Oligodendrocyte-derived extracellular vesicles for therapy of multiple sclerosis

Abstract

To provide a strategy for treating multiple sclerosis (MS) that does not require determination of the specific myelin components targeted by the immune system in multiple sclerosis.SOLUTION: In one aspect, a method of treating and / or preventing multiple sclerosis in a subject in need thereof is provided, the method comprising the step of administering to the subject an effective amount of an oligodendrocyte-derived extracellular vesicle (OI-EVs).SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Description of Research or Development Funded by the Federal Government This invention was made with government support under Grant No. 5-RO1-AI106026-13 awarded by the National Institutes of Health (NIH). The United States government has certain rights in this invention.

Background Art

[0002] Background of the Invention Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) in which the immune system attacks components of CNS myelin produced by oligodendrocytes. Myelin contains multiple components, and it is not known which of them are the targets of the autoimmune response in MS patients. The development of antigen-specific therapies for MS is difficult because of the lack of knowledge about the myelin components that are targeted by the immune system, the variability among patients, and the high likelihood that the specificity of the autoimmune response changes during the course of the disease. Many antigen-specific therapies have been proposed to date. However, none of them have shown promising results in the clinic. Therefore, there is a need in the art for strategies to treat MS that do not require the determination of specific myelin components that are targeted by the immune system. This disclosure addresses this need.

Summary of the Invention

[0003] In one aspect, there is provided a method of treating and / or preventing multiple sclerosis (MS) in a subject in need thereof, the method comprising administering to the subject an effective amount of oligodendrocyte-derived extracellular vesicles (Ol-EVs). In some embodiments, the Ol-EVs comprise myelin antigen (Ag). In some embodiments, the myelin Ag comprises myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and / or myelin proteolipid protein (PLP). In some embodiments, the method is Ag-specific. In some embodiments, the administering step induces immunosuppressive monocytes. In some other embodiments, the immunosuppressive monocytes express PD-L1. In some embodiments, the administering step does not cause a harmful or undesirable effect on the subject's immune system. In yet other embodiments, the oligodendrocyte-derived extracellular vesicles are formulated in a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is administered intravenously, subcutaneously, intradermally, transdermally, orally, or nasally. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the MS is chronic MS or relapsing-remitting MS.

[0004] In another aspect, there is provided a pharmaceutical composition comprising oligodendrocyte-derived extracellular vesicles (Ol-EVs) and at least one pharmaceutically acceptable carrier. In some embodiments, the Ol-EVs comprise myelin antigen (Ag). In some embodiments, the myelin Ag comprises myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and / or myelin proteolipid protein (PLP). In some other embodiments, the composition is formulated for intravenous, subcutaneous, intradermal, transdermal, oral, or nasal administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The following detailed description of selected aspects of the present invention will be better understood when read in conjunction with the accompanying drawings. Selected aspects are shown in the drawings for purposes of illustrating the invention. However, the invention should not be understood as being limited to the exact arrangements and means shown in the aspects of the drawings.

[0006]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 2-1

Figure 2-2

Figure 3-1

Figure 3-2

Figure 3-3

Figure 3-4

Figure 4-1

Figure 4-2

Figure 4-3

Figure 5-1

Figure 5-2

Figure 5-3

Figure 5-4

Figure 6-1

Figure 6-2

Figure 6-3

Figure 7

Figure 8-1

Figure 8-2

Figure 9

Figure 10-1

Figure 10-2

Figure 10-3

Figure 11

Figure 12-1

Figure 12-2

Figure 13-1

Figure 13-2

Figure 13-3

Figure 14

Figure 15-1

Figure 15-2

Figure 16

Figure 17

Figure 18

Mode for Carrying Out the Invention

[0007] Detailed Description Autoimmune diseases such as multiple sclerosis (MS) develop due to a failure in peripheral immune tolerance to certain self-antigens (Ag). A large number of approaches for specifically suppressing autoimmune neuroinflammation have been demonstrated in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. One such approach is intravenous (i.v.) tolerance induction by injection of myelin Ag used for EAE induction. However, translation of this experimental strategy and similar experimental strategies to MS therapy has been hampered by the uncertainty of the relevant myelin Ag in MS patients. To address this problem, a novel therapeutic approach relying on oligodendrocyte (Ol)-derived extracellular vesicles (Ol-EV) that naturally contain multiple types of myelin Ag has been developed. Intravenously injected Ol-EV suppressed the disease preventively and therapeutically in a myelin Ag-dependent manner in several EAE models. This treatment was safe and restored immune tolerance by inducing apoptosis of immunosuppressive monocytes and autoreactive encephalitogenic CD4+ T cells. Finally, the results described herein also show that human Ol releases EVs containing the most relevant myelin Ag, which serves as the basis for using EVs in MS therapy. These findings introduce a novel approach for specifically suppressing central nervous system autoimmunity to myelin Ag.

[0008] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the selected methods and materials are described herein. The following specialized terms are used when describing and claiming the present invention.

[0009] It should also be understood that the specialized terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0010] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or a plurality of elements.

[0011] As used herein, "about" when referring to a measurable value, such as an amount, a period of time, etc., means an inclusion of variations of ±20%, ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value when such variations are suitable for carrying out the disclosed method.

[0012] A disease or disorder is "alleviated" if the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences such symptoms, or both, is reduced.

[0013] As used herein, the terms "composition" or "pharmaceutical composition" refer to a mixture of at least one compound useful in the present invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates the administration of the compound to a patient or subject. There are multiple techniques for administering the compound in the art, including, but not limited to, intravenous administration, subcutaneous administration, oral administration, aerosol administration, parenteral administration, ocular administration, pulmonary administration, and topical administration.

[0014] An "effective amount" or "therapeutically effective amount" of a compound is an amount of the compound sufficient to produce a beneficial effect on the subject to which the compound is administered. An "effective amount" of a delivery vehicle is an amount sufficient to effectively bind or deliver the compound.

[0015] As used herein, "extracellular vesicles" means particles enclosed by a protein-lipid membrane that are secreted by almost all cells and contain proteins, lipids, DNA, and various RNAs. The term extracellular vesicles encompasses both exosomes (30 nm - 100 nm) and microvesicles (100 nm - 1 μm).

[0016] As used herein, the term "oligodendrocyte-derived extracellular vesicles" refers to extracellular vesicles produced by or isolated from oligodendrocytes.

[0017] The terms "patient", "subject", "individual", etc. are used interchangeably herein and refer to any animal that is the subject of the methods described herein, or to its cells, either in vitro or in situ. In certain non-limiting embodiments, the subject is a non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, cows, pigs, cats, dogs, mice, and rats. In certain non-limiting embodiments, the patient, subject, or individual is human.

[0018] As used herein, the term "pharmaceutically acceptable" refers to materials, such as carriers or diluents, that do not abrogate the biological activity or properties of a compound and are relatively non-toxic. That is, the material can be administered to an individual without causing undesirable biological effects or interacting harmfully with any of the components of the composition in which it is included.

[0019] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle involved in carrying or transporting a compound useful in the present invention into or to a patient so that the compound useful in the present invention can perform its intended function, such as a liquid or solid diluent, stabilizer, dispersant, suspending agent, diluent, excipient, thickening agent, solvent, or encapsulating material. Typically, such constructs are transported or carried from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation containing the compound useful in the present invention and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents that are compatible with the activity of the compound useful in the present invention and physiologically acceptable to the patient. Auxiliary active compounds may also be incorporated into the composition. "Pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful in the present invention.Other additional components that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0020] As used herein, "treating a disease or disorder" means reducing the frequency with which the symptoms of the disease or disorder experienced by a patient appear. Diseases and disorders are used synonymously herein.

[0021] As used herein, the term "treatment" or "process of treating" encompasses prophylaxis and / or therapy. Thus, the compositions and methods of the present invention are not limited to therapeutic uses and can be used for prophylactic uses. Thus, "treating a condition, disorder, or state" or "treatment of a condition, disorder, or state" means (i) preventing or delaying the appearance of clinical symptoms of a condition, disorder, or state that may develop in a subject who has or may be predisposed to the condition, disorder, or state but has not yet experienced or manifested clinical or preclinical symptoms of the condition, disorder, or state, (ii) inhibiting the condition, disorder, or state, i.e., stopping or reducing the development of the disease or at least one of its clinical or preclinical symptoms, or (iii) alleviating the disease, i.e., causing regression of the condition, disorder, or state, or at least one of its clinical or preclinical symptoms.

[0022] Range: Throughout this application, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is for mere convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, a description of a range should be considered to specifically disclose all possible subranges as well as the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, as well as the individual numerical values within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0023] Description Multiple sclerosis (MS) is the most common autoimmune demyelinating disease of the central nervous system (CNS) (1, 2). Since all current MS therapies target the immune system in an Ag-nonspecific manner, the long-standing goal in MS research has been an MS therapy based on the restoration of antigen (Ag)-specific peripheral immune tolerance (3). A prerequisite for Ag-specific therapy is knowledge of the relevant self-Ags that are the targets of the autoimmune response. The onset of MS is widely believed to be caused by autoimmunity against myelin Ags produced by oligodendrocytes (Ol). However, the relevant Ags in MS still remain speculative and these Ags may vary between patients and over time in the same patient (4). Based on findings in MS experimental models, several approaches for inducing Ag-specific tolerance have been proposed and some of these have been tested in clinical trials (3, 4). It has previously been reported that Ag-specific immune tolerance is induced and the disease is remitted upon administration (5 - 10) of encephalitogenic peptides, or peptides conjugated to nanoparticles or apoptotic cells, via various routes [intravenous (i.v.), oral, nasal, etc.]. Tolerance induction mechanisms include the induction of immunotolerogenic dendritic cells (DC) and immunosuppressive macrophages, a decrease in pathogenic Th1 and Th17 cell responses (11), and the induction of regulatory T (Treg) and type 1 regulatory T (Tr1) cells (12). Although i.v. tolerance induction has shown significant therapeutic effects in experimental autoimmune encephalomyelitis (EAE), the safety of this approach remains a concern because i.v.-injected myelin Ag may exacerbate rather than remit the disease (3, 4, 13).

