular vesicles (PEVs) containing mitochondria. The PEVs are collected by obtaining blood from one or more donors in step 1010, adding an anticoagulant and a buffer to the blood to form a mixture in step 1020, separating the mixture into a supernatant and platelet-rich plasma (PRP) in step 1030, collecting the PRP in step 1040, stimulating the collected PRP in step 1050, thereby displacing the extracellular vesicles from the platelets in the PRP, and collecting the extracellular vesicles as PEVs in step 1060, although many of these steps related to collecting PEVs are described in other embodiments and are therefore similar. In some embodiments, the method further comprises administering an effective amount of PEVs to the patient's eye in step 1070, thereby treating the ocular disorder. In some embodiments, the PEV is collected at a site different from the site where the procedure is performed, hi some embodiments, the PEV is collected on-site or off-site.

[0045] As used herein, the term "on-site" refers to a location where the administering step is performed or will be performed. The location can be in the same room, office, or ward as the room, office, or ward where the administering step is performed or will be performed. The location can be in the same building as the building where the administering step is performed or will be performed. The location can be in the same building complex that includes multiple buildings, at least one of the multiple buildings is the location where the administering step is performed or will be performed. The building complex can have the same affiliation (business or organization) or at least one of the multiple buildings can have a different affiliation.

[0046] As used herein, the term "off-site" refers to a location external to where the administration step is performed or will be performed. The external location can be a room or lab separate from the building and building complex (if the building is part of a building complex) where the administration step is performed or will be performed.

[0047] In some embodiments, the blood is stored for about 4 days or more. In some embodiments, the blood is stored for about 1 year. In some embodiments, the PEVs are frozen during storage. In some embodiments, the frozen PEVs are stored in combination with a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides. In some embodiments, the anticoagulant is the anticoagulant acid citrate dextrose (ACD). In some embodiments, the buffer is Tyrode's buffer at about pH 6 to about pH 7, preferably about pH 6.5. In some embodiments, the separation is performed by centrifugation. In some embodiments, for the stimulating step, the collected PEVs are subjected to a centrifugation step of 1000 μl of Ca. 2+ In some embodiments, the immune complex comprises heat-aggregated IgG. The concentration of the heat-aggregated Ig used in the stimulation step is preferably about 0.1 mg / mL to about 2.5 mg / mL, more preferably about 0.5 mg / mL. 2+ The concentration of is about 1 mM to about 25 mM, more preferably about 5 mM. In some embodiments, the collected PRP is stimulated by freeze-thaw cycles.

[0048] In some embodiments, after the PEV is administered intraocularly, the PEV contacts at least one cell of the eye. In some embodiments, after the PEV contacts the cell, the PEV is internalized into the cell. As used herein, the terms "contact with" and "contacting with" refer to a composition comprising mitochondria being located in sufficient proximity to a cell to cause at least internalization of the mitochondria into the cell.

[0049] In some embodiments, the effective amount for treating an ocular disorder corresponds to the amount of PEVs internalized, which ranges from about 3 PEVs / cell to about 100 PEVs / cell for at least one cell, as shown for RPEC and bEND in Figures 6A-6B. In some embodiments, the effective amount ..., about 10 PEVs / cell, about 30 PEVs / cell, or about 100 PEVs / cell for at least one cell. Effective amounts will vary depending on the route of administration, the possibility of co-administration with another therapeutic agent, the possibility or method of co-administration with another therapeutic treatment, the type of delivery device used, and the use of any excipients, as will be recognized by one of skill in the art.

[0050] In some embodiments, the eye disorder to be treated is age-related macular degeneration (AMD). AMD is an eye disorder that is one of the main causes of vision loss, especially in developed countries, with a prevalence of up to approximately 40%. AMD is characterized by mitochondrial dysfunction that affects the retina, which is caused by oxidative stress from reactive oxygen species (ROS). In AMD, ROS are produced at high levels in RPE cells, causing damage to mtDNA. This damage can accumulate over time to the point where insufficient mtDNA repair mechanisms cause mitochondrial cell death, which in turn leads to RPE cell death. Because RPE cells support the health of photoreceptors, RPE cell death leads to the death of the photoreceptors that RPE cells support, causing vision loss.

