Treatment and prevention of hemophilic arthropathy with mir10a

Abstract

Compositions comprising microRNA 10a (miR10a) and methods for treatment and prevention of hemophilic arthropathy and associated symptoms, such as neovascularization, synovial hyperplasia, cartilage degeneration, and chondrocyte apoptosis. The miR10a may be encapsulated by a liposome, nanoparticle, or preferably, an extracellular vesicle (EV).

Description

Atty. Dkt. No. 4842-130WO1TREATMENT AND PREVENTION OF HEMOPHILIC ARTHROPATHY WITH miRlOa AND COMPOSITIONS COMPRISING miRlOaSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with government support under grant numbers HL107483 and HL169255 awarded by the National Heart, Lung, and Blood Institute of the U.S. National Institutes of Health. The government has certain rights in the invention.CROSS REFERENCE OF RELATED APPLICATION

[0001] This application claims the benefit of United States provisional patent application No. 63 / 755,829 filed February 7, 2025, the disclosure of which is incorporated herein by reference.FIELD OF THE INVENTION

[0002] The invention is in the field of biochemistry, cellular biology, and medicine relating to the inhibition of hemophilic arthropathy using microRNA 10a (miRlOa) and compositions, including extracellular vesicles (EVs), comprising miRlOa.BACKGROUND

[0003] Hemophilia A occurs in 1 in 5,000 male live births, and Hemophilia B occurs in 1 in 20,000 male live births in the U.S. The number of hemophilia patients in the U.S. has been estimated to be 20,000 (and >400,000 worldwide). Annual treatment costs in the U.S. per patient have been estimated at $100,000 to $500,000 (with extreme cases costing $21 million / year). Total annual medical costs to treat hemophilia in the U.S. have been estimated to be $4.6 billion ($230,000 per patient). Global annual medical cost for hemophilia care is believed to be at least $12 billion. Hemophilia treatment with clotting factors has been categorized as on demand or prophylactic; gene therapy is currently in clinical trials. A number of recombinant FVIII and FIX therapeutic agents have recently been approved or are in late-phase clinical development (SW Pipe, Hematology, Am Soc Hematol Educ Prog. 2016; 2016 (l):650-656). The long term goals of prophylaxis have been:• Prevention of sequelae of chronic disease• Prevention of pain and sufferingAtty. Dkt. No. 4842-130WO1• Improvement in quality of life for the patient and his family• Reduction in long-term societal costs through prevention of disability, improved outcome and maximization of human potential(See, for example, K. Fischer et al, Haemophilia, 2003, 9(4): 376-81 and Haemophilia, 2003, 9 Suppl 7:75-81.)

[0004] Hemophilic arthropathy (HA) is a debilitating joint disease that develops as a consequence of frequent bleeding in the joints of hemophilia patients. HA leads to joint deformities, cartilage degeneration, and bone destruction. Prophylactic treatment of hemophilia patients with clotting factor replacement reduces joint bleeding and HA. Although the use of prophylactic clotting factor replacement considerably reduces the risk of joint bleeding, many hemophilia patients continue to develop the joint disease because of breakthrough bleeding and the development of inhibitors. Recombinant factor Vila (rFVIIa) or activated prothrombin complex concentrates are commonly used as bypassing agents for treating hemophilia patients with inhibitors. Research over the past two decades has led to the development of several non-traditional agents for treating hemophilia, including the development of bispecific antibodies that mimic FVIII. Although these agents improve hemostasis in hemophilia patients or experimental models, they do not fully prevent breakthrough bleeding and have additional limitations.

[0005] EPCR is a member of the Class I MHC family of receptors. EPCR is constitutively expressed in endothelium and acts as the receptor for anti-coagulant protein C. EPCR promotes the activation of protein C, and the activated protein C (APC) inactivates clotting factors Factor Va (FVa) and Factor Villa (FVIIIa) by cleaving them. EPCR is also expressed on many other cell types. The present inventors’ group and others have established that Factor Vila (FVlIa), the clotting factor that initiates the activation of the coagulation cascade upon binding to tissue factor (TF), binds to EPCR. (See: Ghosh S et al., J Biol Chem, 2007, 282: 11849-57; Preston RJ et al., J Biol Chem, 2006, 281: 28850-7; Lopez-Sagaseta J et al., J Thromb Haemost, 2007, 5: 1817-24). FVIIa binding to EPCR on the endothelium or in cells expressing TF induces cell signaling by activating protease activated receptor-1 (PARI) either directly or by enhancing TF-FVIIa-FXa cleavage of PARI (Sen P et al., Blood, 2011, 117: 3199-208; Disse, J et al., J Biol Chem, 2011, 286: 5756-67).

[0006] Recent studies have shown that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells via the EPCR-PAR1 axis, and these FVIIa-released EVs were found to exhibit prohemostatic activity (See: Das et al., Blood, 2021, 137(24): 3428-Atty. Dkt. No. 4842-130WO13442). Most of the EVs detected in the blood of healthy subjects are derived from platelets and red blood cells, and only a small fraction of EVs are from endothelial cells. Various pathological conditions, including coronary syndrome, antiphospholipid syndrome, and sickle cell disease, increase the release of endothelial cell-derived EVs (eEVs) into the circulation. In many cases, eEVs have been found to be detrimental, as they induce inflammation and enhanced endothelial dysfunction. However, studies showed that FVIIa-released eEVs were enriched with anti-inflammatory micro RNAs (miRs), predominantly miRlOa, and the transfer of miRlOa from the eEV’s cargo to recipient monocytes and naive endothelial cells was responsible for anti-inflammatory and barrier protective responses, respectively (See: Das et al., Blood, 2021, 139(1): 118-133). Since inflammation plays a crucial role in the pathogenesis of HA, the current invention investigated the effects of miRlOa and FVIIa-released eEVs in attenuating HA.

[0007] There is a long-felt but unmet need to treat and inhibit recurring joint bleeding in hemophilia patients.SUMMARY OF THE INVENTION

[0008] In some aspects, the present disclosure provides a method for treatment and prevention of an inflammatory disorder. In some aspects, the present disclosure provides a method for treatment and prevention of hemophilic arthropathy (HA). FVIIa-released eEVs containing miRlOa markedly suppress joint bleed-induced HA in hemophilia. Incorporation of miRlOa inhibitor into FVIIa-released eEVs reversed the protective effect of FVIIa-released eEVs, indicating that the protective effect is dependent on miRlOa. More importantly, loading control eEVs with miRlOa mimic conferred the protective effect to control eEVs. Additional studies showed that FVIIa-released eEVs and control eEVs loaded with miRlOa mimic markedly suppressed the joint bleed-induced IL-6 expression and vascular leakage in the synovium. Administration of various eEV preparations had no significant effect on prolonged bleeding time of hemophilia mice. Overall, the present disclosures provides methods for treating hemophilia with miRlOa to attenuate joint bleed-induced HA.

[0009] Some aspects of the present disclosure are directed to methods of treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy, the method comprising administering to the subject a therapeutically effective amount of a microRNA 10a (miRlOa). Some aspects of the present disclosure are directed to methods of suppressing joint edema or joint bleeding in a subject, the method comprising administeringAtty. Dkt. No. 4842-130WO1to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing or preventing macrophage infiltration into a joint of a subject, the method comprising administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing or preventing neovascularization in a joint of a subject, the method comprising administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing or preventing synovial hyperplasia in a subject, the method comprising administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing or preventing cartilage degeneration in a subject, the method comprising administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing or preventing chondrocyte apoptosis in a subject, the method comprising administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing expression of IL-6 in a subject’s synovial fluid, the method comprising administering to the subject a therapeutically effective amount of miRlOa.

[0010] In some aspects, the methods comprise administering the miRlOa at a dose sufficient to reach a concentration of about 0.1 to 10 nM. In some aspects, the methods comprise administering the miRlOa parenterally. In some aspects, the methods comprise administering the miRlOa by intra-articular injection. In some aspects, the methods comprise administering the miRlOa intravenously.

[0011] In some aspects, the miRlOa is administered alone. In some aspects, the miRlOa is administered in a liquid composition. In some aspects, the liquid composition comprises the miRlOa in a solution. In some aspects, the miRlOa is encapsulated by an extracellular vesicle (EV). In some aspects, the EV is a biological EV. In some aspects, the EV is a synthetic EV. In some aspects, the miRlOa is administered in a nanoparticle. In some aspects, the miRlOa is administered in a lipid nanoparticle (LNP). In some aspects, the miRlOa is administered in a liposome.

[0012] Some aspects of the present disclosure are directed to a method of manufacturing a medicament comprising EVs comprising miRlOa for use in treating a subject having an inflammatory disorder, such as hemophilic arthropathy, or a subject at risk of developing hemophilic arthropathy, the method comprising: a) culturing a cell in a culture medium comprising one or more growth factors, and b) isolating the EV from a supernatant of the culture medium from step a).Atty. Dkt. No. 4842-130WO1

[0013] In some aspects, the one or more growth factors are selected from a vascular endothelial growth factor (VEGF), an endothelial cell growth factor (EGF), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF), and combinations thereof. In some aspects, the cell in an endothelial cell and the EV Is derived from the endothelial cell (eEV). In some aspects, the endothelial cell is a human umbilical vein endothelial cell (HUVEC). In some aspects, step a) comprises culturing the cell in endothelial basal medium. In some aspects, the culture medium comprises one or more of ascorbic acid, hydrocortisone hemisuccinate, heparin sulfate, L-glutamine, and combinations thereof. In some aspects, step a) comprises culturing the cell for about 24-96 hours, about 24-72 hours, about 24-48 hours, or about 48-72 hours. In some aspects, step a) comprises culturing the cell for about 48 hours. In some aspects, the culture medium further comprises Factor Vila (FVIIa). In some aspects, the method further comprises transfecting the cell with a miRlOa mimic before step a).