[0024] In the tests described herein, a novel therapeutic approach for restoring immune tolerance in CNS autoimmunity has been developed by using oligodendrocyte (Ol)-derived extracellular vesicles (Ol-EVs) that naturally contain the most relevant myelin Ags (14). EVs are particles enclosed by a protein-lipid membrane that are secreted by almost all cells and play a crucial role in intercellular communication (15, 16). In numerous studies, EVs have been used for the therapy of experimental autoimmune diseases, and their safety and potential for clinical use have been reported (17 - 20). Intravenous (i.v.) injection of Ol-EVs has been shown to preventively and therapeutically suppress the clinical disease in chronic and relapsing-remitting EAE models. Considering that Ol-EVs lacking the myelin Ag used for EAE induction did not suppress EAE, the effect of Ol-EVs is myelin Ag-dependent. The beneficial effect of Ol-EVs in EAE was monocyte-dependent because monocytes upregulated PD-L1 expression in an IL-10-dependent manner, leading to apoptosis of encephalitogenic CD4+ T cells.

[0025] Overall, the studies herein describe a novel therapeutic approach for specifically treating autoimmune demyelinating diseases of the CNS. The studies described herein have shown that intravenous injection of oligodendrocyte-derived extracellular vesicles has an antigen-specific therapeutic effect in animal models of multiple sclerosis, demonstrating the potential of this novel approach for human disease therapy.

[0026] Although not intended to be bound by theory, the present invention is based in part on the discovery that administration of oligodendrocyte-derived extracellular vesicles can treat multiple sclerosis by inducing tolerance to one or more myelin antigens. Oligodendrocyte-derived extracellular vesicles contain multiple myelin proteins, and thus administration to a subject simultaneously induces tolerance to any antigen that can be a target of MS-related autoimmune attacks. Accordingly, in one aspect, the present invention provides a method of treating or preventing multiple sclerosis in a subject in need thereof, the method comprising administering to the subject an effective amount of oligodendrocyte-derived extracellular vesicles (Ol-EV). In another aspect, the present invention provides a method of inducing tolerance to a myelin antigen in a subject, the method comprising administering to the subject an effective amount of oligodendrocyte-derived extracellular vesicles (Ol-EV).

[0027] In certain embodiments, the Ol-EV comprises a myelin antigen (Ag). In certain embodiments, the myelin Ag is selected from the group consisting of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and myelin proteolipid protein (PLP).

[0028] In certain embodiments, the Ol-EV comprises exosomes. In certain embodiments, the Ol-EV comprises microvesicles. In certain embodiments, the Ol-EV comprises exosomes and microvesicles.

[0029] In certain embodiments, the step of administering the Ol-EV treats MS specifically for the Ag.

[0030] In certain embodiments, the step of administering induces immunosuppressive monocytes. In certain embodiments, the step of administering induces immunosuppressive monocytes in an IL-10-dependent manner.

[0031] In certain embodiments, the monocytes are PD-L1-expressing monocytes.

[0032] In certain embodiments, the administering step does not cause a detrimental or unwanted effect on the immune system of the subject.

[0033] In various embodiments, oligodendrocyte-derived extracellular vesicles are formulated in a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition is administered intravenously, subcutaneously, intradermally, transdermally, orally, or nasally. In various embodiments, the subject is a mammal. In various embodiments, the subject is a human. In various embodiments, the oligodendrocyte-derived extracellular vesicles are derived from human oligodendrocytes.

[0034] In various embodiments, multiple sclerosis is chronic multiple sclerosis. In various embodiments, multiple sclerosis is relapse-remitting multiple sclerosis.

[0035] In various embodiments, oligodendrocyte-derived extracellular vesicles are derived from an in vitro culture of oligodendrocytes. In various embodiments, oligodendrocyte-derived extracellular vesicles are derived from an in vitro culture of human oligodendrocytes. Without being bound by theory, it is believed that oligodendrocyte-derived extracellular vesicles derived from an in vitro culture of oligodendrocytes may have different characteristics than vesicles released by oligodendrocytes in vivo. In some embodiments, the EVs used in the therapy express relatively high levels of several myelin proteins but do not express major histocompatibility complex proteins. In some embodiments, the source cells of the EVs are genetically altered to optimize the quality of the EVs.

[0036] In various embodiments, oligodendrocyte-derived extracellular vesicles may be obtained by performing a first centrifugation step on an oligodendrocyte cell culture supernatant, filtering the resulting supernatant, ultracentrifuging the filtrate, and collecting the pellet-like extracellular vesicles (Casella G et al, 2018. PMD: 30017878; Colombo F et al., 2018. PMD: 29467770).

[0037] In various embodiments, oligodendrocyte-derived extracellular vesicles do not express major histocompatibility complex proteins. In various embodiments, oligodendrocytes derived from extracellular vesicles do not express MHC class II molecules. In various embodiments, MHC class I molecule expression can be blocked using any means known in the art. In various embodiments,

[0038] In another aspect, a pharmaceutical composition is provided that includes oligodendrocyte-derived extracellular vesicles and at least one pharmaceutically acceptable carrier. In another aspect, isolated oligodendrocyte-derived extracellular vesicles are provided. In various embodiments, the isolated oligodendrocyte-derived extracellular vesicles are for use in the treatment of multiple sclerosis (MS). In yet another aspect, a pharmaceutical composition for use in the treatment of multiple sclerosis (MS) is provided, the pharmaceutical composition including oligodendrocyte-derived extracellular vesicles and a pharmaceutically acceptable carrier. In another aspect, the use of oligodendrocyte-derived extracellular vesicles in the treatment of multiple sclerosis (MS) is provided. In various embodiments, the multiple sclerosis is chronic multiple sclerosis. In various embodiments, the multiple sclerosis is relapsing-remitting multiple sclerosis.

[0039] In certain embodiments, oligodendrocyte-derived extracellular vesicles (Ol-EVs) contain myelin antigen (Ag). In certain embodiments, the oligodendrocyte-derived extracellular vesicles are derived from human oligodendrocytes. In various embodiments, the myelin Ag includes myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and / or myelin proteolipid protein (PLP). In various embodiments, the composition comprising the oligodendrocyte-derived extracellular vesicles includes exosomes. In certain embodiments, the composition includes microvesicles. In certain embodiments, the composition includes exosomes and microvesicles. In various embodiments, the composition is formulated for intravenous, subcutaneous, intradermal, transdermal, oral, or nasal administration. In various embodiments, the composition is formulated for intravenous administration.

[0040] It has also been demonstrated herein that the effect of Ol-EVs depends on the myelin Ag present in the Ol-EVs and not on other components specifically produced by the Ol-EVs. Thus, in another aspect, there are provided extracellular vesicles derived from cells that contain myelin antigen (Ag). The cells may be cells other than oligodendrocytes. In certain embodiments, the cells express the myelin antigen. In certain embodiments, the cells are engineered to express the myelin antigen. In various embodiments, the myelin antigen is myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or myelin proteolipid protein (PLP). In various embodiments, the cells are engineered to express one or more types of myelin antigens. In various embodiments, the cells are engineered to express at least one myelin antigen selected from myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and myelin proteolipid protein (PLP). In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells.

[0041] Another aspect provides a method of treating or preventing multiple sclerosis in a subject in need thereof, the method comprising administering to the subject an effective amount of extracellular vesicles (EVs) comprising a myelin antigen. Another aspect of the invention provides a method of inducing tolerance to a myelin antigen in a subject, the method comprising administering to the subject an effective amount of extracellular vesicles (EVs) comprising a myelin antigen.

[0042] In various embodiments, the EVs are derived from cells that express a myelin antigen. In certain embodiments, the cells are engineered to express a myelin antigen. In certain embodiments, the cells are engineered to express a myelin antigen at a high level. In certain embodiments, the cells do not express or have reduced expression of major histocompatibility proteins (e.g., MHC class I, MHC class II molecules). In certain embodiments, the cells are genetically engineered to reduce or eliminate the expression of major histocompatibility proteins (e.g., MHC class I, MHC class II molecules).

[0043] In certain embodiments, the EVs comprise a myelin antigen (Ag). In certain embodiments, the myelin Ag is selected from the group consisting of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and myelin proteolipid protein (PLP).

[0044] In certain embodiments, the EVs comprise exosomes. In certain embodiments, the Ol-EVs comprise microvesicles. In certain embodiments, the EVs comprise exosomes and microvesicles.

[0045] In certain embodiments, the administering step does not cause a detrimental or undesirable effect on the subject's immune system.

[0046] In various aspects, the extracellular vesicles are formulated in a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier. In various aspects, the pharmaceutical composition is administered intravenously, subcutaneously, intradermally, transdermally, orally, or nasally. In various aspects, the subject is a mammal. In various aspects, the subject is a human. In various aspects, the extracellular vesicles are derived from human cells.

[0047] In various aspects, the multiple sclerosis is chronic multiple sclerosis. In various aspects, the multiple sclerosis is relapsing-remitting multiple sclerosis.