[0051] In some embodiments, the eye disorder treated is retinitis pigmentosa (RP). RP is a genetic eye disorder that causes severe vision loss and is characterized by rod degeneration. In some embodiments, the eye disorder is Leber's hereditary optic neuropathy (LHON), a genetic mitochondrial genetic disorder that is manifested by three major mtDNA mutations found in 90% of cases. From this mtDNA mutation, LHON primarily affects retinal ganglion cells (RGCs), causing their degeneration and resulting in vision loss. In some embodiments, the eye disorder is diabetic retinopathy, which is characterized by dysfunction of endothelial cells of the retinal microvessels and supporting cells of the retina, such as Müller cells. In diabetic retinopathy, dysfunction of endothelial cells causes their increased permeability, which can lead to vascular leakage. This vascular leakage can lead to peripheral edema and thus other associated retinal diseases, such as diabetic macular edema. In some embodiments, the eye disorder is glaucoma.

[0052] In some embodiments, the cells that receive the PEV include retinal pigment epithelial cells. In some embodiments, the cells that receive the PEV include retinal ganglion cells. In some embodiments, the cells that receive the PEV are located in the macula periphery of the retina of the eye.

[0053] Specific tissues and organs can be specifically targeted by complexing PEVs with specific receptors or coatings that promote "homing" to certain cell types. For example, U.S. Patent No. 10,537,594, the contents of which are hereby incorporated by reference, illustrates the use of the asialoglycoprotein (AsG) receptor system to target mitochondria to hepatocytes. Similar systems can be used to target other tissues or organs.

[0054] Various techniques can be employed to promote mitochondrial internalization into cells, both in vitro and in vivo. It is believed that high levels of mitochondrial internalization are feasible in certain diseased tissues or in elderly people, since the tissue is severely deficient in mitochondria. Cells in an energy-starved state are expected to activate chemical pathways that allow for easier internalization. When free mitochondria or mitochondrial-loaded stem cells float, the cells signal their need and / or phagocytose the mitochondria. It is also feasible to achieve higher levels of mitochondrial uptake, with timing, frequency, and duration adjustments to ensure that a large number of mitochondria are continuously maintained in the bloodstream and are always available for rapid supply. Precision placement can also be employed by injecting mitochondrial material directly into the organ via an arterial shunt. For example, the hepatic artery can be used to concentrate mitochondria in the liver to preferentially regenerate liver tissue. When done properly, processes that may dilute or backflow mitochondria, such as clonal proliferation, can be omitted.

[0055] Diathermy and exercise by the subject may also promote mitochondrial uptake by cells. Exercise causes skeletal muscles to create more mitochondria. This would be expected to cause the cells to accept more transplants. Research suggests that this effect can also be elicited by heating the muscles with RF radio energy or ultrasound for 2-4 hours per session.

[0056] Calorie restriction / fasting by subjects may also promote mitochondrial uptake. Studies have shown that fasting induces mitochondrial changes, fission / fusion, or mitophagy. Perhaps repeated fasting with fluid infusion induces cells to receive a few extra mitochondria and share new mtDNA with the mitochondrial network more quickly.

[0057] Other techniques can also be employed to promote mitochondrial uptake, such as partial intoxication, chemotherapy, or hypoxia to induce autophagy, followed by multiple cycles of infusion. In addition, metformin, melatonin, or other drugs are believed to stimulate mitochondrial uptake by cells. Cloning can be used to shut down the ability of mitochondria to regenerate themselves, forcing cells to rely solely on infused mitochondria.