[0014] Some aspects of the present disclosure are directed to a method of treating a subject having an inflammatory disorder, such as hemophilic arthropathy, or a subject at risk of developing hemophilic arthropathy, the method comprising: a) culturing a cell in a culture medium comprising one or more growth factors, b) isolating the EV from a supernatant of the culture medium from step a), and c) administering to the subject the isolated EVs from step b).

[0015] In some aspects, the one or more growth factors are selected from a vascular endothelial growth factor (VEGF), an endothelial cell growth factor (EGF), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF), and combinations thereof. In some aspects, the cell in an endothelial cell and the EV Is derived from the endothelial cell (eEV). In some aspects, the endothelial cell is a human umbilical vein endothelial cell (HUVEC). In some aspects, step a) comprises culturing the cell in endothelial basal medium. In some aspects, the culture medium comprises one or more of ascorbic acid, hydrocortisone hemisuccinate, heparin sulfate, L-glutamine, and combinations thereof. In some aspects, step a) comprises culturing the cell for about 24-96 hours, about 24-72 hours, about 24-48 hours, or about 48-72 hours. In some aspects, step a) comprises culturing the cell for about 48 hours. In some aspects, the culture medium further comprises Factor Vila (FVIIa). In some aspects, the method further comprises transfecting the cell with a miRlOa mimic before step a).

[0016] In some aspects, the method further comprises (i) reducing or preventing macrophage infiltration into a joint of the subject, (ii) reducing or preventing neovascularization in a joint of the subject, (iii) reducing or preventing synovial hyperplasia is a joint of the subject, (iv) reducing or preventing cartilage degeneration in a joint of theAtty. Dkt. No. 4842-130WO1subject, (v) reducing or preventing chondrocyte apoptosis in the subject, (vi) reducing expression of IL-6 in the subject’s synovial fluid, (vii) administering a clotting factor, or (viii) a combination of (i)-(vii).

[0017] In some aspects, the method comprises administering the EVs parenterally. In some aspects, the method comprises administering the EVs by intra-aiticular injection. In some aspects, the method comprises administering the EVs intravenously.

[0018] In some aspects, the subject has hemophilia. In some aspects, the subject has hemophilic arthropathy. In some aspects, the subject has hemarthrosis.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0020] FIG. 1 shows needle puncture-induced bleeding in the joints of mice that were administered saline, control extracellular vesicles (Con eEVs), or FVIIa-released eEVs (FVIIa eEVs). Panel A is a graph depicting the change in joint diameter over 14 days after injury. Panel B is a graph depicting visual bleeding score over 14 days after injury. Panel C shows representative images taken 14 days after injury.

[0021] FIG. 2 is a graph comparing the number of iron deposits in the joints of mice administered saline, FVIIa eEVs, FVIIa eEVs loaded with scrambled either miRlOa (FVIIa eEVs / scr miRlOa) or miRlOa inhibitor (FVIIa eEVs / miRlOa Inh), Con eEVs, or Con eEVs loaded with either scrambled mirlOa (Con eEVs / scr miRlOa) or miRlOa mimic (Con eEVs / miRlOa mimic) following needle puncture-induced joint bleeding.

[0022] FIG. 3 shows graphs demonstrating the effects of FVIIa eEV treatment compared to saline or Con eEVs on needle puncture -induced joint bleeding in mice. Panel A shows the histology score based on hematoxylin and eosin (H&E) staining. Panel B shows the macrophage score based on immunostaining for F4 / 80 marker for macrophages. Panel C shows the number of vessels based on staining for endothelial cell marker CD31 for neoangiogenesis. Panel D shows cartilage degeneration based on Alcian blue staining.

[0023] FIG. 4 is a graph comparing cartilage degeneration in the joints of mice administered saline, FVIIa eEVs, FVIIa eEVs / scr niiRlOa, FVIIa eEVs / miRlOa Inh, ConAtty. Dkt. No. 4842-130WO1eEVs, Con eEVs / scr miRlOa, or Con eEVs / miRlOa mimic following needle puncture-induced joint bleeding.

[0024] FIG. 5 is a graph showing synovitis score for mice administered saline. Con eEVs, or FVIIa eEVs following needle puncture -induced joint bleeding. Synovitis score is based on the extent of synovial hyperplasia (0-3 points), vascularity (0-3 points), and the presence of discoloration by hemosiderin, blood (erythrocytes), and synovial villi (0 for absence and 1 for their presence, for each category), and cartilage degeneration (0 to 2 points), resulting in a combined score of 0 to 11 points for increasing pathology.

[0025] FIG. 6 shows needle puncture -induced bleeding in the joints of mice that were administered saline, FVIIa eEVs. FVIIa eEVs / scr miRlOa, or FVIIa eEVs / miRlOa Inh. Panel A is a graph depicting the change in join diameter over 14 days after injury. Panel B is a graph depicting visual bleeding score over 14 days after injury. Panel C shows representative images taken 14 days after injury.

[0026] FIG. 7 shows graphs demonstrating the effects of saline, FVIIa eEVs, FVIIa eEVs / scr miRlOa, or FVIIa eEVs / miRlOa Inh on needle puncture-induced joint bleeding in mice. Panel A shows the histology score based on H&E staining. Panel B shows the macrophage score based on immunostaining for F4 / 80 marker for macrophages. Panel C shows the number of vessels based on staining for endothelial cell marker CD31 for neoangiogenesis. Panel D shows cartilage degeneration based on Alcian blue staining.

[0027] FIG. 8 is a graph showing synovitis score for mice administered saline, FVIIa eEVs, FVIIa eEVs / scr miRlOa, or FVIIa eEVs / miRlOa Inh following needle puncture-induced joint bleeding.

[0028] FIG. 9 shows needle puncture-induced bleeding in the joints of mice that were administered saline, Con eEVs, Con eEVs / scr miRlOa, or Con eEVs / miRlOa mimic. Panel A is a graph depicting the change in join diameter over 14 days after injury. Panel B is a graph depicting visual bleeding score over 14 days after injury. Panel C shows representative images taken 14 days after injury.

[0029] FIG. 10 shows graphs demonstrating the effects of saline, Con eEVs, Con eEVs / scr miRlOa, or Con eEVs / miRlOa mimic on needle puncture-induced joint bleeding in mice. Panel A shows the histology score based on H&E staining. Panel B shows the macrophage score based on immunostaining for F4 / 80 marker for macrophages. Panel C shows the number of vessels based on staining for endothelial cell marker CD31 for neoangiogenesis. Panel D shows cartilage degeneration based on Alcian blue staining.Atty. Dkt. No. 4842-130WO1

[0030] FIG. 11 is a graph showing synovitis score for mice administered saline, Con eEVs, Con eEVs / scr miRlOa, or Con eEVs / miRlOa mimic following needle puncture-induced joint bleeding.

[0031] FIG. 12 is a graph comparing the number of apoptotic chondrocytes in the joints of mice administered saline, FVIIa eEVs, FVIIa eEVs / scr miRlOa, FVIIa eEVs / miRlOa Inh, Con eEVs, Con eEVs / scr miRlOa, or Con eEVs / miRlOa mimic following needle puncture-induced joint bleeding.

[0032] FIG. 13 is a graph showing the level of IL-6 expression in synovial fluid of mice administered saline, FVIIa eEVs, FVIIa eEVs / scr miRlOa, FVIIa eEVs / miRlOa Inh, Con eEVs, Con eEVs / scr miRlOa, or Con eEVs / miRlOa mimic following needle puncture-induced joint bleeding.

[0033] FIG. 14 is a graph showing the level of fluorescence measured in tissue sections from knee joints of mice injected with fluorescin dextran.

[0034] FIG. 15 shows graphs comparing the average time to hemostasis and amount of blood loss in mice subjected to vein incision-induced bleeding following administration of saline, FVIIa eEVs, FVIIa eEVs / scr miRlOa, FVIIa eEVs / miRlOa Inh, Con eEVs, Con eEVs / scr miRlOa, or Con eEVs / miRlOa mimic.DETAILED DESCRIPTION OF THE INVENTION

[0035] The present disclosure provides compositions and methods for treating or preventing the development of arthropathy in hemophilic subjects as well as reducing, attenuating or preventing various biological reactions associated with arthropathy, including reducing joint swelling, macrophage infiltration, iron deposition and / or blood vessel formation (angiogenesis; neoangiogenesis). In one aspect, the present disclosure provides a method of treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy, comprising administering to the subject a therapeutically effective amount of a microRNA 10a (miRlOa). In other aspects, the present disclosure provides methods of suppressing joint edema or joint bleeding in a subject, reducing or preventing neovascularization in a joint of a subject, reducing or preventing synovial hyperplasia in a subject, reducing or preventing cartilage degeneration in a subject, reducing or preventing chondrocyte apoptosis in a subject, or reducing expression of IL-6 in a subject’s synovial fluid. In still other aspects, the present disclosure provides compositions comprising miRlOaAtty. Dkt. No. 4842-130WO1and / or extracellular vesicles (EVs) for use in the treatment or prevention of hemophilic arthropathy, and methods of manufacturing thereof.

[0036] The compositions of the present disclosure may be used for the treatment or prevention of additional inflammatory disorders. Examples of inflammatory disorders treatable by the compositions of the present disclosure include, for example and without limitation, arthritis (e.g., psoriatic arthritis, reactive arthritis, rheumatoid arthritis, gouty arthritis, osteoarthritis), scleroderma, systemic lupus erythematosus, dermatomyositis, gout, and arthropathy. In some embodiments, the present disclosure provides a method of treating or preventing an inflammatory disorder, comprising administering to the subject a therapeutically effective amount of a microRNA 10a (miRlOa).I. Definitions

[0037] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0038] 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. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0039] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an extracellular vesicle,” is understood to represent one or more extracellular vesicles. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0040] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such asAtty. Dkt. No. 4842-130WO1“A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0041] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower).