[0048] In another aspect, there is provided a pharmaceutical composition comprising extracellular vesicles comprising a myelin antigen and at least one pharmaceutically acceptable carrier. In another aspect, there is provided an isolated extracellular vesicle comprising a myelin antigen. In various aspects, the isolated extracellular vesicles are for use in the treatment of multiple sclerosis (MS). In yet another aspect, there is provided a pharmaceutical composition for use in the treatment of multiple sclerosis (MS), the pharmaceutical composition comprising extracellular vesicles comprising a myelin antigen and a pharmaceutically acceptable carrier. In another aspect, there is provided the use of extracellular vesicles in the treatment of multiple sclerosis (MS), wherein the extracellular vesicles comprise a myelin antigen. In various aspects, the myelin antigen is myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or myelin proteolipid protein (PLP). In various aspects, the extracellular vesicles comprise one or more myelin antigens selected from myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or myelin proteolipid protein (PLP). In various aspects, the multiple sclerosis is chronic multiple sclerosis. In various aspects, the multiple sclerosis is relapsing-remitting multiple sclerosis.

[0049] Administration / Dosing In a clinical setting, the delivery system for the compositions described herein can be introduced into a subject by any of a number of methods, each of which is well known in the art. For example, the pharmaceutical formulation of the composition may be administered by inhalation or, for example, systemically by intravenous injection.

[0050] The dosing schedule may affect what constitutes an effective amount. For example, a therapeutic formulation may be administered to a subject before or after the onset of symptoms associated with the disease or condition. Further, several divided doses as well as staggered doses may be administered daily or continuously. Alternatively, the dose may be administered by continuous infusion or by bolus injection. Further, the dosage of the therapeutic formulation may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0051] The composition of the present invention can be administered to a subject, preferably a mammal, more preferably a human, in an effective dose and for a period effective to treat a disease or disorder in the subject using known procedures. The effective amount of the composition required to achieve a therapeutic effect will vary according to factors such as the time of administration, the duration of administration, other drugs, compounds, or materials used in combination with the composition, the condition of the disease or disorder, the age, sex, weight, condition, general health, and medical history of the subject being treated; as well as similar factors well known in the medical field. The dosing schedule may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily. Or the dose may be proportionally reduced as indicated by the exigencies of the treatment situation. One of ordinary skill in the art will be able to study the relevant factors and make a determination regarding the effective amount of the composition without undue experimentation. The formulations may be mixed with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral administration, parenteral administration, nasal administration, intravenous administration, subcutaneous administration, enteral administration, or any other suitable mode of administration known in the art. The formulations may be sterilized and, if desired, may be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts for influencing osmotic pressure, buffers, coloring substances, flavoring substances, and / or aromatic substances. These may, if desired, be combined with other active agents, such as other analgesics.

[0052] The routes of administration of any composition of the present invention include oral administration, nasal administration, rectal administration, intravaginal administration, parenteral administration, buccal administration, sublingual administration, or topical administration. Compounds or agents (e.g., extracellular vesicles (EVs)) for use in the present invention may be formulated for administration by any suitable route, such as oral administration or parenteral administration, e.g., transdermal administration, transmucosal administration (e.g., sublingual administration, lingual administration, (transbuccal) buccal administration, (transurethral) urethral administration, vaginal administration (e.g., transvaginal administration and perivaginal administration), (intranasal) nasal administration, and (transrectal) rectal), intravesical administration, intralung administration, intraduodenal administration, intragastric administration, intrathecal administration, subcutaneous administration, intramuscular administration, intradermal administration, intraarterial administration, intravenous administration, intratracheal administration, inhalation administration, and topical administration.

[0053] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powders or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, and the like. It should be understood that the formulations and compositions useful in the present invention are not limited to the specific formulations and compositions described herein.

[0054] Oral administration In the case of oral application, tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gel caps are particularly suitable. Compositions for oral use can be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose; granulating and disintegrating agents such as corn starch; binders such as starch; and lubricants such as magnesium stearate. Tablets may or may not be coated and may be coated using known methods for the accuracy or delay of active ingredient release. Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert diluent.

[0055] In the case of oral administration, the compounds of the present invention may be in the form of tablets or capsules prepared by conventional means using pharmaceutically acceptable excipients such as binders (e.g., polyvinylpyrrolidone, hydroxypropylcellulose, or hydroxypropylmethylcellulose); diluents (e.g., corn starch, lactose, crystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). If desired, the tablets may be coated by appropriate methods and coating materials such as the OPADRY™ film coating system available from Colorcon, West Point, Pa (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type, and OPADRY™ White, 32K18400). Liquid preparations for oral administration may be in the form of solutions, syrups, or suspensions. The liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl p-hydroxybenzoate or propyl p-hydroxybenzoate or sorbic acid).

[0056] Parenteral administration In the case of parenteral administration, the compounds or agents of the present invention (e.g., extracellular vesicles (EVs)) may be formulated for injection or infusion, e.g., intravenous, intramuscular, or subcutaneous injection or infusion, and may be formulated for administration in bolus doses and / or continuous infusion. Optionally, suspensions, solutions, or emulsions dissolved in an oily vehicle or an aqueous vehicle containing other formulatory agents such as suspending agents, stabilizers, and / or dispersing agents may be used.

[0057] Xu Fang Formulations and Drug Delivery Systems In certain embodiments, the formulations of the present invention may be short-term formulations, rapid-offset formulations, as well as sustained release formulations, such as, but not limited to, sustained release formulations, delayed release formulations, and pulsatile release formulations.

[0058] The term “sustained release” is used in its conventional sense and refers to a pharmaceutical formulation that releases a drug gradually over an extended period of time and, although not necessarily, may result in a substantially constant drug blood concentration over an extended period of time. The period may be as long as one month or more and should result in a longer release than an equivalent dose of the drug administered in bolus form.

[0059] In the case of sustained release, the compound may be formulated using a suitable polymeric material or hydrophobic material that imparts sustained release properties to the compound. Thus, the compound for use in the methods of the present invention may be administered, for example, by injection in the form of microparticles, or by implantation in the form of a cachet or disk.

[0060] In certain embodiments, the compounds of the present invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0061] The term “delayed release” is used in its conventional sense and, as used herein, refers to a pharmaceutical formulation that first releases a drug after some delay following administration of the drug and, although not necessarily, may include a delay of from about 10 minutes to about 12 hours.

[0062] The term “pulsatile release” is used in its conventional sense and, as used herein, refers to a pharmaceutical formulation that releases a drug in a manner that results in a pulsed drug plasma profile following administration of the drug.

[0063] The term "immediate release" is used in its conventional meaning and refers to a pharmaceutical formulation that releases a drug immediately after administration.

[0064] As used herein, "short-term" refers to about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any and all whole or partial increments thereof, and any period including these.

[0065] As used herein, "rapid onset of disappearance" refers to about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any and all whole or partial increments thereof, and any period including these.

[0066] Drug administration The therapeutically effective amount or dose of the compounds or agents (e.g., extracellular vesicles (EVs)) of the present invention depends on the age, sex, weight of the patient, the current medical condition of the patient, and the progression of the disease or disorder intended herein in the patient being treated. One of ordinary skill in the art can determine an appropriate dosage according to these factors and other factors.

[0067] An appropriate dosage of the compounds of the present invention may be in the range of about 0.001 mg to about 5,000 mg / day, for example, about 0.01 mg to about 1,000 mg, for example, about 1 mg to about 500 mg, for example, about 5 mg to about 250 mg / day. This dosage may be administered as a single dose or as multiple doses, for example, 1 to 4 times or more per day. When multiple doses are used, the amount of each dose may be the same or different. For example, a daily dose of 1 mg may be administered as two 0.5 mg doses at an interval of about 12 hours between doses.

[0068] The amount of the compound administered on a daily basis may be understood to be administered daily, every other day, every two days, every three days, every four days, or every five days in non-limiting examples. For example, when administered every other day, a dose of 5 mg per day is initiated on Monday, and on Wednesday, a first dose of 5 mg is administered on the following day, and on Friday, a second dose of 5 mg is administered on the following day.

[0069] The actual dosage level of the cells in the pharmaceutical formulation of the present invention may be varied so as to obtain an amount of the composition effective to achieve the desired therapeutic response for a particular subject, composition, and method of administration without toxicity to the subject.

[0070] The toxicity and therapeutic efficacy of such treatment regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, determination of the LD 50 (dose that causes death in 50% of the population) and the ED 50 (dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and is expressed as the ratio of LD 50 to ED 50 . When formulating a range of dosages for use in humans, data obtained from cell culture assays and animal studies are optionally used. The dosage of such a compound preferably falls within a range of circulating concentrations that includes the ED 50 with minimal toxicity. Optionally, the dosage varies within this range depending on the dosage form used and the route of administration utilized.

[0071] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

[0072] Although the present invention has been disclosed with respect to specific embodiments, it is apparent that other embodiments and variations of the present invention can be devised by those skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Example

[0073] Experimental Example The present invention will be further described in detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples, but rather should be construed as encompassing any and all variations that become apparent as a result of the disclosure provided herein.

[0074] Without further elaboration, it is believed that one of ordinary skill in the art can, using the foregoing description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. Accordingly, the following practical examples are specifically pointed out as selected embodiments of the present invention and should not be construed as limiting the remainder of the disclosure.

[0075] The materials and methods used in these experiments will now be described.

[0076] Mouse B10.PL, SJL, C56BL / 6 WT, B6.Ly5.1(CD45.1 + )、RAG1 - / - 、2D2、OT-II、Zbtb46 iDTR、ROSA26-stop-Tdtomato、IL-10Rβ - / - 、IL-10 - / -and Rosa26-LSL-Cas9 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were maintained under specific pathogen-free conditions with a 12 / 12 h light / dark cycle and ad libitum feeding throughout the experimental procedures, with a maximum of 5 mice per cage. Every effort was made to minimize mouse suffering. The experimental protocol using mice was approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University.