[0058] Some embodiments relate to a method for increasing cellular respiration, the method comprising transducing a cell with a PEV isolated according to the methods described herein, and generating ATP from the PEV.

[0059] The isolated PEV comprises functional mitochondria. In some embodiments, the term "functional mitochondria" refers to mitochondria that consume oxygen. In another embodiment, functional mitochondria have an intact outer membrane. Other embodiments include mitochondrial fragments, mitochondrial DNA, or segments thereof, and mRNA encoding mitochondrial gene products. In some embodiments, functional mitochondria are intact mitochondria. In another embodiment, functional mitochondria consume oxygen at an increased rate over time. In another embodiment, mitochondrial functionality is measured by oxygen consumption. In another embodiment, mitochondrial oxygen consumption can be measured by any method known in the art. According to some embodiments, functional mitochondria are mitochondria that exhibit an increased rate of oxygen consumption in the presence of ADP and substrates such as, but not limited to, glutamate, malate, or succinate. Each possibility represents a separate embodiment of the invention. In another embodiment, functional mitochondria are mitochondria that generate ATP. In another embodiment, functional mitochondria are mitochondria that have the ability to manufacture their own RNA and proteins and are self-renewing structures. In another embodiment, functional mitochondria produce mitochondrial ribosomes and mitochondrial tRNA molecules.

[0060] As used herein, the term "intact mitochondria" refers to mitochondria that include outer and inner membranes, the intermembrane space, the cristae (formed by the inner membrane), and the matrix. In another embodiment, intact mitochondria contain mitochondrial DNA. In another embodiment, intact mitochondria contain active respiratory chain complexes I-V embedded in the inner membrane. In another embodiment, intact mitochondria consume oxygen.

[0061] According to another embodiment, mitochondrial membrane integrity can be determined by any method known in the art. In a non-limiting example, mitochondrial membrane integrity is measured using tetramethylrhodamine methyl ester (TMRM) or tetramethylrhodamine ethyl ester (TMRE) fluorescent probes. Each possibility represents a separate embodiment of the invention. Mitochondria observed under a microscope and showing TMRM or TMRE staining have an intact outer mitochondrial membrane.

[0062] As used herein, the term "mitochondrial membrane" refers to a mitochondrial membrane selected from the group consisting of the inner mitochondrial membrane, the outer mitochondrial membrane, or a combination thereof.

[0063] In some embodiments, the functional mitochondria are partially purified mitochondria. As used herein, the term "partially purified mitochondria" refers to mitochondria that have been separated from other cellular components, where the weight of mitochondria comprises 20-80%, preferably 30-80%, and most preferably 40-70% of the combined weight of mitochondria and other subcellular fractions, as exemplified in Hartwig et al., Proteomics, 2009, (9):3209-3214, the disclosure of which is hereby incorporated by reference. Each possibility represents a separate embodiment of the present invention.

[0064] According to another embodiment, the partially purified mitochondria do not contain intact cells. According to another embodiment, the composition of the present invention does not contain intact cells. According to another embodiment, the composition of the present invention does not contain mitochondrial aggregates or aggregates, or cell debris, or components larger than 5 μm. Each possibility represents a separate embodiment of the present invention. According to another embodiment, the composition of the present invention is devoid of particulate matter larger than 5 μm. As used herein, the term "particulate matter" refers to intact cells, cell debris, mitochondrial aggregates, cell debris aggregates, or combinations thereof. Each possibility represents a separate embodiment of the present invention. As used herein, a composition devoid of exogenous particulate matter larger than 5 μm has a concentration of particulate matter larger than 5 μm of 1 μM or less, preferably less than 0.5 μM, and most preferably less than 0.1 μM.

[0065] According to some embodiments, non-denatured cells, cell debris, or aggregates are removed from the compositions of the present invention. According to some embodiments, the compositions of the present invention are filtered through a filter of 5 μm or less to remove any non-denatured cells, cell debris, or aggregates, as exemplified herein below. Without being bound by any theory or mechanism, the use of compositions containing mitochondrial aggregates according to the methods of the present invention may be less effective or even harmful to the subject. According to another embodiment, the compositions of the present invention do not include liposomes or any other particulate carriers. Each possibility represents a separate embodiment of the present invention.