[0042] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 0.01 to 2.0” should be interpreted to include not only the explicitly recited values of about 0.01 to about 2.0. but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 0.5, 0.7, and 1.5, and sub-ranges such as from 0.5 to 1.7, 0.7 to 1.5, and from 1.0 to 1.5, etc. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described. Additionally, it is noted that all percentages are in weight, unless specified otherwise.

[0043] In understanding the scope of the present disclosure, the terms “including” or “comprising” and their derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The temr “consisting essentially of,” as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps. It is understood that reference to any one of these transition terms (i.e. “comprising,” “consisting,” or “consisting essentially”) provides direct support forAtty. Dkt. No. 4842-130WO1replacement to any of the other transition term not specifically used. For example, amending a term from “comprising” to “consisting essentially of’ or “consisting of’ would find direct support due to this definition for any elements disclosed throughout this disclosure. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0044] As used herein, the term “subject” means a mammal to which a composition of the present disclosure can be administered. In some aspects, the subject is a human subject.

[0045] As used herein, “miRlOa” refers to microRNA 10a produced by cells and “miRlOa mimic” refers to a synthetic microRNA 10a. Although their origin differs, miRlOa and miRlOa mimic have the same sequence and structure, and the terms may be used interchangeably.

[0046] As used herein, a plurality of compounds, elements, or steps may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[0047] The term “exemplary” is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a more concrete fashion.

[0048] Furthermore, certain molecules, constructs, compositions, elements, moieties, excipients, disorders, conditions, properties, steps, or the like may be discussed in the context of one specific embodiment or aspect or in a separate paragraph or section of this disclosure. It is understood that this is merely for convenience and brevity, and any such disclosure is equally applicable to and intended to be combined with any other embodiments or aspects found anywhere in the present disclosure and claims, which all form the application and claimed invention at the filing date. For example, a list of constructs, molecules, method steps, kits, or compositions described with respect to a construct, molecule, isolated polypeptide, activatable molecule, composition, or method is intended to and does find direct support for embodiments related to constructs, molecules, isolated polypeptides, activatable molecules, compositions, formulations, and methods described in any other part of this disclosure, even if those methodAtty. Dkt. No. 4842-130WO1steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.IL Methods of the Disclosure

[0049] Some aspects of the present disclosure are directed to a method of treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy. In some aspects, the present disclosure includes administering to the subject a therapeutically effective amount of a microRNA 10a (miRlOa). Some aspects of the present disclosure are directed to methods of suppressing joint edema or joint bleeding in a subject by administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing or preventing macrophage infiltration into a joint of a subject by administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing or preventing neovascularization in a joint of a subject by administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing or preventing synovial hyperplasia in a subject by administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing or preventing cartilage degeneration in a subject by administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing or preventing chondrocyte apoptosis in a subject by administering to the subject a therapeutically effective amount of miRlOa. Some aspects of the present disclosure are directed to methods of reducing expression of IL-6 in a subject’s synovial fluid by administering to the subject a therapeutically effective amount of miRlOa.

[0050] Some aspects of the present disclosure are directed to a method of treating a subject having an inflammatory disorder, comprising administering to the subject a therapeutically effective amount of a microRNA 10a (miRlOa). In some aspects, the subject has arthritis (e.g., psoriatic arthritis, reactive arthritis, rheumatoid arthritis, gouty arthritis, osteoarthritis), scleroderma, systemic lupus erythematosus, dermatomyositis, gout, or arthropathy.

[0051] In some aspects, the subject has hemophilia. In some aspects, the subject has hemophilic arthropathy. In some aspects, the subject is at risk of developing hemophilic arthropathy. In some aspects, the subject has hemarthrosis.Atty. Dkt. No. 4842-130WO1

[0052] In some aspects, miRlOa is administered as a sole active agent. In some aspects, miRlOa is administered as an adjuvant with one or more additional therapeutics. In some aspects, miRlOa is administered in combination with a clotting factor. The three clotting factor replacement therapy types are standard half-life (SHL), extended half-life (EHL), and ultra-long half-life (UHL; hemophilia A only). In some aspects, miRlOa is administered to improve efficacy of a clotting factor. In some aspects, miRlOa is administered to reduce the amount of a clotting factor administered.

[0053] In some aspects, the method comprises reducing or preventing macrophage infiltration into a joint of the subject. In some aspects, the method comprises reducing or preventing neovascularization in a joint of the subject. In some aspects, the method comprises reducing or preventing synovial hyperplasia in a joint of the subject. In some aspects, the method comprises reducing or preventing cartilage degeneration in a joint of the subject. In some aspects, the method comprises reducing or preventing chondrocyte apoptosis in the subject. In some aspects, the method comprises reducing expression of IL-6 in the subject’s synovial fluid.

[0054] In some aspects, the method comprises administering the miRlOa at a dose sufficient to reach a plasma concentration of about 0.1 to 10 nM. In some aspects, the method comprises administering the miRlOa at a dose sufficient to reach a plasma concentration of about 0.2 nM to about 10 nM, about 0.3 nM to about 10 nM, about 0.4 nM to about 10 nM, about 0.5 nM to about 10 nM, about 0.6 nM to about 10 nM, about 0.7 nM to about 10 nM, about 0.8 nM to about 10 nM, about 0.9 nM to about 10 nM, about 1 nM to about 10 nM, about 2 nM to about 10 nM, about 3 nM to about 10 nM, about 4 nM to about 10 nM, about 5 nM to about 10 nM, about 6 nM to about 10 nM, about 7 nM to about 10 nM, about 8 nM to about 10 nM, or about 9 nM to about 10 nM. In some aspects, the method comprises administering the miRlOa at a dose sufficient to reach a plasma concentration of about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, about 0.1 nM to about 0.9 nM, about 0.1 nM to about 0.8 nM, about 0.1 nM to about 0.7 nM, about 0.1 nM to about 0.6 nM, about 0.1 nM to about 0.5 nM, about 0.1 nM to about 0.4 nM, about 0.1 nM to about 0.3 nM, or about 0.1 nM to about 0.2 nM. In some aspects, the method comprises administering the miRlOa at a dose sufficient to reach a plasma concentration of about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM,Atty. Dkt. No. 4842-130WO1about 0.8 nM, about 0.9 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, or about 10 nM.

[0055] In some aspects, the method comprises administering the miRlOa at a dose sufficient to reach a synovial fluid concentration of about 0.1 to 10 nM. In some aspects, the method comprises administering the miRlOa at a dose sufficient to reach a synovial fluid concentration of about 0.2 nM to about 10 nM, about 0.3 nM to about 10 nM, about 0.4 nM to about 10 nM, about 0.5 nM to about 10 nM, about 0.6 nM to about 10 nM, about 0.7 nM to about 10 nM, about 0.8 nM to about 10 nM, about 0.9 nM to about 10 nM, about 1 nM to about 10 nM, about 2 nM to about 10 nM, about 3 nM to about 10 nM, about 4 nM to about 10 nM, about 5 nM to about 10 nM, about 6 nM to about 10 nM, about 7 nM to about 10 nM, about 8 nM to about 10 nM, or about 9 nM to about 10 nM. In some aspects, the method comprises administering the miRlOa at a dose sufficient to reach a synovial fluid concentration of about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, about 0.1 nM to about 0.9 nM, about 0.1 nM to about 0.8 nM, about 0.1 nM to about 0.7 nM, about 0.1 nM to about 0.6 nM, about 0.1 nM to about 0.5 nM, about 0.1 nM to about 0.4 nM, about 0.1 nM to about 0.3 nM, or about 0.1 nM to about 0.2 nM. In some aspects, the method comprises administering the miRlOa at a dose sufficient to reach a synovial fluid concentration of about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, or about 10 nM.

[0056] In some aspects, the method comprises administering the miRlOa parenterally. For example, the miRlOa may be administered intravenously (IV), subcutaneously (SQ), intradermally (ID), intraperitoneally (IP), or intramuscularly (IM). In some aspects, the method comprises administering the miRlOa by intra-articular injection. In some aspects, the method comprises administering the miRlOa intravenously.

[0057] In some aspects, the miRlOa is administered in a liquid composition, e.g., a solution. In some aspects, the miRlOa is administered in a solution comprising one or more of a buffer, a salt, a preservative, a stabilizer, a pH adjusting agent, and a solubilizing agent. In some aspects, the miRlOa is administered in a pharmaceutical composition comprising a pharmaceutically acceptable excipient.Atty. Dkt. No. 4842-130WO1

[0058] In some aspects, the miRlOa is encapsulated by a delivery vehicle. For example, the delivery vehicle may be an extracellular vesicle (EV), a lipid nanoparticle (LNP), or a liposome. In some aspects, the miRlOa is encapsulated by a liposome using methods known in the art. In some aspects, the miRlOa is encapsulated by a LNP using methods known in the art. In some aspects, the miRlOa is encapsulated by an EV using methods known in the art.

[0059] Some aspects of the present disclosure are directed to a method of treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy by administering to the subject a therapeutically effective amount of EVs comprising miRlOa. Some aspects of the present disclosure are directed to use of miRlOa in a method of treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy by administering to the subject a therapeutically effective amount of the miRlOa, wherein the miRlOa is encapsulated in EVs.