[0077] HEK cells HEK cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% EV-depleted fetal bovine serum (FBS), penicillin, streptomycin (100 U / ml), and 2 mM L-glutamine. To collect all cell culture supernatants for EV isolation, FBS depleted of EVs by ultracentrifugation overnight at 4 °C at 110,000 g was added to the medium. All cells were maintained at 5% CO 2 , 37 °C.

[0078] PDGFRα + Cell isolation Mouse whole brains were harvested from 5-day-old C56BL / 6 and Rosa26-LSL-Cas9 pups, dissociated manually, and enzymatically digested using a neural dissociation kit (Miltenyi). The suspension was quenched with DMEM (Gibco) supplemented with 10% EV-depleted FBS and centrifuged at 1200 rpm for 5 min. The tissue was then homogenized by passing it through an 18-gauge needle and then filtered through a 70 μm cell strainer (Fisher) to remove remaining debris. PDGFRα+ cells were isolated from this cell suspension using positive selection with a magnetic beads separation kit (Miltenyi).

[0079] Culture of OPCs and mature Ols PDGFRα + Cells were plated in an OPC differentiation medium consisting of DMEM / F12, N-2, B-27, Glutamax (2 mM), SHH (200 ng / mL), β-FGF, PDGF-AA (20 ng / mL), and Normycin, and incubated at 37 °C in 5% CO 2 2. After 3 - 5 days, the medium was replaced with fresh Ol maturation medium consisting of DMEM / F12, N-2, and B-27, supplemented with Glutamax (2 mM), T3 (40 ng / mL), SHH (200 ng / mL), Noggin (100 ng / mL), cAMP (50 μM), TGF (100 ng / mL), and NT3 (10 ng / mL). Cells were maintained in the Ol maturation medium for up to 3 weeks, with the medium being changed every 5 days.

[0080] Human OPCs derived from H9 human ESCs (Millipore) approved by the NIH were cultured for 3 weeks and differentiated into mature Ols according to the Millipore protocol.

[0081] Cell transduction OPCs and HEK cells were transduced with a lentivirus encoding Cre recombinase (Lv-Cre; Addgene #12106) or a lentivirus encoding mouse MOG (Lv-MOG, Origene). Briefly, approximately 2x10 6 cells were transduced with Lv-Cre or Lv-MOG in complete medium supplemented with 10% EV-depleted serum for HEK cells. In contrast, for OPCs, the same medium described in the previous section was used. EVs were purified from the cell culture supernatant of HEK cells after 2 - 3 days and from mature Ols after 2 - 3 weeks.

[0082] MOG - / - Generation of Ols PDGFRα+ cells were isolated from the brains of Rosa26-LSL-Cas9 offspring. OPCs were transduced with a lentivirus expressing Cre and MOG sgRNA or scrambled sgRNA. Mature MOG - / -Ol was obtained by puromycin selection. MOG knockout was confirmed by PCR and Duoset ELISA (LSBio) in both Ol and Ol-EV.

[0083] MOG + Generation of HEK cells HEK cells were transduced with lentivirus encoding MOG. MOG + HEK cells were obtained by puromycin selection in complete medium supplemented with 10% EV-depleted serum. MOG expression was confirmed by immunofluorescence and Duoset ELISA (LSBio) in both HEK cells and HEK-EV.

[0084] CRISPR / CAS9 lentiCRISPR v2 was purchased from Addgene (plasmid number 52961). The Cre gene was amplified using TIFF2025081585000002.tif12169 and used to replace the Cas9 sequence in lentiCRISPR v2 via XbaI and BamHI enzyme sites (the XbaI site was then removed after ligation). Then, a new XbaI site was introduced behind the KpnI site for multiple sgRNA expression. The final plasmid was named Lenti-sgRNA backbone-EFS-Cre-P2A-puro.

[0085] MOG sgRNA was digested using Benchling (https: / / www.benchling.com / crispr / ), oligos were synthesized from IDT, and annealed at room temperature to obtain sgRNA. The sgRNA fragments were inserted separately into pLenti-sgRNA backbone-EFS-Cre-P2A-puro via BsmBI. The sgRNA activity was analyzed in the N2A-Cas9 cell line, and the sgRNAs with high activity were selected for further use.

[0086] (Table 1) Sequences of sgRNA oligos and detection primers TIFF2025081585000003.tif82170

[0087] EV purification EVs were purified from cell culture supernatants using a standardized protocol (17). The supernatants were collected and centrifuged at 300 g for 10 minutes to remove cells and debris. The resulting supernatant was further clarified with a 0.45 μm syringe filter (Millex, Millipore) and then ultracentrifuged at 100,000 g for 2 hours to pellet the EVs. The pellet was resuspended in lysis buffer containing protease inhibitor, 0.1 μm filtered PBS, or fixative according to the purpose of EV use.

[0088] Nanoparticle tracking analysis (NTA) of EVs EVs were resuspended in 0.1 μm filtered PBS and diluted 1:100 or 1:1000. Samples were analyzed using NTA 3.1 Build 3.1.46 software and an NS 300 instrument (Malvern Instruments, MA).

[0089] Mass spectrometry and data processing Liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis was performed using a Q Exactive HF mass spectrometer (ThermoFisher Scientific) coupled with a Nano-ACQUITY UPLC system (Waters). Samples were digested in-gel with trypsin and injected onto a UPLC Symmetry trap column (180 μm i.d. x 2 cm, packed with 5 μm C18 resin; Waters). Trypsin peptides were separated by reverse-phase HPLC on a BEH C18 nanocapillary analytical column (75 μm i.d. x 25 cm, 1.7 μm particle size; Waters) using a 240 min gradient formed by solvent A (0.1% formic acid dissolved in water) and solvent B (0.1% formic acid dissolved in acetonitrile). The eluted peptides were analyzed by a mass spectrometer set to repeatedly scan at m / z 400 - 2000 in positive ion mode. Full MS scans were collected at 60,000 resolution, followed by data-dependent MS / MS scans at 15,000 resolution for the 20 most abundant ions above a minimum threshold of 10,000. Peptide match was set to preferred. Isotope option was excluded. Charge-state screening was enabled to reject unassigned, singly charged ions. Peptide sequences were identified using MaxQuant 1.6.2.3 (39). MS / MS spectra were searched against the UniProt mouse protein database (October 2017) and a common contaminants database using full trypsin specificity with up to two missed cleavages, static carboxamidomethylation of Cys, variable oxidation of Met, and protein N-terminal acetylation. The "match between runs" function was used to help transfer identifications across multiple experiments to minimize missing values.A consensus identification list was generated and the false discovery rate for protein and peptide identification was set at 1%.

[0090] EAE induction and scoring EAE was induced as previously described ( 11 , 40 , 41 ). The EAE immunization protocol is summarized in Table 2 .

[0091] Mice were weighed and scored daily for clinical signs. Clinical assessment of EAE was performed according to the following scoring criteria: 0=healthy; 1=floppy tail; 2=ataxia and / or hind limb paresis; 3=hind limb paralysis and / or forelimb paresis; 4=tetraparalysis; and 5=moribund or dead (42).

[0092] Table 2 EAE model TIFF2025081585000004.tif109170

[0093] Bone marrow chimera B6.Ly5.1(CD45.1 + ) Congenic hosts were lethally irradiated with 2x2.5Gy doses with an 8-hour interval between doses and 5x10 6 CD45.2 + Bone marrow cells were reconstituted by tail vein injection. Mice were reconstituted for 6 weeks before use.

[0094] DT ablation One day before iv injection of EVs, diphtheria toxin (DTX; Sigma-Aldrich) was administered ip at 1 μg / 20 g mouse in 200 μl PBS. Mice were injected twice with DTX.

[0095] PD-L1 blockade and Ly6g depletion One day before EV injection, WT and R26-stop-Tdtomato EAE mice were injected i.p. with 200 μg / mouse of αPD-L1 Ab (clone 10F.9G2, BioXCell) or 200 μg / mouse of αLy6g Ab (clone 1A8, BioXCell). Mice were injected with Ab twice for each treatment.

[0096] i.v. administration of self-Ag and EV As previously described (11), i.v. tolerance was induced. Briefly, after disease onset, each mouse was given 200 μg of MOG 35-55 , 400 μg of MBP Ac(1-11) , 100 μg of PLP 139-151 , or at least 10 10 individual EVs dissolved in PBS, for a total of 3 times every 3 days. Control mice were given only PBS.

[0097] Ag-specific recall response EAE mice were sacrificed, and their draining lymph nodes and spleens were dissociated with a 70 μm strainer and a single cell suspension was prepared in IMDM supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U), streptomycin (10 μg / mL), L-glutamine (0.3 mg / mL), and 2-mercaptoethanol (55 μM). After treatment with RBC lysis buffer (Biolegend, CA, USA), the cells were extensively washed with complete IMDM by centrifugation at 1,300 rpm for 5 minutes at 4 °C, and the cell density was adjusted to 2x10 6 / mL. 100 μL of the adjusted cell suspension was added to each well of a 96-well plate. MOG 35-55 was added to a final concentration of 20 μg / mL. The cells were incubated at 37 °C for 72 hours. For negative controls, the cells were cultured without MOG 35-55 . Cell culture supernatants were collected and stored at -20 °C until use, and cell proliferation and cytokine production were analyzed by flow cytometry.

[0098] Reconstitution of WT and RAG1 - / - mice 2 x 10 cells from the spleen and CNS of Rosa26-stop-tdtomato EAE mice 6 sorted Tdtomato+CD11b + CD11c + Ly6c + cells were given i.v. to WT EAE mice. 3 x 10 - / - total CD4+ T cells isolated with magnetic beads from the spleen of WT and PD1 6 mice were used i.v. to reconstitute RAG1- / - mice. Mice were immunized for EAE induction 72 hours after adoptive transfer.