[0066] According to another embodiment, the weight of mitochondria in the partially purified mitochondria comprises at least 20% of the combined weight of mitochondria and other subcellular fractions. According to another embodiment, the weight of mitochondria in the partially purified mitochondria comprises 20%-40% of the combined weight of mitochondria and other subcellular fractions. According to another embodiment, the weight of mitochondria in the partially purified mitochondria comprises 40%-80% of the combined weight of mitochondria and other subcellular fractions. According to another embodiment, the weight of mitochondria in the partially purified mitochondria comprises 30%-70% of the combined weight of mitochondria and other subcellular fractions. According to another embodiment, the weight of mitochondria in the partially purified mitochondria comprises 50%-70% of the combined weight of mitochondria and other subcellular fractions. According to another embodiment, the weight of mitochondria in the partially purified mitochondria comprises 60%-70% of the combined weight of mitochondria and other subcellular fractions. According to another embodiment, the weight of mitochondria in the partially purified mitochondria accounts for less than 80% of the combined weight of mitochondria and other subcellular fractions.

[0067] The following examples are presented so that the invention might be more fully understood. The specific techniques, conditions, materials, proportions, and reported data set forth to illustrate the principles of the invention are exemplary and should not be construed as limiting the scope of the invention. EXAMPLES

[0068] This example illustrates that PEVs are harvested from mouse blood according to some embodiments.

[0069] The following steps were carried out:

[0070] 1. Blood was collected from a mouse donor. In this particular protocol, the donor was a male DsRed mouse (a transgenic mouse expressing the red fluorescent protein variant DsRed). MST under the control of the chicken β-actin promoter was linked to the cytomegalovirus (CMV) immediate early enhancer. Here, 3 x 1 mL (1 mL / mouse) of blood was used.

[0071] 2. ACD (20%) was added as an anticoagulant and 40% Tyrode's buffer (pH 6.5) was also added to the blood. The blood mixture (20% ACD + 40% Tyrode's buffer (TB) (pH 6.5)) was then centrifuged at 500g for 3 minutes. The PRP and buffy coat were then collected and centrifuged at 300g for 2 minutes.

[0072] 3. PRP was collected and 20% ACD + 10 mM EDTA was added, followed by a centrifugation step at 1300 g for 5 minutes.

[0073] 4. Each pellet was suspended in 0.1 mL of TB (pH 6.5) and 0.9 mL of TB (pH 7.4) was added.

[0074] 5. Platelets were pooled, counted using a cellometer and diluted to 10e8 / mL in TB7.4.

[0075] 6. A total of 900 million platelets were obtained and 5 mM CaCl2 was added prior to stimulation.

[0076] 7. Platelets were stimulated overnight (16 hours) at room temperature with 0.5 mg / mL heat-aggregated IgG. Heat-aggregated IgG was prepared by agglutinating human IgG (25 mg / mL, MPBIO) at 62° C. for 1 hour.

[0077] 8. Stimulation was stopped by adding 10 mM EDTA.

[0078] 9. The stimulated platelets were centrifuged at 300 g for 5 minutes to remove any residual platelets or cells.

[0079] 10. The supernatant was collected and the PEVs were analyzed using a flow cytometer.

[0080] 11. The remaining platelets were evaluated and contamination was less than 1%.

[0081] 12. The obtained PEV was diluted three times with PBS and centrifuged at 18°C ​​and 18000g for 90 min.

[0082] 13. The pellet was resuspended in 0.3 mL of PBS and the PEVs were counted by flow cytometry. The concentration was estimated to be 1.5 x 10e9 PEVs / ml.