[0060] In some aspects, the method comprises administering the EVs at a dose sufficient to reach a miRlOa plasma concentration of about 0.1 to 10 nM. In some aspects, the method comprises administering the EVs at a dose sufficient to reach a miRlOa plasma concentration of about 0.2 nM to about 10 nM, about 0.3 nM to about 10 nM, about 0.4 nM to about 10 nM, about 0.5 nM to about 10 nM, about 0.6 nM to about 10 nM, about 0.7 nM to about 10 nM, about 0.8 nM to about 10 nM, about 0.9 nM to about 10 nM, about 1 nM to about 10 nM, about 2 nM to about 10 nM, about 3 nM to about 10 nM, about 4 nM to about 10 nM, about 5 nM to about 10 nM, about 6 nM to about 10 nM, about 7 nM to about 10 nM, about 8 nM to about 10 nM, or about 9 nM to about 10 nM. In some aspects, the method comprises administering the EVs at a dose sufficient to reach a miRlOa plasma concentration of about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, about 0.1 nM to about 0.9 nM, about 0.1 nM to about 0.8 nM, about 0.1 nM to about 0.7 nM, about 0.1 nM to about 0.6 nM, about 0.1 nM to about 0.5 nM, about 0.1 nM to about 0.4 nM, about 0.1 nM to about 0.3 nM, or about 0.1 nM to about 0.2 nM. In some aspects, the method comprises administering the EVs at a dose sufficient to reach a miRlOa plasma concentration of about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, or about 10 nM.Atty. Dkt. No. 4842-130WO1

[0061] In some aspects, the method comprises administering the EVs at a dose sufficient to reach a miRlOa synovial fluid concentration of about 0.1 to 10 nM. In some aspects, the method comprises administering the EVs at a dose sufficient to reach a miRlOa synovial fluid concentration of about 0.2 nM to about 10 nM, about 0.3 nM to about 10 nM, about 0.4 nM to about 10 nM, about 0.5 nM to about 10 nM, about 0.6 nM to about 10 nM, about 0.7 nM to about 10 nM, about 0.8 nM to about 10 nM, about 0.9 nM to about 10 nM, about 1 nM to about 10 nM, about 2 nM to about 10 nM, about 3 nM to about 10 nM, about 4 nM to about 10 nM, about 5 nM to about 10 nM, about 6 nM to about 10 nM, about 7 nM to about 10 nM, about 8 nM to about 10 nM, or about 9 nM to about 10 nM. In some aspects, the method comprises administering the EVs at a dose sufficient to reach a miRlOa synovial fluid concentration of about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, about 0.1 nM to about 0.9 nM, about 0.1 nM to about 0.8 nM, about 0.1 nM to about 0.7 nM, about 0.1 nM to about 0.6 nM, about 0.1 nM to about 0.5 nM, about 0.1 nM to about 0.4 nM, about 0.1 nM to about 0.3 nM, or about 0.1 nM to about 0.2 nM. In some aspects, the method comprises administering the EVs at a dose sufficient to reach a miRlOa synovial fluid concentration of about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, or about 10 nM.

[0062] In some aspects, the method comprises administering the EVs parenterally. For example, the EVs may be administered intravenously (IV), subcutaneously (SQ), intradermally (ID), intraperitoneally (IP), or intramuscularly (IM). In some aspects, the method comprises administering the EVs by intra-articular injection. In some aspects, the method comprises administering the EVs intravenously.

[0063] In some aspects, the method comprises administering the EVs to the subject at a dose of about 1 x 108to about 1 x 1011EVs / kg. In some aspects, the method comprises administering the EVs at a dose of about 1 x 108EVs / kg, about 2 x 108EVs / kg, about 3 x 108EVs / kg, about 4 x 108EVs / kg, about 5 x 108EVs / kg, about 6 x 108EVs / kg, about 7 x 10sEVs / kg, about 8 x 108EVs / kg, about 9 x 108EVs / kg, about 1 x 109EVs / kg, about 2 x 109EVs / kg, about 3 x 109EVs / kg, about 4 x 109EVs / kg, about 5 x 109EVs / kg, about 6 x 109EVs / kg, about 7 x 109EVs / kg, about 8 x 109EVs / kg, about 9 x 109EVs / kg, about 1 x 1010EVs / kg, about 2 x 1010EVs / kg, about 3 x 1010EVs / kg, about 4 x 1010EVs / kg, about 5 x 1010Atty. Dkt. No. 4842-130WO1EVs / kg, about 6 x IO10EVs / kg, about 7 x IO10EVs / kg, about 8 x IO10EVs / kg, about 9 x IO10EVs / kg, or about 1 x 1011EVs / kg.

[0064] The EVs of the present disclosure may be produced by a method known in the art. In some aspects, the EVs of the present disclosure are produced by a method comprising: a) culturing a cell in a culture medium comprising one or more growth factors, and b) isolating the EVs from a supernatant of the culture medium from step a).

[0065] Some aspects of the present disclosure are directed to a method of manufacturing a medicament for use in treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy, wherein the medicament comprises EVs comprising miRlOa, the method comprising: a) culturing an isolated cell in a culture medium comprising one or more growth factors, and b) isolating the EVs from a supernatant of the culture medium from step a). In some aspects, the subject has hemophilia. In some aspects, the subject has hemophilic arthropathy. In some aspects, the subject is at risk of developing hemophilic arthropathy. In some aspects, the subject has hemarthrosis.

[0066] In some aspects, the isolated human cell is an endothelial cell. In some aspects, the isolated human cell is a human umbilical vein endothelial cell (HUVEC).However, the type of cell used in the cell culture methods of the present disclosure are not particularly limited, and may be any suitable cell type known in the art to produce EVs.

[0067] In some aspects, the culture medium is an endothelial basal medium.However, the type of culture medium used in the cell culture methods of the present disclosure is not particularly limited. A person of ordinary skill in the art will understand that the culture medium used will vary depending on the type of cell. In some aspects, the culture medium comprises one or more of ascorbic acid, hydrocortisone hemisuccinate, heparin sulfate, L-glutamine, and combinations thereof. Additional cell culture additives known in the art may be used and are well within the knowledge of a person of ordinary skill in the art.

[0068] In some aspects, the one or more growth factors are selected from a vascular endothelial growth factor (VEGF), an endothelial cell growth factor (EGF), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF), and combinations thereof. In some aspects, the one or more growth factors include one or more VEGF, for example VEGF- A, VEGF-B, VEGF-C, VEGF-D, and / or PIGF. In some aspects, the one or more growth factors include one or more EGF, for example EGF, amphirgulin, betacellulin, epigen, epiregulin, HB-EGF, neuregulins, and / or TGF-alpha. In some aspects, the one or more growth factors include one or more FGF, for example FGF-10, FGF-4, FGF-9, FGF-17, FGF-8, FGF-12, FGF-16, FGF-18, FGF-19, FGF-23, FGF-6, FGF-3, FGF-5, FGF-20, FGF-22, FGF-7, FGF-1,Atty. Dkt. No. 4842-130WO1FGF-2, and / or bFGF. In some aspects, the one or more growth factors include one or more IGF, for example, IGF-I and / or IGF-II.

[0069] In some aspects, the method comprises culturing the isolated human cell for about 24-96 hours, about 24-72 hours, about 24-48 hours, or about 48-72 hours. In some aspect, the method comprises culturing the isolated human cell for about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, about 90 hours, or about 96 hours. In some aspects, the method comprises culturing the isolated human cell for about 48 hours.

[0070] In some aspects, the method comprises culturing the isolated human cell in the presence of Factor Vila (FVIIa). Thus, in some aspects, the culture medium comprises FVIIa. In some aspects, the method comprises transfecting the isolated human cell with a miRlOa mimic before step a). The method of transfection is not particularly limited, and any method of transfection known in the art may be used.

[0071] Some aspects of the present disclosure are directed to a method of treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy, the method comprising a) culturing an isolated cell in a culture medium comprising one or more growth factors, b) isolating EVs from a supernatant of the culture medium from step a), and c) administering to the subject the isolated EVs from step b). In some aspects, the subject has hemophilia. In some aspects, the subject has hemophilic arthropathy. In some aspects, the subject is at risk of developing hemophilic arthropathy. In some aspects, the subject has hemarthrosis.

[0072] In some aspects, the isolated human cell is an endothelial cell. In some aspects, the isolated human cell is a human umbilical vein endothelial cell (HUVEC).However, the type of cell used in the cell culture methods of the present disclosure are not particularly limited, and may be any suitable cell type known in the art to produce EVs.

[0073] In some aspects, the culture medium is an endothelial basal medium.However, the type of culture medium used in the cell culture methods of the present disclosure is not particularly limited. A person of ordinary skill in the art will understand that the culture medium used will vary depending on the type of cell. In some aspects, the culture medium comprises one or more of ascorbic acid, hydrocortisone hemisuccinate, heparin sulfate, L-glutamine, and combinations thereof. Additional cell culture additives known in the art may be used and are well within the knowledge of a person of ordinary skill in the art.

[0074] In some aspects, the one or more growth factors are selected from a vascular endothelial growth factor (VEGF), an endothelial cell growth factor (EGF), a fibroblastAtty. Dkt. No. 4842-130WO1growth factor (FGF), an insulin-like growth factor (IGF), and combinations thereof. In some aspects, the one or more growth factors include one or more VEGF, for example VEGF-A, VEGF-B, VEGF-C, VEGF-D, and / or PIGF. In some aspects, the one or more growth factors include one or more EGF, for example EGF, amphirgulin, betacellulin, epigen, epiregulin, HB-EGF, neuregulins, and / or TGF-alpha. In some aspects, the one or more growth factors include one or more FGF, for example FGF-10, FGF-4, FGF-9, FGF-17, FGF-8, FGF-12, FGF-16, FGF-18, FGF-I9. FGF-23, FGF-6, FGF-3, FGF-5, FGF-20, FGF-22, FGF-7, FGF-1, FGF-2, and / or bFGF. In some aspects, the one or more growth factors include one or more IGF, for example, IGF-I and / or IGF-II.

[0075] In some aspects, the method comprises culturing the isolated human cell for about 24-96 hours, about 24-72 hours, about 24-48 hours, or about 48-72 hours. In some aspect, the method comprises culturing the isolated human cell for about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, about 90 hours, or about 96 hours. In some aspects, the method comprises culturing the isolated human cell for about 48 hours.

[0076] In some aspects, the method comprises culturing the isolated human cell in the presence of Factor Vila (FVIIa). Thus, in some aspects, the culture medium comprises FVIIa. In some aspects, the method comprises transfecting the isolated human cell with a miRlOa mimic before step a). The method of transfection is not particularly limited, and any method of transfection known in the art may be used.