[0099] Histological evaluation At least 5 mice / group were perfused for 10 minutes through the left ventricle of the heart with saline containing 0.5 mM EDTA and then fixed with cold 4% paraformaldehyde (PFA; Sigma-Aldrich). Spinal cords and brains from EAE mice were dissected and post-fixed with 2% PFA overnight. The following stains were used: Kluver Barrera (demyelination), Bielshowsky (axonal injury). The number of perivascular inflammatory infiltrates was counted and expressed as the number of inflammatory infiltrates per mm 2 The demyelinated area and axonal loss were expressed as a percentage of the damaged area.

[0100] Cryoelectron microscopy 3 microliters of EV sample was applied onto a 200 mesh copper grid (Quantifoil R1.2 / 1.3) that had been subjected to glow discharge treatment for 60 seconds. Excess solution was blotted with filter paper for 6 seconds using a Vitrobot Mark IV (FEI Netherlands) at 4 °C, and the grid was immediately plunge-frozen by quickly immersing it into liquid ethane at -165 °C. Cryo-EM data for both samples were collected using a Tecnai F 200 KeV TEM microscope operated at 200 keV. Images were detected at a magnification of 25,000X using a Falcon III direct electron detector. Individual dose fractionated frames collected at a rate of 40 frames / s during a 4-second exposure were averaged to create each micrograph. The frames were motion corrected to form a single micrograph. The collected micrographs were in focus and in the range of 2.0 - 4.0 μm.

[0101] Fluorescence microscope Ol was fixed with 4% PFA at 4 °C for 15 minutes, quenched with 0.1 M glycine, and processed for indirect immunofluorescence. A Nikon NX1 (Nikon Microsystems) confocal microscope was used for image acquisition. Images were analyzed using ImageJ software (GraphPad). Anti-MBP (ThermoFisher) and anti-MOG (Millipore) were used as primary antibodies.

[0102] Spinal cord sections of EAE mice were washed twice with PBS 1X and incubated for up to 1 hour at room temperature in blocking solution PBS with or without 0.1% Triton (depending on what Ag is) and 10% serum of the secondary Ab species. The primary antibody was diluted with blocking mix (1% serum) and incubated overnight at +4°C. A Nikon NX1 (Nikon Microsystems) confocal microscope was used for image acquisition. Images were analyzed using ImageJ software (GraphPad). Anti-CD11b (Abcam) and anti-arginase 1 (GeneTex) were used as primary antibodies.

[0103] ELISA Mouse and human MBP and PLP1 in EV pellets were measured by ELISA (Biomatik and LSBio). MOG in WT Ol, Ol-EV, MOG- / -Ol, and MOG- / -Ol-EV ELISA (LSBio) was measured.

[0104] Measurement of MOG-specific Ig in the sera of EAE mice ELISA plates were coated overnight at 4°C with 10 μg / ml MOG dissolved in PBS 35-55 peptide. The plates were blocked with 2% BSA dissolved in PBS for 2 hours at 37°C. Sera were diluted 1:100 with blocking buffer and added to the plates for incubation overnight at 4°C. Sera from WT C57BL / 6 mice injected with Ol-EV were applied to the plates without prior dilution. Peroxidase-conjugated goat α-mouse secondary Ab (Thermo Scientific) and tetramethylbenzidine (BioFX Laboratories) were used for 30 minutes at room temperature to detect bound αMOG Ab derived from the sera.

[0105] Western blot analysis 20 μg of cellular protein and 5 - 10 μg of EV were diluted with Laemmli buffer and loaded onto an 8 - 14% polyacrylamide gel. The purified EV was resuspended in lysis buffer supplemented with protease inhibitor cocktail (Sigma - Aldrich). Protein concentration was measured by BCA (Micro BCA, Pierce). Mouse anti - mouse flotillin 1 (BD Bioscience), rabbit anti - Alix (Millipore), goat anti - Tsg101 (Millipore), mouse anti - MOG (Millipore), and rabbit Gapdh (Cell Signaling) were used as primary antibodies.

[0106] Isolation of CNS - infiltrating leukocytes Brain and spinal cord tissues were incubated with 0.4 mg / mL type IV collagenase (Sigma - Aldrich) at 37 °C for 30 min and dissociated using a 19 - gauge needle to obtain a homogeneous cell suspension. Finally, CNS cells were concentrated by centrifugation on a Percoll gradient as previously described (43).

[0107] Flow cytometry and cell sorting Flow cytometry was performed using a FACSaria II (Becton Dickinson) and analyzed by FlowJo software (Tree Star). Fluorescent dye - conjugated Mabs specific for CD45 (clone 30 - F11), CD45.1 (A20), CD11b (M1 / 70), CD3 (17A2), CD8α (53 - 6.7), CD4 (RM4 - 5), CD19 (1D3 / CD19), CD11c (N418), PDCA1 (927), Ly6c (AL - 21), F4 / 80 (MB8), Ly6g (1A8), MHC - II (M5 / 114.15.2), PD - 1 (29F.1A12), PD - L1 (10F.9G2), caspase 3 (catalog number 550480), CCR2 (47503), MBP (P82H9 FITC), MOG (sc - 166172 PE), and PLP (ab28486) were purchased from BD Biosciences, R&D, Biolegend, Santa Cruz, or Abcam.

[0108] For intracellular staining, cells were stimulated with phorbol 12-myristate 13-acetate (50 ng / ml, Sigma-Aldrich) and ionomycin (500 ng / ml, Sigma-Aldrich) in the presence of GolgiPlug (1:1000, BD Pharmigen) for 4 hours, permeabilized using the Cytofix / Cytoperm Plus kit (BD Bioscience), and stained with the following fluorochrome-conjugated MAbs obtained from Biolegend and BD Pharmingen: CNPase (836408 alexa fluor 647), GM-CSF (MP1-22E9), IL-17A (TC11-18H10.1), IL-10 (JES5-16E3), IFN-γ (XMG1.2), Zbtb46 (U4-1374). Dead cells were excluded using L / D BD Pharmingen.

[0109] qPCR Total RNA was extracted from OPCs, mature Ols, moDCs, and neutrophils using the RNeasy Mini Kit (Qiagen). Genomic DNA was removed by treatment with DNase I (Qiagen). cDNA synthesis was performed using the ThermoscriptTM RT-PCR system (Invitrogen). Pdgfrα (Mm00440701_m1); ng2 (Mm00507257_m1); sox10 (Mm01300162_m1); olig2 (Mm01210556_m1); mobp (Mm02745649_m1); mag (Mm00487538_m1); plp1 (Mm01297210_m1); mog (Mm01279062_m1); cnp (Mm01306641_m1); mbp (Mm01262037_m1); galc (Mm01337517_m1); Arg-1 (Mm00475988_m1); pd-l1 (Mm03048248_m1); stat3 (Mm01219775_m1); irf1 (Mm01288580_m1); il-10 (Mm00439614_m1); tim-3 (Mm00454540_m1); pd-l2 (Mm00451734_m1); tgf-β (Mm01178820_m1); tgf-α (Mm00446232_m1); icosL (Mm00497237_m1); il-27 (Mm00461162_m1); casp3 (Mm01195085_m1); ccl2 (Mm00441242_m1); tnf-α (Mm00443258_m1); il-23 (Mm00518984_m1); inos (Mm00440502_m1); il-1β (Mm00434228_m1); cd-80 (Mm00711660_m1); cd-86 (Mm00444540_m1), and gapdh (4352339E). mRNA levels were measured by real-time RT-PCR (Applied Biosystems, Invitrogen). The relative change in gene expression was calculated using the 2-ΔΔCT method (44).

[0110] Statistical analysis Statistical analysis was performed using GraphPad Prism 8 software. Statistical evaluations were presented as mean ± s.d. or mean ± s.e.m. as appropriate. Results were analyzed using two-way or one-way ANOVA and post hoc tests were performed with Bonferroni and independent two-sided Student's t-tests. Statistical significance was * p < 0.05; ** p < 0.001; *** ranked as p < 0.0001.

[0111] The results of this experiment will now be explained.

[0112] Example 1: Mature Ols release EVs containing the most relevant myelin Ags To generate Ol-EVs, mouse CNS PDGFR+ cells were harvested, differentiated into oligodendrocyte progenitor cells (OPCs), and finally into mature Ols (21). After 3 weeks of culture, more than 60% of the OPCs became mature Ols (CNPase + and GalC high ) and expressed myelin proteins: myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and myelin proteolipid protein (PLP) (Figure 1A and Figures 8A - 8E). OPCs and mature Ols produced a large amount of EVs with an average diameter of 240 nm when confirmed by cryo-EM and nanoparticle tracking analysis (NTA) (Figure 1B and Figure 8G). Mass spectrometry of Ol-EVs and major EV markers according to the minimal information for studies of extracellular vesicles (MISEV) guidelines (22) are shown in Figure 1C and Figure 8H. Multiple myelin proteins including MBP, MOG, and PLP were also detected (Figure 1D), and these levels were quantified by ELISA (Figure 1E).

[0113] To determine whether Ol-EV could be harmful to mice, Ol-EV was administered i.v. to naive C57BL / 6 mice at 3-day intervals for a total of six injections. As a control, HEK cell-derived EV (HEK-EV) was injected. No effects of Ol-EV and HEK-EV injections on mice were observed (Figure 1F), and antibodies against MOG (contained in the injected Ol-EV) were not detected 1 month after the start of Ol-EV administration (Figure 1G). Overall, these data indicate that mature Ols release EVs containing multiple myelin Ags and that Ol-EV is well tolerated in vivo.