[0083] 14. PEVs can be tagged with CD41 tag to allow them to be counted in a flow cytometer. When so tagged, PEVs make up approximately 40% of the total (CD41+PEVs). A dot plot representing the PEV population is illustrated in Figure 11. (DsRed axis indicates PEVs). EXAMPLES

[0084] This example illustrates that PEVs can be internalized by retinal cells according to some embodiments.

[0085] The following steps were carried out:

[0086] 1. Immortalized mouse retinal pigment epithelial cells (RPEC) and brain endothelial cells (bEND) were seeded overnight prior to PEV incubation (approximately 20,000 cells / well (RPEC) and 16,000 cells / well (bEND)).

[0087] 2. PEV was collected from mouse donors following the steps outlined in Example 1.

[0088] RPECs were preincubated with or without PEVs (approximately 3, 10, 30, or 100 mitochondria per cell + PEVs) for either 3, 18, 24, or 36 hours in Prigrow III supplemented with 3.1% penicillin-streptomycin (pH 7.4) and 1%, 5%, or 10% FBS (non-heat activated).

[0089] bEND was preincubated with or without PEVs (approximately 3, 10, 30, or 100 mitochondria per cell + PEVs) for 24 hours in DMEM supplemented with 4.1% penicillin-streptomycin (pH 7.4) and 1%, 5%, or 10% FBS (non-heat activated).

[0090] 5. RPEC and bEND were washed and placed in XF medium supplemented with 2 mM glutamine, 1 mM pyruvate, and 8 mM D-glucose, and 1% FBS (pH 7.4).

[0091] 6. After washing, RPEC and bEND in XF medium were centrifuged at 300g for 5 and 60 minutes at 37°C without CO2.

[0092] 2A-B are confocal images showing stained nuclei 202 [DAPI (4',6-diamidino-2-phenylindole)] and cell membrane 204 of RPEC 200, and PEV 210 (DsRed). As shown in FIG. 2A-B (40× and 20× magnification, respectively), PEV 210 is largely internalized by RPEC 200. As shown in FIG. 3, this internalization can be stable for well over 24 hours. FIG. 4 shows another group of RPEC 400, its nuclei 402 stained with DAPI, demonstrating its ability to internalize administered PEV / mitlet 410 (DsRed) after 3 and 6 hours. The left panel of FIG. 4 shows RPEC 400, including its stained nuclei 402 and its cell membrane 404. Also shown in the left panel is stained PEV / mitlet 410. The right panel of Figure 4 shows 1) stained nuclei 402 of RPEC 400 from the adjacent left panel; and 2) stained PEV / mitolet 410 from the adjacent left panel. Panels from Figure 4 were acquired at 40x.

[0093] To verify mitochondrial internalization in RPEC 500, XZ and YZ scans of fluorescently labeled mitochondria 510 (here indicated by the labeled arrowhead) from PEV were performed using confocal microscopy, as shown in FIG. 5. The nuclei 502 of RPEC 500 were stained with DAPI. The cell membrane 504 of RPEC 500 was stained with CellMask™, as shown in FIG. 5, where the XZ plane is perpendicular to the YZ plane. The XZ, YZ scan shows how the highest intensity from a point source of fluorescently labeled mitochondria 510 (see arrowhead) is located within the RPEC 500. The results of this internalization are shown in FIG. 6A. FIG. 6B shows that bEND also internalized mitochondria delivered by PEV. Thus, these results demonstrate that PEV can deliver durable mitochondria into RPEC and bEND.

[0094] To evaluate the activity of mitochondria delivered by PEV, the Seahorse XF assay (testing for mitochondrial stress) was performed on RPECs pre-incubated with PEV for 24 hours. Oxygen consumption rate (OCR) was measured between different pre-incubation parameters for RPECs (PEV 0, 3, 10, 30, or 100 cells / cell) as presented in step 3 of this example, with 5% FBS considered to be the preferred level for the serum used (higher serum levels such as 10% FBS may induce cell division).