[0077] In some aspects, the method comprises administering the EVs parenterally. For example, the EVs may be administered intravenously (IV), subcutaneously (SQ), intradermally (ID), intraperitoneally (IP), or intramuscularly (IM). In some aspects, the method comprises administering the EVs by intra-articular injection. In some aspects, the method comprises administering the EVs intravenously.

[0078] In some aspects, the method comprises administering the EVs at a dose of about 1 x 108to about 1 x 1011EVs / kg. In some aspects, the method comprises administering the EVs at a dose of about 1 x 108EVs / kg, about 2 x 10sEVs / kg, about 3 x 108EVs / kg, about 4 x 108EVs / kg, about 5 x 108EVs / kg, about 6 x 108EVs / kg, about 7 x 108EVs / kg, about 8 x 108EVs / kg, about 9 x 108EVs / kg, about 1 x 109EVs / kg, about 2 x 109EVs / kg, about 3 x 109EVs / kg, about 4 x 109EVs / kg, about 5 x 109EVs / kg, about 6 x 109EVs / kg, about 7 x 109EVs / kg, about 8 x 109EVs / kg, about 9 x 109EVs / kg, about 1 x 1010EVs / kg, about 2 x 1010EVs / kg, about 3 x 1010EVs / kg, about 4 x 1010EVs / kg, about 5 x 1010EVs / kg,Atty. Dkt. No. 4842-130WO1about 6 x IO10EVs / kg, about 7 x IO10EVs / kg, about 8 x IO10EVs / kg, about 9 x IO10EVs / kg, or about 1 x 1011EVs / kg.

[0079] In some aspects, the method comprises reducing or preventing macrophage infiltration into a joint of the subject. In some aspects, the method comprises reducing or preventing neovascularization in a joint of the subject. In some aspects, the method comprises reducing or preventing synovial hyperplasia in a joint of the subject. In some aspects, the method comprises reducing or preventing cartilage degeneration in a joint of the subject. In some aspects, the method comprises reducing or preventing chondrocyte apoptosis in the subject. In some aspects, the method comprises reducing expression of IL-6 in the subject’s synovial fluid.III. Compositions of the Disclosure

[0080] In some aspects, the present disclosure provides a composition comprising a microRNA 10a (miRlOa). In some aspects, the composition comprising the miRlOa is a liquid composition. In some aspects, a liquid composition may comprise one or more of a buffer, a salt, a preservative, a stabilizer, a pH adjusting agent, and a solubilizing agent. In some aspects, the miRlOa is formulated with a pharmaceutically acceptable carrier.

[0081] In some aspects, the miRlOa is encapsulated by a delivery vehicle. For example, the delivery vehicle may be an extracellular vesicle (EV), a lipid nanoparticle (LNP), or a liposome. In some aspects, the miRlOa is encapsulated by a liposome using methods known in the art. In some aspects, the miRlOa is encapsulated by a LNP using methods known in the art. In some aspects, the miRlOa is encapsulated by an EV. In some aspects, the EV is produced by a method comprising: a) culturing an isolated cell in a culture medium comprising one or more growth factors, and b) isolating the EVs from a supernatant of the culture medium from step a). In some aspects, the isolated human cell is an endothelial cell. In some aspects, the isolated human cell is a human umbilical vein endothelial cell (HUVEC). However, the type of cell used in the cell culture methods of the present disclosure are not particularly limited, and may be any suitable cell type known in the art to produce EVs. In some aspects, the culture medium is an endothelial basal medium. However, the type of culture medium used in the cell culture methods of the present disclosure is not particularly limited. A person of ordinary skill in the art will understand that the culture medium used will vary depending on the type of cell. In some aspects, the culture medium comprises one or more of ascorbic acid, hydrocortisone hemisuccinate, heparin sulfate, L-Atty. Dkt. No. 4842-130WO1glutamine, and combinations thereof. Additional cell culture additives known in the art may be used and are well within the knowledge of a person of ordinary skill in the art.

[0082] In some aspects, the one or more growth factors are selected from a vascular endothelial growth factor (VEGF), an endothelial cell growth factor (EGF), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF), and combinations thereof. In some aspects, the one or more growth factors include one or more VEGF, for example VEGF- A, VEGF-B, VEGF-C, VEGF-D, and / or PIGF. In some aspects, the one or more growth factors include one or more EGF, for example EGF, amphirgulin, betacellulin, epigen, epiregulin, HB-EGF, neuregulins, and / or TGF-alpha. In some aspects, the one or more growth factors include one or more FGF, for example FGF-10, FGF-4, FGF-9, FGF-17, FGF-8, FGF-12, FGF-16, FGF-18, FGF-19, FGF-23, FGF-6, FGF-3, FGF-5, FGF-20, FGF-22, FGF-7, FGF-1, FGF-2, and / or bFGF. In some aspects, the one or more growth factors include one or more IGF, for example, IGF-I and / or IGF-II.

[0083] In some aspects, the EV is produced by a method comprising culturing the isolated human cell for about 24-96 hours, about 24-72 hours, about 24-48 hours, or about 48-72 hours. In some aspect, the method comprises culturing the isolated human cell for about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, about 90 hours, or about 96 hours. In some aspects, the method comprises culturing the isolated human cell for about 48 hours.

[0084] In some aspects, the EV is produced by a method comprising culturing the isolated human cell in the presence of Factor Vila (FVIIa). Thus, in some aspects, the culture medium comprises FVIIa. In some aspects, the EV is produced by a method comprising transfecting the isolated human cell with a miRlOa mimic before step a). The method of transfection is not particularly limited, and any method of transfection known in the art may be used.EXAMPLESExample 1 : Materials and Methods

[0085] Reagents

[0086] Human recombinant FVIIa (NovoSeven) was purchased from UT Health pharmacy (Tyler, TX). miRlOa mimic, miRlOa inhibitor, and scrambled (scr) miRlOa were obtained from Sigma-Aldrich (St. Louis, MO). Rat anti-mouse F4 / 80 antibody was from CellAtty. Dkt. No. 4842-130WO1Signaling (Danvers, MA) and polyclonal rabbit anti-mouse CD31 antibody was from Abeam (Cambridge, MA). Alcian blue stain was from Sigma-Aldrich (St. Louis, MO). TUNEL staining kit was from Promega (Madison, WI). Iron staining kit was obtained from American Mastertech Scientific, Inc. (Lodi, CA). The blocking solution and other histochemical reagents were from Dako (Agilent; Santa Clara, C A). ELISA kit for measuring mouse IL-6 was from Invitrogen (Thermo Fisher Scientific. Waltham, MA).

[0087] Cells

[0088] Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Walkersville, MD) and cultured in endothelial basal medium supplemented with endothelial cell-specific growth factors (Life Line Cell Technology, Frederick, MD).

[0089] Animals

[0090] FVIIT / _mice were bred in-house. FVIIkAmice in the B6 / 129S background (Jackson laboratories) were backcrossed with C57BL / 6J mice for more than 10 generations to generate FVIIT / _mice in the C57BL / 6J genetic background. Animal protocols employed in the study were reviewed and approved by the Institutional Animal Care and Use Committee and all studies were conducted according to the animal welfare guidelines outlined in the Guide for the Care and Use of Laboratory Animals.

[0091] Generation of EVs and their isolation and quantification

[0092] HUVECs were treated with a control vehicle or FVIIa (25 nM) for 6 h and EVs released into overlying supernatant media were isolated as described in Das et al.(Blood, 2021, 137(24): 3428-3442). eEVs were quantified by nanoparticle tracking analysis (NTA) as described in Das et al. (Blood, 2022, 139(1): 118-133). To incorporate miRlOa inhibitor or miRlOa mimic into eEVs, endothelial cells were transfected with miRlOa inhibitor, miRlOa mimic, or the corresponding scrambled (scr) sequences by using the Lipofectamine RNAiMAX reagent (Thermo Fisher) in serum-free conditions. Cells were cultured for 48 h before they were treated with a control vehicle or FVIIa to generate EVs.

[0093] Induction of joint bleed by a needle puncture and treatments

[0094] Intraarticular bleeding into knee joints was induced by a needle puncture injury, as described by Sun et al. (Blood, 2008, 112(12): 4532-4541) and Hakobyan et al. (Haemophilia, 2008, 12(4): 804-809). One day before the injury, the hair over both knee joints was removed by using hair removal cream. Joint capsule of the right knee of anesthetized FVIIT / _mice was punctured with a 30x0.5-G needle below the patella to induce bleeding into the joint. The left knee of the same mouse, which was not subjected to the needle puncture, was used as a control (uninjured). For pain management, mice were treatedAtty. Dkt. No. 4842-130WO1with analgesics as described by Magisetty et al. (Blood, 2020, 135(25): 2211-2223). Mice were administered control- or FVIIa-released eEV (2xl09eEVs / mouse) intravenously (IV) via the tail vein at 30 min, 24 h, and 72 h following the knee injury. At specified intervals following the injury, mice were euthanized, the skin was removed from over the knee joints, and the knee joints were photographed and processed for histology staining, immunohistochemistry, or synovial fluid collection.

[0095] Evaluation of hemarthrosis

[0096] Knee diameter, before the injury and every alternate day for 2 weeks following the injury, was measured using electronic Vernier calipers, and the percent change in joint diameter was calculated. The knee diameter before the injury was subtracted from the diameter following the injury, and differences in the knee diameter were plotted as percentage change in the joint diameter. Gross examination of knee joints was performed every alternate day to assess visually the extent of blood leaking into joints and knee mobility. A visual bleeding score (VBS) was assigned to score the extent of injury (0, normal knee and absence of blood; 1, normal knee, presence of blood; 2, distended but not a tense knee, presence of blood; 3, tense and distended knee, presence of blood). The joint diameter evaluation and the assignment of VBS were performed in a blinded fashion where the evaluator was not aware of the experimental treatments or mice groups.