[0114] Example 2: i.v. injection of Ol-EV suppresses disease in several active EAE models To determine whether Ol-EV could restore immune tolerance in EAE, the effects of Ol-EV were tested in three active EAE models with clinical disease of chronic (MOG 35-55 / C57BL / 6, MBP Ac(1-11) / B10.PL) and relapsing-remitting (PLP 139-151 / SJL) courses. Syngeneic Ol-EV (Ol-EV / i.v.) dissolved in PBS was administered i.v. three times before the onset of clinical disease or 3 days after disease onset. Control mice were injected i.v. with PBS (sham treatment), immune peptides dissolved in PBS, or HEK-EV dissolved in PBS. Ol-EV significantly ameliorated clinical disease in both the prophylactic and therapeutic regimens in all three EAE models, while HEK-EV had no effect (Figures 2A-2F). The therapeutic effect persisted until at least 2 weeks after the last injection, when the mice were sacrificed. In the PLP 139-151 / SJL EAE model, Ol-EV treatment had a significant therapeutic effect but was somewhat less efficient than the other two EAE models in suppressing the ongoing disease. The relative resistance of PLP 139-151 / SJL EAE to i.v. tolerance induction has been reported by other investigators (23). The therapeutic efficacy of Ol-EV in adoptive EAE was also examined. In this adoptive EAE, MOG derived from donor EAE mice35-55 Specific CD4 + T cells were transplanted into recipient naive C57BL / 6 mice. Ol-EV treatment halted EAE progression (Figs. 9A - 9C), and thus, similar therapeutic effects were shown in both active EAE and adoptive EAE. In contrast to i.v. administration, subcutaneously (s.c.) injected Ol-EV did not ameliorate EAE (Figs. 10A, 10B). This suggests that the i.v. route may be extremely important for inducing tolerance by Ol-EV.

[0115] Consistent with the remission of clinical disease, Ol-EV treatment protected EAE mice from neuropathological signs, demyelination, and axonal injury (Figs. 10C, 10D). Furthermore, Ol-EV treatment reduced the number of infiltrating CD45 + and CD4 + cells in the CNS, and splenocytes significantly reduced the recall response to immune peptides (Figs. 10E - 10H).

[0116] Significant therapeutic benefits were obtained in EAE by i.v. injection of free encephalitogenic peptides, but repeated injection of these may induce anaphylactic shock and death in a number of mice (24). Overall, the effect of Ol-EV was similar to that of the peptides included as positive controls in these experiments. However, Ol-EV / i.v. was found to be safer than peptide / i.v. (Fig. 2G).

[0117] These data indicate that i.v. injection of Ol-EV suppresses ongoing clinical disease in multiple EAE models.

[0118] Example 3: The suppressive effect of Ol-EV in EAE is myelin Ag-dependent To elucidate the mechanism by which Ol-EV suppresses EAE, first, MOG 35-55The temporal effect of Ol-EV on blood T cells was confirmed using specific T cell receptor transgenic 2D2 mice. When Ol-EV was injected i.v. into 2D2 mice, the number of CD4 + T cells decreased in peripheral blood (Figure 3A) and spleen (Figures 11A - 11C), but the reaction rate was much slower than that of MOG 35-55 / i.v. injection. CD4+ T cells became caspase 3 + positive 24 hours after Ol-EV injection. In contrast, MOG 35-55 induced active caspase 3 expression only 6 hours later (Figures 3B, 3C). These data suggest that apoptosis of CD4 + T cells was Ag-specific as it was not induced by HEK-EV treatment. The delayed effect of Ol-EV compared to free peptides is likely due to the time required to process and present the full-length MOG protein from the vesicles. In contrast, presentation of the injected MOG 35-55 peptide occurs through a different pathway and is faster than Ol-EV. Perhaps the much larger amount of the injected free peptide compared to the peptides generated by processing of the MOG protein from the vesicles also facilitates the rapid response to this peptide.

[0119] To further explore the effect of Ol-EV on T cell activation, CFSE-labeled naive CD4 + T cells specific for MOG(2D2) or OVA(OT-II) were adoptively transferred into CD45.1 + naive mice, and 2 days later the inventors injected Ol-EV i.v. When confirmed by production of IFN-γ and IL-17A (Figures 3D, 3F) and CFSE dilution (Figures 3G, 3H), the effect of Ol-EV was Ag-specific as it induced only activation and proliferation of MOG-specific CD4 + T cells and did not induce activation and proliferation of OVA-specific CD4 + T cells. Furthermore, Ol-EV induced significant PD-1 expression in 2D2 but not in OT-II CD4 + T cells (Figures 3E, 3I). MBPAc(1-11) CD4 in T cell receptor transgenic mice + Similar results were obtained using T cells (25) (data not shown), demonstrating that these effects were not restricted to MOG-specific T cells. Overall, these data suggest that in vivo treated, CD4 + It can be seen that Ol-EVs deliver myelin Ag that is presented to T cells.

[0120] Finally, to determine whether Ol-EV / iv suppresses EAE in a myelin Ag-dependent manner, we used C57BL / 6 / MOG 35-55 -EAE mice were injected with MOG-deficient Ol-EVs. Ac(1-11) We injected MBP-deficient Ol-EVs into EAE mice. MOG-deficient Ol-EVs were generated using the CRISPR / Cas9 system. Ol-EVs were derived from Ols of Cas9-transgenic mice transduced with lentivirus containing MOG-specific sgRNA and Cre. In contrast, control Ol-EVs were derived from Ols of Cas9-transgenic mice transduced with lentivirus containing scrambled sgRNA and Cre (Figures 12A-12C). MOG knockout in Ols and the resulting EVs was confirmed by PCR and ELISA (Figures 12D, 12E). MBP-deficient Ol-EVs showed a significant reduction in MBP expression. - / - The Ol-EVs were generated from Ol-transplant mice, which are “shiverer mice,” a type of mouse model of EAE (26). In both EAE models, myelin Ag-deficient Ol-EVs did not suppress disease (Fig. 3J and K), demonstrating that Ol-EV / iv suppresses EAE in an Ag-dependent manner.

[0121] To test whether the inhibitory effect of Ol-EVs on EAE was solely dependent on myelin Ags and not on other components present in Ol-EVs that are specifically produced by Ol, we engineered HEK cells to express mouse MOG. We confirmed that the EVs of these cells also contained MOG (Figures 13A-13C). Next, we used C57BL / 6 / MOG 35-55HEK / MOG-EV or Ol-EV was injected into EAE mice. Both treatments had a similar inhibitory effect on EAE (Figs. 13D - 13F). From this, it was confirmed that the effect of Ol-EV depends on the myelin Ag present in Ol-EV, but not on other components specifically produced by Ol.

[0122] Example 4: Ol-EV / i.v. is preferentially taken up by monocytes and neutrophils Cultured Ol expresses very low levels of MHC class II molecules as shown by flow cytometry analysis (Fig. 14). Or, Ol-EV does not express these molecules when confirmed by mass spectrometry (data not shown). This is often seen in Ol under non-inflammatory conditions (27, 28). Therefore, the possibility that Ol-EV directly presents myelin Ag to CD4 + T cells is eliminated. A hypothesis was established that i.v.-injected Ol-EV is taken up by phagocytic APCs, and the phagocytic APCs process these proteins and present them to encephalitogenic Th cells on MHC class II.

[0123] To clearly determine which cells take up Ol-EV / i.v. and present myelin Ag, Cre recombinase-containing Ol-EV was produced by transducing OPCs with Cre-expressing lentivirus (data not shown). Naïve Rosa26.stop.Td-tomato reporter mice were injected i.v. with Cre + Ol-EV, and the mice were sacrificed at various times (6 hours, 24 hours, and 48 hours) after injection (Figs. 15A, 15B). The majority of Td-tomato + cells were phagocytes in the spleen and blood, such as monocytes (43%), neutrophils (28%), and different DC subsets (26%). In contrast, only 4% of B cells were Td-tomato + positive, and there were few CD3 + T cells that were Td-tomato + positive (Figs. 15C, 15D). Td-tomato +Cells were not found in either the lymph nodes (LNs) or the central nervous system (CNS). This demonstrated that Ol-EVs did not reach the LNs and did not cross the intact blood-brain barrier (BBB). However, in the CNS of EAE mice, the integrity of the BBB was compromised (29), and substantially all monocyte-derived DCs (moDCs; CD11b + CD11c + Ly6c high CCR2 + Ly6g - ) and neutrophils (CD11b + CD11c - Ly6c + Ly6g + )-containing numerous Td-tomato + cells were found. In contrast, only a small fraction of microglia (CD45 int CD11b + Ly6c - ) were Td-tomato + (Figure 4C). Td-tomato + cells were not found in the lymphoid population (CD4 + , CD8 + , and CD19 + ) nor in neurons, astrocytes, or Ols (data not shown). Similar to naive mice (Figures 15A, 15B), the majority of Td-tomato + cells in the spleen were moDCs and neutrophils, and a minority of B cells were Td-tomato + (Figure 4D). These data indicate that in EAE mice, the cells that take up Ol-EV / i.v. are mainly monocytes / moDCs, classical DCs (cDCs), and neutrophils found in the peripheral blood, spleen, and CNS.

[0124] To determine which myeloid cell populations, moDCs, neutrophils, and / or cDCs mediate the suppression of EAE by Ol-EVs, neutrophils were depleted using anti-Ly6g Ab during Ol-EV treatment of EAE mice (the number of neutrophils in the blood decreased by approximately 75%; FIGS. 16A and 16B). Consistent with the finding that neutrophil depletion after disease onset does not affect EAE (30), neutrophil depletion itself did not affect the disease course (FIG. 4E). Surprisingly, neutrophil depletion did not affect the suppression of EAE by Ol-EVs (FIG. 4E). This suggests that neutrophils do not mediate the effect of Ol-EVs.