[0095] With respect to the OCR readings in Figures 7A-D, similar (otherwise slightly lower) basal respiration levels are shown in Figure 7A between RPECs that were preincubated with PEV and those that were not (control). Basal respiration levels refer to the energy requirements of RPECs under baseline conditions. The increase in spare respiratory capacity of RPECs when preincubated with PEV is shown in Figure 7B, a result suggesting an improved ability of RPECs to respond to energy requirements (i.e., improved cellular adaptability or plasticity). Figure 7C appears to show that ATP generation is slightly enhanced in some of the RPECs that were preincubated with PEV. Figure 7D shows that proton leak does not appear to be an issue for RPECs that internalized PEV compared to RPECs that did not internalize PEV. EXAMPLES

[0096] Naked mitochondria were extracted from live liver and administered to various amounts of RPEC (1, 10, 100, 1000, and 10000 ng) in different wells. The oxygen consumption rate for each group was determined by Seahorse XF assay. As shown in FIG. 8, each group of RPEC administered with naked mitochondria showed a significant increase in their respective basal respiration compared to control RPEC that did not receive any naked mitochondria (control RPEC showed an OCR of about 4 pmol / min). As shown, the increase in basal respiration of RPEC administered with naked mitochondria was at least two-fold compared to the control.

[0097] Appendix The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0098] As used herein, the term "and / or" is intended to expressly indicate either an alternative or an alternative sense.

[0099] As used herein, the word "comprise" or variations such as "comprises" or "comprising" are understood to imply the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0100] The following explanations of terms and methods are provided to better describe the present disclosure and to provide guidance to those of skill in the art in practicing the present disclosure. The singular forms "a," "an," and "the" refer to one or more, unless the context clearly dictates otherwise. For example, the term "comprising a nucleic acid molecule" includes one or more nucleic acid molecules and is considered equivalent to the common phrase "comprising at least one nucleic acid molecule." The term "or" refers to a single element or a combination of two or more elements of a set of alternative elements listed, unless the context clearly dictates otherwise. As used herein, "comprises" means "includes." Thus, "comprising A or B" means "including A, B, or A and B," and does not exclude additional elements. Unless otherwise specified, in the event that this definition differs from other possible definitions, the definition provided herein takes precedence.

[0101] It should be recognized that all combinations of the above concepts and additional concepts discussed in more detail below (such concepts presented shall not be mutually inconsistent) are considered part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are considered part of the inventive subject matter disclosed herein. It should also be recognized that the terminology explicitly employed in this specification may also appear in any disclosure incorporated by reference, but that the terminology is to be consistent with the meaning most consistent with the particular concept disclosed herein.

[0102] Throughout this specification, when a term such as "one example," "another example," "one example," etc. is used, it means that a particular element (e.g., a feature, structure, and / or characteristic) described in connection with that example is included in at least one example described herein, but may or may not be present in other examples. In addition, it is understood that elements described with respect to any example can be combined in any suitable manner in various examples, unless the context clearly dictates otherwise. Although several examples have been described in detail, it is understood that the disclosed examples may be modified. Thus, the above description is considered to be non-limiting.

[0103] It is understood that any feature, material, characteristic, or group described in connection with a particular embodiment or example applies to any other embodiment or example described in this section or anywhere in the specification, except to the extent that it is inconsistent therewith. All of the features disclosed herein (including any accompanying claims, abstract, and drawings), and / or all steps of any method or process disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. Protection is not limited to the details described in any of the above cases. Protection extends to any novel feature or any novel combination of the features disclosed herein (including any accompanying claims, abstract, and drawings), or any novel step or any novel combination of the steps of any method or process disclosed.