[0097] Histology and immunohistochemistry of knee joints and synovitis scoring

[0098] For histology and immunohistochemistry, knee joints (the femur and tibia / fibula area, ~1 cm each direction from the joint) were collected into 10% neutral buffered formalin fixative. After 48 h of fixation, knee joints were decalcified for 16 to 20 h in DECAL (StatLab, McKinney, TX), processed in graded alcohol, and embedded in paraffin. Five-micrometer thin sections were cut, and the sections were stained with hematoxylin and eosin (H&E) for general histological analysis and Alcian blue or Safranin O / Fast Green for proteoglycans and glycosaminoglycans to evaluate cartilage degeneration. Joint tissue sections were immunostained for CD31 to assess neo-angiogenesis and F4 / 80 for macrophage infiltration. Apoptosis in knee joints was evaluated by staining the knee joint sections with a TUNEL stain. To measure iron deposits in the injured synovium, the joint sections were stained with Prussian blue stain to detect tissue iron. Staining pattern in all tissue sections were scored as described by Magisetty et al. (Blood, 2022, 139(18): 2830-2841). Synovitis score is a cumulative score of synovial hyperplasia (0-3 points), vascularity (0-3 points), and the presence of discoloration by hemosiderin, blood (erythrocytes), andAtty. Dkt. No. 4842-130WO1presence of synovial villi (0 for absence and 1 for their presence in each category), and cartilage degeneration (0 to 2 points). The range of cumulative score was 0 to 11.

[0099] Imaging

[0100] Tissue sections were viewed and imaged using Olympus microscope system equipped with 4x / 0.13, 10x / 0.30, 20x / 0.50, 40x / 0.75 objective lenses, DP27 camera, and Cellsens software. All sections were viewed and imaged using 4x / 0.13 objective lens and selective areas were imaged using either 20x / 0.50 or 40x / 0.75 objective lenses.

[0101] Assessment of Vascular Leakage

[0102] Vascular leakage into knee joints was quantified by extravasation of fluorescein dextran as described by Magisetty et al. (Thromb Haemost, 2019, 119(8): 1283-1294). Briefly, at the end of 14 day following the knee injury, mice were injected with fluorescein dextran (20 pg / Kg in 100 pl; MW 70,000, anionic, lysine fixable) intravenously via the tail vein. Three hours after the dextran administration, mice were exsanguinated and perfused with ice-cold saline, and knee joints were excised. Joints were processed for tissue sectioning. After deparaffinizing tissue sections, they were imaged using Biotek Lionheart FX Automated Fluorescence Microscope and the fluorescence intensity of the images was quantified. Joint tissues from mice not injected with fluorescein dextran were used to measure auto fluorescence, and the autofluorescence values were subtracted from fluorescence intensities measured in joint tissues of experimental animals to quantify vascular leakage.

[0103] Assessment of hemostasis using the saphenous vein incision-induced bleeding

[0104] Immediately after the administration of eEVs, acute bleeding was induced by the incision of the saphenous vein. The number of hemostatic plugs formed in 30 min duration were recorded and the average time to achieve hemostasis was calculated from these data, as described previously by Keshava et al. (J Thromb Haemost, 2016, 14(3): 546-550) and Keshava et al. (Blood Adv, 2017, 1(15): 1206-1214). Throughout the 30-min experimental time period, blood coming from the wound site was adsorbed on Kim-wipes periodically, and hemoglobin was extracted by soaking the wipes in 20 ml of solution of ABX Lysebio (France) for 2 h or more. Hemoglobin was also extracted from known volumes of freshly collected mouse blood to generate a standard curve for calculating the volume of blood loss.

[0105] Data analysis

[0106] In most of the experiments, 6 to 10 animals were used in each group, and the data were shown as mean ± SEM. Statistical significance among the groups was analyzed byAtty. Dkt. No. 4842-130WO1two-way analysis of variance, repeated measures followed by Tukey’s post hoc multiple comparison test for percent change in the joint diameter (joint edema) and the visual bleeding score, For all other studies, one-way analysis of variance followed by Tukey’s post hoc multiple comparison test was used.Example 2: Administration of FVIIa-released eEVs reduces joint edema and bleeding in hemophilia A mice following hemarthrosis

[0107] Joint bleeding in FVIII-mice was induced with a needle puncture of the joint and mice were administered saline, control eEVs (Con eEVs), or FVIIa-released eEVs (FVIIa eEVs) (2 xlO9eEVs / mice) via tail vein injection at 30 min, 24 h, and 72 following the injury. Mice were monitored for 14 days to evaluate joint edema by measuring the joint diameter and determining the visual bleeding score. The knee joint diameter, before the injury and following the injury on alternate days, was measured using electronic calipers. The diameter of the knee joint before the injury was subtracted from the diameter following the injury, and the differences in the knee joint diameter were plotted as the percentage change in the joint diameter (FIG. 1, panel A). Joint bleeding was evaluated by physical examination of knee joints and assigning an arbitrary score (0 to 3; 0, normal knee and absence of blood; 1, normal knee, and presence of blood; 2, distended but not a tense knee, and presence of blood; 3, tense and distended knee, and presence of blood) (FIG.1, panel B). Assessment of joint bleeding showed no statistically significant differences in the visual bleeding score (VBS) among mice treated with saline, control eEVs, or FVIIa-released eEVs on day 2. However, the VBS was markedly reduced thereafter in mice treated with FVIIa-released eEVs, reaching no observable VBS on day 12. In contrast, the VBS score was decreased only minimally in mice treated with saline or control eEVs.

[0108] Representative photographs of knee joints at the end of day 14 following the injury are presented in FIG. 1, panel C. Gross examination of knee joints on day 14 showed presence of readily observable blood in the joints of FVIIT7’ mice administered with saline or control eEVs whereas the blood in the articular space was mostly resolved in mice treated with FVIIa-released eEVs. Analysis of iron deposition in the synovium by Prussian blue staining as an indicator of recurrent joint bleeding showed prominent iron deposition in the joint tissue of injured knee of FVIII7-mice treated with saline or control eEVs. Minimal iron deposition was found in the injured knees of mice treated with FVIIa-released eEVs (FIG.2).Atty. Dkt. No. 4842-130WO1Example 3: Treatment of hemophilia mice with FVIIa-released and not control eEVs reduces joint bleed-induced synovitis and HA

[0109] Joint bleeding in FVIIT / _mice was induced with a needle puncture and mice were treated with control or FVIIa-released eEVs as described in Example 2. At the end of day 14 following the injury, mice were euthanized, knee joints were excised and fixed, and joint tissue sections were processed for histopathological analysis by staining with H&E (FIG. 3, panel A), immunostaining for F4 / 80 marker for macrophages (FIG. 3, panel B), immunostaining for endothelial cell marker CD31 for neoangiogenesis (FIG.3. panel C), and Alcian blue for catilage (FIG. 3, panel D). The joint tissue pathology was quantified by scoring H&E-stained sections on a 0 to 6 scale for synovial hyperplasia [(0, normal, four cell layers thick; 1, four to five cell layers thick; 2, six to seven cell layers thick; 3, more than seven layers thick), the presence of RBC (0, absent; 1, present), villus formation (0, absent; 1, present), and discoloration by hemosiderin (0, absent; 1, present)]. Macrophage infiltration was quantified on a 0-4 scale (0, absence of macrophages; 1, scattered macrophages; 2, line of macrophages; 3, a cluster of macrophages; 4, sheets of macrophages. The number of blood vessels were counted in four independent microscopic fields at 40x magnification, and averaged. Cartilage degeneration was scored on a scale of 0-2 (0, absence of cartilage degeneration; 1, partial loss of proteoglycan content and pannus formation; 2. complete cartilage degeneration / absence of proteoglycans, pannus formation, and femur remodeling).

[0110] Histological examination of joints tissue sections from mice subjected to joint bleeding on day 14 by H&E staining showed hypercellularity in the joint space of saline- or control eEV-treated F VI II7mice. The joint space was filled with multiple layers of proliferating synovial fibroblasts, which led to the expansion of the synovial and stromal linings, and the formation of synovial villi (FIG. 3, panel A). The presence of red blood cells in the synovium was readily observable. Histological examination of joint tissue sections from injured mice treated with FVIIa-released eEVs showed no or only minimal synovial hypertrophy and no readily detectable red blood cells in the joint space. Joint tissue sections form the injured mice administered with FVIIa-released eEVs showed a clear synovial lining with minimal thickening. Further analysis of joint tissue sections showed massive infiltration of macrophages and extensive neoangiogenesis in the joint tissue of injured mice treated with saline or control eEVs (FIG. 3, panels B and C). Macrophage infiltration and neoangiogenesis in the synovium were minimal if mice were treated with FVIIa-released eEVs. Analysis of joint tissue sections for cartilage by the Alcian blue staining revealed aAtty. Dkt. No. 4842-130WO1complete loss of cartilage in the injured knee of mice treated with saline or control eEVs, but the cartilage appeared to be intact and no signs of cartilage degeneration in mice treated with FVIIa-released eEVs (FIG. 3, panel D). Staining joint tissues with Safranin O / Fast Green, which also stains glycosaminoglycans and proteoglycans, further supported the observation that FVIIa-released eEVs protects against joint bleed-induced cartilage erosion (FIG.4). The global scoring of synovitis pathology revealed that synovitis is significantly lower in mice treated with FVIIa-released eEVs compared to mice treated with saline or control eEVs (FIG.5).Example 4: miRlOa inhibitor reverses the protective effect of FVIIa-released eEVs

[0111] To investigate whether protective effect of FVIIa-released eEVs on HA is mediated by miRlOa in eEVs, FVIIa-released eEVs were packed with either scrambled miRlOa or miRlOa inhibitor. Joint bleeding in FVIir / _mice was induced with a needle puncture and the mice were administered saline, FVIIa-released eEVs (FVIIa eEVs), scrambled miRlOa containing FVIIa-released eEVs (FVIIa eEVs / scr miRlOa) or miRlOa inhibitor containing FVIIa-released eEVs (FVIIa eEVs / miRlOa Inh) (2 xlO9eEVs / mice) via tail vein injection at 30 min, 24 h, and 72 h following the injury. Joint diameter (FIG. 6, panel A) and VBS (FIG. 6, panel B) were measured as described in Example 2. Incorporation of miRlOa inhibitor into FVIIa-released eEVs fully reversed the protective effect of FVIIa-released eEVs in reducing joint edema and recurrent bleeding following the initial injury. Incorporation of scr miRlOa had no significant effect on the protective effects of FVIIa-released eEVs on joint edema or recurrent joint bleeding. Gross examination of knee joints on day 14 showed that the blood in the articular space is mostly resolved at day 14 in mice treated with FVIIa-released eEVs. However, a significant amount of blood was still seen in the joint space of FVIII7’ mice if the administered FVIIa-released eEVs were made to contain miRlOa inhibitor (FIG. 6, panel C). The above data was further supported by Prussian blue staining of joint tissue sections for iron deposition (FIG. 2).