[0125] Next, the role of cDCs (CD11c + MHCII + Zbtb46 + ) was investigated. First, to limit the effect of diphtheria toxin (DTX) on cDCs, radiation-induced Zbtb46-DTR (CD45.2 + )→CD45.1 + bone marrow chimeric mice were generated (31) (FIGS. 16C and 16D), and 6 weeks after reconstitution, EAE was induced in these mice. Starting after disease onset, DTX was then injected i.p. every other day during EV treatment. It was confirmed that DTX treatment reduced splenic cDCs (FIGS. 16E and 16F). cDC depletion was also not required for the suppression of EAE by Ol-EVs (FIG. 4F). Since depletion of monocytes suppresses EAE onset, the role of monocytes cannot be directly tested (32). Collectively, these data suggest that these cells mediate the suppression of EAE by Ol-EVs, as almost all monocytes / moDCs in the inflamed CNS acquire Ol-EVs and have the ability to present myelin Ag in the context of MHC class II.

[0126] Example 5: Ol-EV / i.v. induces immunosuppressive monocytes Given the data indicating that monocytes / moDCs mediate the effect of Ol-EVs in EAE, these phenotypes were then examined. Ol-EVs were injected i.v. into EAE mice, and their spleens and CNS Td-tomato+ Single cells were FACS sorted (using the same strategy as shown in FIGS. 4A and 4B), and their mRNAs were analyzed. Treatment with Ol-EV induced significant upregulation of several regulatory genes, particularly Arg1, Pdl1, Il10, Irf1, Havcr2 (tim-3), and Stat3, compared to the control (FIG. 5A). Interestingly, monocytes derived from the CNS also significantly reduced the expression of some pro-inflammatory mediators (Ccl2, Tnf, Inos, Il23a, and Il1b), which play important roles in the development of EAE (33), while monocytes derived from the spleen did not (FIG. 5A). Some of these findings were verified by immunostaining for the corresponding proteins. IL-10 + and PD-L1 + percent monocytes were significantly higher in both the spleen and CNS (FIGS. 5B, 5C, and 17A, 17B), and the number of Arg1 + CD11b + cells was higher in the spinal cord of EAE mice treated with Ol-EV (FIGS. 18A, 18B).

[0127] Similar to naive 2D2 mice injected i.v. with Ol-EV (FIGS. 3A - 3K), EAE mice treated with Ol-EV had a higher percentage of apoptotic (caspase-3 + and PD-1 + ) encephalitogenic CD4 + T cells in both the spleen and CNS (FIGS. 5D - 5G and 13H, 13I). We tested whether there was a correlation between the number of immunosuppressive monocytes (PD-L1 + CCR2 + Ly6c + ) and the number of apoptotic T cells (caspase-3 + PD-1 + CD4 + ), and a strong positive correlation was found (FIG. 5H). This supports the idea that the interaction between monocytes and encephalitogenic T cells causes T cell apoptosis and disease remission. Whether Ol-EV / i.v. affects Foxp3 + CD25 +Whether it affects the number or frequency of Tregs was also examined, and no difference was found compared to the control (Figures 13J and 13K). From this, it is suggested that Tregs do not mediate the suppressive effect of Ol-EV in EAE.

[0128] Finally, to functionally verify the immunosuppressive phenotype of Ol-EV-induced moDCs, Cre + FACS-sorted CNS-derived Td-tomato + moDCs derived from EAE mice treated with Ol-EV were transplanted into mice with ongoing disease (Figure 5I). A single transfer of Td-tomato + moDCs induced a rapid recovery from the disease. In contrast, the disease course did not change when control Td-tomato + moDCs derived from EAE mice treated with Cre+HEK-EV were transferred (Figure 5I).

[0129] These data suggest that treating EAE mice with Ol-EV / i.v. causes monocytes / moDCs to acquire an immunosuppressive phenotype and remit the disease by inducing the death of encephalitogenic T cells.

[0130] Example 6: Ol-EV / i.v.-induced PD-L1 in monocytes is of great significance for EAE suppression Considering the importance of PD-1 and its ligand in immune tolerance (34 - 36), it was investigated whether Ol-EV suppresses EAE via the PD-1 / PD-L1 interaction. Anti-PD-L1 Ab was injected i.v. 24 hours before Ol-EV injection after disease onset. At the time of anti-PD-L1 treatment, EAE mice developed severe disease that did not respond to Ol-EV / i.v. treatment (Figure 5J). In contrast, blocking PD-L2 with Ab did not prevent EAE suppression by Ol-EV (Figure 18C). To confirm the importance of PD-L1 in the Ol-EV effect without using anti-PD-L1 Ab, PD-1 - / - or WT CD4+ T cells were transplanted into RAG1 - / - mice and MOG was used for EAE induction 35-55Immunized and injected with Ol-EV i.v. after disease onset. Vesicles suppressed EAE in mice transfected with WT CD4 + T cells, but not in mice transfected with PD-1 - / - CD4 + T cells (Figure 18D).

[0131] Overall, these data demonstrated that the PD-1 / PD-L1 interaction is crucial for the therapeutic effect of Ol-EV in EAE, but PD-L2 is not.

[0132] Example 7: Ol-EV induces PD-L1 in an IL-10-dependent manner Ol-EV / i.v. induced IL-10 expression in splenic and CNS monocytes (Figures 5A - 5J and 6A - 6G). Since IL-10 has immunomodulatory functions, IL-10 may contribute to the suppression of EAE by Ol-EV, for example, by inducing PD-L1 expression (6). To test this, first, EAE was induced in mice lacking the IL-10 receptor β subunit (IL-10Rb - / - ), and Ol-EV or HEK-EV was injected i.v. at disease onset. In the absence of IL-10Rb, Ol-EV did not suppress EAE (Figures 6A, B), and the number of leukocytes isolated from the CNS of IL-10Rb - / - mice treated with Ol-EV did not decrease as in WT mice (Figure 6C). Next, to investigate which cell population produces IL-10 and induces PD-L1 on monocytes upon Ol-EV treatment, WT or IL-10 - / - CD4+ T cells were used to create mismatch co-cultures with APCs derived from MOG 35-55 immunized mice, and Ol-EV or HEK-EV was added (Figures 6D and 6F). Myeloid APCs (CD11b + CD11c + MHCII + CD19 -IL-10 deficiency in monocytes / macrophages (cells) prevented PD-L1 induction on myeloid cells by Ol-EV. In contrast, IL-10 deficiency in CD4+ T cells had no effect. From these data, it can be seen that Ol-EV induces IL-10 in monocytes / DCs and then induces PD-L1 expression in an autocrine manner.

[0133] Example 8: Human Ol releases EVs containing myelin Ag A prerequisite for using Ol-EV / i.v. as an MS therapy is that human Ol (hOl) releases EVs containing multiple types of myelin Ag. To confirm whether this is the case, human OPCs derived from NIH-approved H9 human ESCs (Millipore) were differentiated into mature hOl, and EVs were collected from the culture supernatant. hOl released a large amount of EVs, and the average diameter was 300 nm when confirmed by cryo-EM (Figure 7A). The proteome profiles of hOPC-derived EVs and hOl-EVs were analyzed by mass spectrometry, and these protein profiles were found to be significantly different (Figures 7B, 7C). Similar to mouse Ol-EV, hOl-EV contained a significant amount of myelin proteins such as MBP and MOG (Figure 7D), whereas OPC-derived EVs contained a much smaller amount of these proteins.

[0134] From these data, it can be seen that hOl differentiated in vitro releases EVs containing a significant amount of myelin Ag. This serves as proof of principle that hOl-EV is essentially similar to mouse Ol-EV, and thus it is possible that MS patients may also have the same beneficial effects as those of mouse Ol-EV in EAE.

[0135] Example 9: Current MS therapies non-specifically target the immune system and have potentially serious side effects because they suppress the immune system systemically (4). The long-standing goal in MS research has been to devise Ag-specific therapies that suppress only the harmful immune responses while leaving the rest of the immune system intact. The prerequisite for Ag-specific therapies is to identify the target Ag. The autoimmune responses in MS are thought to target myelin proteins such as MOG, MBP, and PLP that are produced by Ol (41). The relevant myelin Ags are not necessarily the same among MS patients and it is also thought that over time, the specificity of the autoimmune response may shift from the initial myelin Ag epitope to another epitope or Ag (41). The idea that the Ag specificity of this pathogenic response progresses is called "epitope spreading" and it has also been proposed that newly generated responses against additional myelin Ags contribute to disease relapse and chronicity (24). Overall, the identity of the relevant Ags in MS remains unknown and may vary among patients and over time. Despite success in experimental animal models, the lack of knowledge about this Ag has hindered the development of Ag-specific MS therapies. Some of the therapies tested in animals address the issue of antigen complexity but not the approach that requires knowledge of the Ag that causes the disease (4). Based on the findings in EAE, several approaches have been proposed for inducing Ag-specific tolerance in MS (4). One of these approaches is to induce tolerance by i.v. injection of encephalitogenic peptides released or by peptides conjugated to nanoparticles or apoptotic cells (5-10). I.v. tolerance induces immune tolerance-inducing APCs, reduces pathogenic Th1 and Th17 cell responses (11, 42), and suppresses EAE by inducing Treg and Tr1 cells (37). We have recently shown that the induction of i.v. tolerance in ongoing EAE depends on IL-27 (11) and galectin-1 (42).