[0104] Moreover, certain features described in the present disclosure in the context of an individual implementation may also be implemented in combination in a single implementation. Conversely, various features described in the context of a single implementation may be implemented separately in multiple implementations or in any suitable subcombination. In addition, although features may be described above as functioning in a particular combination, one or more features from the claimed combination may in some cases be separated from the combination, and the combination may be claimed as a subcombination or a variation of the subcombination.

[0105] In addition, although operations may be depicted in the figures or described herein in a particular order, such operations need not be performed in the particular order or sequential order depicted, or all operations may be performed to achieve a desired result. Other operations not depicted or described may be incorporated in the example methods and processes. For example, one or more additional operations may be performed before, after, simultaneously with, or during any of the described operations. Furthermore, operations may be rearranged or resequenced in other implementations. Those skilled in the art will recognize that in some cases, the actual steps taken in the illustrated and / or disclosed processes may differ from those shown in the figures. Depending on the case, certain steps of the steps described above may be removed, or other steps may be added. Furthermore, the features and attributes of the specific examples disclosed above may be combined in different ways to form additional examples, all of which are within the scope of the present disclosure.

[0106] In light of the purpose of this disclosure, certain aspects, advantages, and novel features are described herein. Not all such advantages may necessarily be achieved based on any particular case. Thus, for example, a person skilled in the art will recognize that the present disclosure may be embodied or implemented in a manner that achieves one advantage or group of advantages as taught herein, but does not necessarily achieve other advantages that may be taught or suggested herein.

[0107] Conditional language, such as "can," "could," "might," or "may," generally has the intent to convey that certain features, elements, and / or steps are included in some instances and not included in other instances, unless otherwise specified or understood in the context in which it is used. Thus, such conditional language does not generally have the intent to suggest that the features, elements, and / or steps are required in any way in one or more instances, or that one or more instances necessarily include logic for determining whether those features, elements, and / or steps are included or performed in any particular instance, with or without user input or prompting.

[0108] Conjunction language, such as the idiomatic phrase "at least one of X, Y, and Z," is understood based on the context as it is commonly used, unless specifically stated otherwise, to convey that an item, term, etc. can be either X, Y, or Z. Thus, such conjunctive language does not have the general intent to mean that a particular instance requires that there be at least one X, at least one Y, and at least one Z.

[0109] As used herein, language of degree, such as the terms "approximately," "about," "generally," and "substantially," describes a number, amount, or characteristic that is similar to a stated number, amount, or characteristic yet performs a desired function or achieves a desired result.

[0110] The scope of the present disclosure is not intended to be limited by the specific disclosure of preferred examples in this section or elsewhere herein, but rather, the scope of the present disclosure may be defined by the claims as they are presented in this section or elsewhere herein, or as they may be presented in the future. Claim language shall be construed broadly based on the language employed in the claims, and not limited to the examples described herein or during the prosecution of the application, which examples shall be construed as non-exclusive.

[0111] The described embodiments and examples of the present disclosure are illustrative rather than restrictive, and are not intended to be representative of all embodiments or examples of the present disclosure. Thus, the scope is not limited by the specific embodiments and examples described herein. Although the basic novel characteristics of the present disclosure have been shown, described, and emphasized as applied to various specific embodiments thereof, it will also be understood that various omissions, substitutions, and changes in the details of the disclosed methods may be apparent and may be made by one skilled in the art without departing from the spirit of the present disclosure. For example, all combinations of method steps that perform substantially the same function in substantially the same manner to achieve the same results are expressly intended to be within the scope of the present disclosure. In addition, it should also be recognized that the method steps shown or described in connection with any form or embodiment disclosed in this disclosure may be incorporated into any other disclosed or described or suggested form or embodiment as a general matter of design choice. Moreover, various modifications and variations may be made literally and in the legal equivalents recognized without departing from the spirit or scope of the present disclosure as set forth in the following claims. [Explanation of symbols]