[0112] At the end of day 14, mice were euthanized, knee joints were excised and fixed, and joint tissue sections were processed for histopathological and 1HC analysis. All stained joints tissue sections were scored as described in Example 3. Analysis of joint tissue sections of mice treated with FVIIa-released eEVs loaded with miRlOa inhibitor showed the same extensive damage to synovial lining, emergence of synovial villi, and abundance of RBCs in the synovium as observed in joint tissue sections of mice treated with saline (FIG. 7, panel A). Further analysis of joint tissue sections of mice treated with miRlOa inhibitorAtty. Dkt. No. 4842-130WO1containing FVIIa-released eEVs showed, as observed in the joint tissue sections of mice treated with saline, a massive macrophage infiltration into the synovium (FIG. 7, panel B) and extensive neovascularization (FIG. 7, panel C), whereas joint tissue sections of mice administered with FVIIa-released eEVs containing scr miRlOa showed minimal macrophage infiltration and neovascularization. Alcian blue (FIG. 7, panel D) and Safranin O / Fast Green staining (FIG. 4) revealed that mice administered with saline or miRlOa inhibitor containing FVIIa-released eEVs showed marked cartilage disintegration. In contrast, mice treated with FVIIa-released eEVs or FVIIa-released eEVs containing scrambled miRlOa prevented the cartilage degeneration. The global scoring of synovitis pathology revealed miRlOa inhibitor in FVIIa-released eEVs reversed the protective effect of FVIIa-released eEVs in suppressing hemophilic arthropathy (FIG. 8).Example 5: Treatment of hemophilia mice with miRlOa mimic containing eEVs suppresses joint bleed-induced hemophilic arthropathy

[0113] To further demonstrate that miRlOa in eEVs is responsible for providing the protection against the development of HA, FVIIT7’ mice subjected to joint bleed were administered control eEVs or control eEVs loaded with scr miRlOa or miRlOa mimic (2 x 109eEVs / mice) via tail vein injection at 30 min, day 1, and day 3 following the knee injury and evaluated for the development of HA. Measurement of knee joint diameter and VBS revealed that joint edema and joint bleeding was markedly lower in mice treated with control eEVs containing miRlOa mimic compared to mice treated with saline, control eEVs, or control eEVs containing scr miRlOa (FIG. 9, panels A and B). At the end of 14 days, no visually detectable blood was noted in the joint area of the injured mice treated with control eEVs loaded with miRlOa mimic (FIG. 9, panel C). Joint tissue sections of mice treated with control eEVs containing miRlOa mimic showed no or minimal iron deposition compared to mice treated with control eEVs and control eEVs containing scrambled miRlOa (FIG. 2).

[0114] IHC analysis of joint tissue sections further confirmed that treatment of FVIIT mice with control eEVs loaded with miRlOa mimic markedly attenuated the development of HA following hemarthrosis. Treatment of FVII I mice with control eEVs containing miRlOa mimic showed markedly reduced joint bleed-induced synovial hyperplasia (FIG. 10, panel A), macrophage infiltration (FIG. 10, panel B), neovascularization (FIG. 10, panel C), and cartilage degeneration (FIG. 10, panel D) compared to mice treated with saline, control eEVs, or control eEVs loaded with scr miRlOa. Additionally, the global scoring of theAtty. Dkt. No. 4842-130WO1synovitis pathology showed that the synovitis was markedly less severe in mice treated with control eEVs containing miRlOa mimic compared to other groups of mice (FIG. 11).Example 6: Administration of miRlOa containing eEVs reduces joint bleed-induced chondrocyte apoptosis, elaboration of IL-6 levels and vascular leakage in the synovium

[0115] Joint bleeding in FVIIIzmice was induced with a needle puncture of joint and mice were administered saline, control eEVs, control eEVs loaded with scrambled miRlOa or miRlOa mimic, FVIIa-released eEVs, or FVIIa-released eEVs loaded with scrambled miRlOa or miRlOa inhibitor as described in Examples 2-4.

[0116] To evaluate chondrocyte apoptosis, 14 days following the injury, the knee joint was excised, sectioned, and stained with TUNEL. Administration of FVIIa-released eEVs markedly reduced chondrocyte apoptosis, while incorporation of miRlOa inhibitor into FVIIa-released eEVs negated the protective effect (FIG. 12). In contrast to FVIIa-released eEVs, the administration of control eEVs had no significant effect on joint bleed-induced chondrocyte apoptosis. However, treatment of mice with control eEVs loaded with miRlOa mimic also markedly reduced chondrocyte apoptosis induced by joint bleed (FIG. 12).

[0117] To evaluate IL-6 levels, mice were euthanized at day 7 post -injury and synovial fluids were collected. IL-6 levels were then quantified using ELISA (FIG. 13). Treatment of FVIII / _mice with saline or control eEVs did not affect IL-6 levels in the synovium. In contrast, the administration of FVIIa-released eEVs completely blocked IL-6 expression in the synovium following hemarthrosis. However, treatment of FVIII / _mice with FVIIa-released eEVs containing miRlOa inhibitor did not block joint bleed-induced IL-6 expression levels in the synovium. Additional experiments showed treatment of mice with control eEVs containing miRlOa mimic markedly curtailed the joint bleed-induced IL-6 levels in the synovium.

[0118] To evaluate vascular leakage, 14 days after the knee injury, mice were injected with fluorescein dextran (70000 molecular weight, 20 mg / kg) via the tail vein, and 3 hours later, mice were perfused and euthanized. Knee joints were collected, sectioned, and analyzed by fluorescence microscopy at 4x magnification. The fluorescence intensity of tissue sections was quantified and corrected to autofluorescence using joint tissue sections obtained from mice that were not administered with fluorescein dextran (FIG. 14). Hemarthrosis markedly increased vascular leakage into knee joints and the surrounding tissue. Treatment of mice with FVIIa-released eEVs or control eEVs loaded with miRlOa mimic significantly reducedAtty. Dkt. No. 4842-130WO1the vascular leakage in the injured knee. Incorporation of miRlOa inhibitor into FVIIa-released eEVs attenuated the barrier protective effect of FVIIa-released eEVs.Example 7: miRlOa in eEVs does not affect hemostasis in FVIIF7" mice

[0119] In order to establish that miRlOa-depedent protective effects of eEVs on the joint bleed-induced HA primarily conies from the anti-inflammatory effects of miRlOa and not from any potential effect of miRlOa in correcting impaired hemostasis in hemophilia mice, we evaluated the hemostatic effect of various eEV preparations in FVIlF mice using saphenous vein incision-induced bleeding model. Joint bleeding in FVIII7-mice was induced with a needle puncture of joint and mice were administered saline, control eEVs, control eEVs loaded with scrambled miRlOa or miRlOa mimic, FVIIa-released eEVs, or FVIIa-released eEVs loaded with scrambled miRlOa or miRlOa inhibitor as described in Examples 2-4. Immediately, mice were subjected to saphenous vein incision-induced bleeding and the number of hemostatic plugs formed in a 30 min duration were recorded. During the 30-min period, blood coming from the wound site was collected onto Kim wipes through the capillary action. Hemoglobin was extracted and measured. Blood loss was determined using the reference curve of hemoglobin obtained from the known volumes of mouse blood. There were no significant differences in blood loss among the different groups. Incorporation of miRlOa inhibitor into FVIIa-released eEVs or miRlOa mimic loading to control eEVs did not alter the hemostatic potential of FVIIa-released eEVs or control eEVs, respectively, indicating that miRlOa in eEVs does not influence hemostasis (FIG. 15).OTHER EMBODIMENTS

[0120] While the subject matter of this disclosure has been described and shown in considerable detail with reference to certain illustrative embodiments, including various combinations and sub-combinations of features, those skilled in the art will readily appreciate other embodiments and variations and modifications thereof as encompassed within the scope of the present disclosure. Moreover, the descriptions of such embodiments, combinations, and sub-combinations is not intended to convey that the claimed subject matter requires features or combinations of features other than those expressly recited in the claims.Accordingly, the scope of this disclosure is intended to include all modifications and variations encompassed within the spirit and scope of the following appended claims.Section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

Atty. Dkt. No. 4842-130WO1CLAIMSWhat is claimed is:

1. A method of treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy, the method comprising administering to the subject a therapeutically effective amount of a microRNA 10a (miRlOa).

2. A method of suppressing joint edema or joint bleeding in a subject, the method comprising administering to the subject a therapeutically effective amount of a miRlOa.

3. A method of reducing or preventing macrophage infiltration into a joint of a subject, the method comprising administering to the subject a therapeutically effective amount of a miRlOa.

4. A method of reducing or preventing neovascularization in a joint of a subject, the method comprising administering to the subject a therapeutically effective amount of a miRlOa.