[0136] From clinical trials testing the effect of s.c. delivery of modified MBP peptides, this approach has been shown to potentially worsen the disease in some MS patients (43). From testing in MS patients, i.v. injection of immunodominant MBP peptides (500 mg every 6 months over 24 months) into progressive MS patients (n = 32) was found to be safe (44). At 24 months, this treatment was only significantly beneficial in patients (n = 20) with HLA haplotype DR2 and / or DR4. Long-term follow-up of these responsive patients found that the median time to progression was 78 months compared to 18 months for placebo treatment. In another study, single i.v. injection of autologous leukocytes conjugated to seven immunodominant myelin peptides was also safe (9). Findings from these studies suggest that i.v. delivery of myelin Ag may be safe and beneficial for MS patients. Based on findings in experimental animals, injection of myelin Ag in the form of particles (cells, nanoparticles) is a safer approach than injection of soluble free Ag, and this is thought to also apply to Ol-Ev (45).

[0137] The EV field has rapidly evolved over the past decade (20). EVs are particles enclosed by a protein-lipid membrane that are secreted by almost all cells and play a major role in cell-to-cell communication under both physiological and pathological conditions (15, 16). Several studies have reported the presence of EVs derived from CNS resident cells such as microglia and astrocytes in cerebrospinal fluid and blood, and that their amounts increase during inflammatory conditions such as MS and EAE (46). Ol also releases EVs, but little is known about the role of Ol-EV during homeostasis or during disease.

[0138] Thanks to significant progress in EV biology, EVs are now being studied as a therapy for several diseases (20). EVs have been used in many studies of experimental autoimmune disease therapies (17 - 19) and have been reported to be effective in regulating EAE inflammation by targeting microglia / macrophages, inducing immune tolerance - inducing DCs, and inducing Tregs (18 - 20). We have shown that in vitro - cultured Ol releases EVs containing the most relevant myelin proteins in both exosomes and microvesicles. In most reports describing EVs as drug - delivery tools, only exosomes have been used because of certain therapeutic advantages (17, 48). Due to different sorting mechanisms of myelin proteins into different classes of Ol - EVs (49), it has been shown that PLP is abundant in exosomes, while MBP and MOG are mainly present in membrane - derived microvesicles (50). We used whole Ol - EVs, exosomes, and microvesicles, and their administration prophylactically and therapeutically suppressed neuroinflammation in an Ag - dependent manner in several EAE models. This treatment had no observable side effects and was safer than injection of free peptides. The injected Ol - EVs were preferentially taken up by phagocytes, monocytes, neutrophils, and cDCs, but only monocytes were found to be essential for Ol - EV - induced tolerance. Monocytes that took up Ol - EVs upregulated the expression of several anti - inflammatory molecules such as PD - L1 and IL - 10 that mediate disease suppression. Finally, we show that hOl also releases EVs containing myelin proteins.

[0139] Ol-EV carries multiple types of myelin Ags and reduces encephalitogenic T cell responses to multiple types of myelin Ags / epitopes and suppresses neuroinflammation in several EAE models. The use of synthetic multi-epitope Ags (combining several myelin epitopes into one artificial protein) has been reported to be more efficient than individual peptides in suppressing EAE (23, 38). The effect of Ol-EV is Ag-dependent and specific. By using MOG-specific and MBP-specific T cell receptor transgenic mice, it was found that Ol-EV induces apoptosis and anergy of autoreactive T cells via the myelin Ags carried by Ol-EV. Furthermore, it has been shown herein that Ol-EV lacking MOG or MBP does not suppress MOG 35-55 -induced or MBP Ac(1-11)-induced EAE.

[0140] The heterogeneity in the size and protein content of EVs is a variable that affects EV uptake by recipient cells, which can occur via multiple pathways (16). Nanoparticles are known to be ingested by scavenger receptor-dependent mechanisms (7). However, although the specificity of EV uptake has not been fully elucidated (15), phagocytic cells, such as monocytes / moDCs, DCs, macrophages, and microglia, have been described to be able to internalize EVs via receptor-mediated endocytosis, phagocytosis, and micropinocytosis (51).

[0141] The role of moDCs in EAE has been extensively described (32). moDCs are generally not present in the healthy CNS but infiltrate the meninges and parenchyma during inflammation and contribute to CNS pathology by acquiring an enhanced ability for Ag processing and presentation. In contrast to several studies that have described the importance of "Ag-capture" by splenic phagocytes to restore immune tolerance in EAE (7), the data herein suggest that moDCs infiltrating the CNS acquire Ol-EV and mediate EAE suppression. Indeed, transfer of CNS-derived moDCs from EAE mice treated with Ol-EV into mice with ongoing disease rapidly suppressed EAE inflammation.

[0142] It has been shown herein that treatment of EAE mice with Ol-EV / i.v. upregulates PD-1 on CD4+ T cells and PD-L1 and PD-L2 on moDCs. Considering the importance of PD-1 and its ligands in immune tolerance (38), we investigated whether Ol-EV suppresses EAE via the PD-1 / PD-L1 and / or PD-1 / PD-L2 interaction. Blocking PD-L1 with Ab abolished EAE suppression by Ol-EV, whereas blocking PD-L2 had no effect. This demonstrated that interaction between PD-1 on T cells and PD-L1 on moDCs results in anergy and apoptosis of encephalitogenic T cells and disease remission. This is consistent with the reported role of PD-L1 in i.v. tolerance induction in EAE (6).

[0143] IL-10 plays an important immunomodulatory role and is an anti-inflammatory cytokine that suppresses inflammatory responses and autoimmunity, including EAE (6). IL-10 has been shown to be required for peptide / i.v. tolerance induction in EAE. Several studies have shown the potential to induce IL-10 in various ways to promote immune tolerance in EAE (6, 7), and that tolerance is lost upon IL-10 blockade (6). Tolerance is lost upon IL-10 blockade (6). It has been demonstrated herein that IL-10 production by APCs, but not by CD4+ T cells, is required for the suppression of EAE by Ol-EV. Apparently, IL-10 induced the expression of PD-L1 on moDCs and suppressed the disease.

[0144] An important mechanism for establishing and maintaining peripheral tolerance relies on Tregs. Therefore, we explored whether Ol-EV / i.v. affects Tregs, but no change in the total number or frequency of Tregs among CD4+ T cells was found. This suggests that Ol-EV / i.v. suppresses EAE through a Treg-independent mechanism, resulting in apoptosis and anergy of T cells through the direct interaction between myelin-specific CD4+ T cells and immune tolerance-inducing moDCs. However, it remains possible that the immune tolerance-inducing phenotype of moDCs can be induced independently of Tregs, with Tregs contributing without expanding themselves. Similar to our findings, a modest contribution of Tregs to Ag-specific i.v. tolerance induction in EAE has been reported in a system using myelin Ag conjugated to microbeads (7). These reported findings support the idea that Tregs may contribute somewhat to the suppression of EAE by Ol-EV but are likely not essential for EAE suppression.

[0145] In conclusion, considering that Ol-EV contains most or perhaps all relevant myelin Ags, it has the potential ability to induce Ag-specific tolerance and suppress diseases caused by immune responses against myelin Ags. Therefore, the use of Ol-EV would avoid the need to identify relevant myelin Ags on a patient-by-patient basis and increase the likelihood that Ol-EV / i.v. could become a widely applicable Ag-specific MS therapy.

[0146] Sequence Information SEQUENCE LISTING <110> Thomas Jefferson University <120> Oligodendrocyte-derived Extracellular Vesicles for Therapy of Multiple Sclerosis <150> US 62 / 953,257 <151> 2019-12-24 <150> US 62 / 857,182 <151> 2019-06-04 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer - Cre gene <400> 1 tactagtggc gcgccaccat gcccaagaag aagagg 36 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer - Cre gene <400> 2 ggatccagcg taatctggaa catcgt 26 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mMOG sgRNA1 forward <400> 3 caccgagcaa gcacctgaat accg 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mMOG sgRNA1 reverse <400> 4 aaaccggtat tcaggtgctt gctc 24 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> mMOG sgRNA2 forward <400> 5 caccgtcacc tctaccgaaa tggca 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> mMOG sgRNA2 reverse <400> 6 aaactgccat ttcggtagag gtgac 25 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mMOG DP forward <400> 7 tcccactctt gtgtcttgga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mMOG DP reverse <400> 8 agcaggtgta gcctccttca 20

Claims

1. A method for treating and / or preventing multiple sclerosis (MS) in a subject in need thereof, comprising administering to the subject an effective amount of oligodendrocyte-derived extracellular vesicles (Ol-EVs).

2. The method of claim 1, wherein the Ol-EV comprises a myelin antigen (Ag).

3. 3. The method of claim 2, wherein the myelin Ag comprises myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and / or myelin proteolipid protein (PLP).

4. The method of claim 1, which is Ag-specific.

5. 2. The method of claim 1, wherein the administering step induces immunosuppressive monocytes.

6. The method of claim 5, wherein the immunosuppressive monocytes express PD-L1.

7. The method of any one of claims 1 to 6, wherein the administering step does not cause adverse or undesirable effects on the subject's immune system.

8. The method of claim 1, wherein the oligodendrocyte-derived extracellular vesicles are formulated in a pharmaceutical composition comprising at least one pharma- ceutically acceptable carrier.

9. 9. The method of claim 8, wherein the pharmaceutical composition is administered intravenously, subcutaneously, intradermally, transdermally, orally, or nasally.

10. The method of claim 1, wherein the subject is a mammal.

11. 11. The method of claim 10, wherein the subject is a human.

12. 2. The method of claim 1, wherein the MS is chronic MS or relapsing-remitting MS.

13. A pharmaceutical composition comprising oligodendrocyte-derived extracellular vesicles (Ol-EVs) and at least one pharma- ceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, wherein the Ol-EV comprises a myelin antigen (Ag).

15. 15. The pharmaceutical composition of claim 14, wherein the myelin Ag comprises myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and / or myelin proteolipid protein (PLP).

16. 16. The pharmaceutical composition of any one of claims 13 to 15, wherein the composition is formulated for intravenous, subcutaneous, intradermal, transdermal, oral, or nasal administration.

Images