[0112] 110 Process 110 120 Process 120 130 Process 130 140 Process 140 150 Process 150 160 Process 160 200 RPEC 202 Stained Nuclei 204 Cell membrane 210 PEV 400 RPEC 402 Stained Nuclei 404 Cell membrane 410 PEV / Mitret 500 RPEC 502 Stained Nuclei 504 Cell membrane 510 Fluorescently Labeled Mitochondria 1010 Process 1010 1020 Process 1020 1030 Process 1030 1040 Process 1040 1050 Process 1050 1060 Process 1060 1070 Process 1070

Claims

1. 1. Platelet-derived extracellular vesicles (PEVs) containing mitochondria for use in the treatment of an ocular disorder or a symptom of said ocular disorder, comprising: Obtaining blood from one or more donors; adding an anticoagulant and a buffer to the blood to form a mixture; separating the mixture into a supernatant and a platelet rich plasma (PRP); harvesting the PRP; stimulating the collected PRP, thereby displacing extracellular vesicles from platelets within said PRP; Collecting the extracellular vesicles as the PEVs. Platelet-derived extracellular vesicles collected by.

2. The PEV of claim 1, collected at a site different from the site where the treatment is performed.

3. 3. The PEV of claim 1 or 2, provided in a syringe configured to deliver the PEV into an eye.

4. The PEV of claim 1 or 2, provided within a port delivery system that provides a PEV implant for sustained delivery of the PEV within the eye.

5. The PEV of any one of claims 1 to 4, which is administered to the vitreous cavity of the eye.

6. The collected PRP is 2+ The PEV of any one of claims 1 to 5, which is stimulated with an immune complex in the presence of.

7. The PEV of claim 6, wherein the immune complex comprises heat-aggregated IgG.

8. The PEV of any one of claims 1 to 5, wherein the collected PRP is stimulated by freeze-thaw cycles.

9. The concentration of the heat-aggregated IgG is about 0.1 mg / mL to about 2.5 mg / mL, and the Ca 2+ The PEV of claim 7, wherein the concentration of said PEV is from about 1 mM to about 25 mM.

10. 10. The PEV of any one of claims 1 to 9, wherein the anticoagulant is the anticoagulant acid citrate dextrose (ACD).

11. The PEV of any one of claims 1 to 10, wherein the buffer is Tyrode's buffer at about pH 6 to about pH 7.

12. The PEV of any one of claims 1 to 11, wherein the separating step is performed by centrifugation.

13. The PEV of any one of claims 1 to 8, wherein the blood has been stored for 4 days or more.

14. The PEV of claim 13, wherein the blood is stored for up to one year.

15. The PEV of any one of claims 1 to 14, wherein the PEV is administered in a manner that contacts at least one cell of the eye.

16. The PEV of claim 15, wherein the PEV is internalized into the cell after contacting the cell.

17. The PEV of claim 16, administered at a dosage of about 3 PEVs / cell to about 100 PEVs / cell.

18. 15. The PEV of claim 13 or 14, which is frozen during storage.

19. 20. The PEV of claim 18, wherein the frozen PEV is stored in combination with a cryoprotectant.

20. 20. The PEV of claim 19, wherein the cryoprotectant is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

21. The PEV of any one of claims 1 to 20, wherein the eye disorder is age-related macular degeneration.

22. The PEV of any one of claims 1 to 20, wherein the eye disorder is retinitis pigmentosa.

23. The PEV of any one of claims 1 to 20, wherein the eye disorder is Leber's hereditary optic neuropathy.

24. 21. The PEV of any one of claims 1 to 20, wherein the eye disorder is diabetic retinopathy.

25. 21. The PEV of any one of claims 1 to 20, wherein the eye disorder is glaucoma.

26. The PEV of any one of claims 15 to 25, wherein the at least one cell comprises a retinal pigment epithelial cell.

27. The PEV of any one of claims 15 to 20 and 23, wherein the at least one cell comprises a retinal ganglion cell.

28. The PEV of any one of claims 15 to 25, wherein the at least one cell is located in the macula periphery of the retina of the eye.

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