5. A method of reducing or preventing synovial hyperplasia in a subject, the method comprising administering to the subject a therapeutically effective amount of a miRlOa.

6. A method of reducing or preventing cartilage degeneration in a subject, the method comprising administering to the subject a therapeutically effective amount of a miRlOa.

7. A method of reducing or preventing chondrocyte apoptosis in a subject, the method comprising administering to the subject a therapeutically effective amount of a miRlOa.

8. A method of reducing expression of IL-6 in a subject’s synovial fluid, the method comprising administering to the subject a therapeutically effective amount of a miRlOa.

9. The method of any one of claims 1-8, comprising administering the miRlOa at a dose sufficient to reach a synovial fluid concentration of about 0.1 to 10 nM.Atty. Dkt. No. 4842-130WO110. The method of any one of claims 1-9, further comprising:i) reducing or preventing macrophage infiltration into a joint of the subject; ii) reducing or preventing neovascularization in a j oint of the subject;iii) reducing or preventing synovial hyperplasia in a joint of the subject;iv) reducing or preventing cartilage degeneration in a joint of the subject; v) reducing or preventing chondrocyte apoptosis in the subject;vi) reducing expression of IL-6 in the subject’s synovial fluid;vii) administering a clotting factor; orviii) a combination of i) - vii).

11. The method of any one of claims 1-10, comprising administering the miRlOa parenterally.

12. The method of claim 11, comprising administering the miRlOa by intra-articular injection.

13. The method of claim 11, comprising administering the miRlOa intravenously.

14. The method of any one of claims 1-13, wherein the miRlOa is administered in a liquid composition.

15. The method of claim 14, wherein the liquid composition comprises one or more of a buffer, a salt, a preservative, a stabilizer, a pH adjusting agent, and a solubilizing agent.

16. The method of any one of claims 1-13, wherein the miRlOa is encapsulated by an extracellular vesicle (EV), a lipid nanoparticle (LNP), or a liposome.

17. The method of claim 16, wherein the miRlOa is encapsulated by an EV.

18. The method of claim 17, wherein the EV is produced by a method comprising:Atty. Dkt. No. 4842-130WO1a) culturing an isolated cell in a culture medium comprising one or more growth factors; andb) isolating the EV from a supernatant of the culture medium from step a).

19. The method of claim 18, wherein the one or more growth factors are selected from a vascular endothelial growth factor (VEGF), an endothelial cell growth factor (EGF), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF), and combinations thereof.

20. The method of claim 18 or 19, wherein the isolated cell is an endothelial cell and the EV is derived from the endothelial cell (eEV).

21. The method of claim 20, wherein the endothelial cell is a human umbilical vein endothelial cell (HUVEC).

22. The method of claim 20 or 21, wherein step a) comprises culturing the cell in endothelial basal medium.

23. The method of any one of claims 18-22, wherein the culture medium comprises one or more of ascorbic acid, hydrocortisone hemisuccinate, heparin sulfate, L-glutamine, and combinations thereof.

24. The method of any one of claims 18-23, wherein step a) comprises culturing the cell for about 24-96 hours, about 24-72 hours, about 24-48 hours, or about 48-72 hours.

25. The method of claim 24, wherein step a) comprises culturing the cell for about 48 hours.

26. The method of any one of claims 19-25, wherein the culture medium further comprises factor Vila (FVIIa).

27. The method of any one of claims 19-25, further comprising transfecting the cell with a miRlOa mimic before step a).

28. The method of any one of claims 1-27, wherein the subject has hemophilia.

29. The method of any one of claims 1-28, wherein the subject has hemophilicarthropathy.Atty. Dkt. No. 4842-130WO130. The method of any one of claims 1-29, wherein the subject has hemarthrosis.

31. Use of microRNA 10a (miRlOa) in a method of treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy, wherein the method comprises administering to the subject a therapeutically effective amount of the miRlOa, wherein the miRlOa is encapsulated in extracellular vesicles (EVs).

32. The use of claim 31, comprising administering the miRlOa at a dose sufficient to reach a concentration of about 0.1 to 10 nM miRlOa.

33. The use of claim 31 or 32, comprising administering the EVs at a dose of about 1 x 108to about 1 x 1011EVs / kg.

34. The use of any one of claims 31-33, comprising administering the EVs parenterally.

35. The use of claim 34, comprising administering the EVs by intra-articular injection.

36. The use of claim 34, comprising administering the EVs intravenously.

37. The use of any one of claims 31-36, wherein the EVs are produced by a method comprising:a) culturing a cell in a culture medium comprising one or more growth factors;andb) isolating the EVs from a supernatant of the culture medium from step a).

38. The use of claim 37, wherein the one or more growth factors are selected from a vascular endothelial growth factor (VEGF), an endothelial cell growth factor (EGF), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF), and combinations thereof.

39. The use of any one of claims 31-38, wherein the EVs are produced from an endothelial cell (eEV).

40. The use of claim 39, wherein the endothelial cell is a human umbilical vein endothelial cell (HUVEC).Atty. Dkt. No. 4842-130WO141. The use of claim 39 or 40, wherein step a) comprises culturing the endothelial cell in endothelial basal medium.

42. The use of any one of claims 39-41, wherein the culture medium comprises one or more of ascorbic acid, hydrocortisone hemisuccinate, heparin sulfate, L-glutamine, and combinations thereof.

43. The use of any one of claims 39-42, wherein step a) comprises culturing the cell for about 24-96 hours, about 24-72 hours, about 24-48 hours, or about 48-72 hours.

44. The use of claim 41 , wherein step a) comprises culturing the cell for about 48 hours.

45. The use of any one of claims 39-44, wherein the culture medium further comprises factor Vila (FVIIa).

46. The use of any one of claims 39-45, wherein the method further comprises transfecting the cell with a miRlOa mimic before step a).

47. The use of any one of claims 31-46, wherein the subject has hemophilia.

48. The use of any one of claims 31 -47, wherein the subject has hemophilic arthropathy.

49. The use of any one of claims 31-48, wherein the subject has hemarthrosis.

50. A method of manufacturing a medicament for use in treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy, wherein the medicament comprises extracellular vesicles (EVs) comprising a microRNA 10a (miRlOa), the method comprising:a) culturing an isolated human cell in a culture medium comprising one or more growth factors; andb) isolating the EVs from a supernatant of the culture medium from step a).

51. The method of claim 50, wherein the one or more growth factors are selected from a vascular endothelial growth factor (VEGF), an endothelial cell growth factor (EGF), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF), and combinations thereof.Atty. Dkt. No. 4842-130WO152. The method of claim 50 or 51, wherein the isolated human cell is an endothelial cell.

53. The method of claim 52, wherein the endothelial cell is a human umbilical vein endothelial cell (HUVEC).

54. The method of claim 52 or 53, wherein step a) comprises culturing the isolated human cell in endothelial basal medium.

55. The method of any one of claims 52-54, wherein the culture medium comprises one or more of ascorbic acid, hydrocortisone hemisuccinate, heparin sulfate, L-glutamine, and combinations thereof.

56. The method of any one of claims 50-55, wherein step a) comprises culturing the isolated human cell for about 24-96 hours, about 24-72 hours, about 24-48 hours, or about 48-72 hours.

57. The method of claim 56, wherein step a) comprises culturing the isolated human cell for about 48 hours.

58. The method of any one of claims 50-57, wherein the culture medium further comprises factor Vila (FVIIa).

59. The method of any one of claims 50-58, further comprising transfecting the isolated human cell with a miRlOa mimic before step a).

60. A method of treating a subject having hemophilic arthropathy or at risk of developing hemophilic artropathy, the method comprising:a) culturing an isolated human cell in a culture medium comprising one or more growth factors;b) isolating extracellular vesicles (EVs) from a supernatant of the culture medium from step a); andc) administering to the subject the isolated EVs from step b).

61. The method of claim 60, wherein the one or more growth factors are selected from a vascular endothelial growth factor (VEGF), an endothelial cell growth factor (EGF), aAtty. Dkt. No. 4842-130WO1fibroblast growth factor (FGF), an insulin-like growth factor (IGF), and combinations thereof.

62. The method of claim 60 or 61, wherein the isolated human cell is an endothelial cell.

63. The method of claim 62, wherein the endothelial cell is a human umbilical vein endothelial cell (HUVEC).

64. The method of claim 62 or 63, wherein step a) comprises culturing the isolated human cell in endothelial basal medium.

65. The method of any one of claims 62-64, wherein the culture medium comprises one or more of ascorbic acid, hydrocortisone hemisuccinate, heparin sulfate, L-glutamine, and combinations thereof.

66. The method of any one of claims 62-65, wherein step a) comprises culturing the isolated human cell for about 24-96 hours, about 24-72 hours, about 24-48 hours, or about 48-72 hours.

67. The method of claim 66, wherein step a) comprises culturing the isolated human cell for about 48 hours.

68. The method of any one of claims 60-67, wherein the culture medium further comprises factor Vila (FVIIa).

69. The method of any one of claims 60-68, further comprising transfecting the isolated human cell with a miRlOa mimic before step a).

70. The method of any one of claims 60-69, further comprising:(i) reducing or preventing macrophage infiltration into a joint of the subject; (ii) reducing or preventing neovascularization in a joint of the subject; (iii) reducing or preventing synovial hyperplasia in ajoint of the subject;(iv) reducing or preventing cartilage degeneration in ajoint of the subject; (v) reducing or preventing chondrocyte apoptosis in the subject;Atty. Dkt. No. 4842-130WO1(vi) reducing expression of IL-6 in the subject’s synovial fluid(vii) administering a clotting factor; or(viii) a combination of i) - vii).

71. The method of any one of claims 60-70, wherein step c) comprises administering the isolated EVs parenterally.

72. The method of claim 71, wherein step c) comprises administering the isolated EVs by intra-articular injection.

73. The method of claim 71, wherein step c) comprises administering the isolated EVs intravenously.

74. The method of any one of claims 60-73, wherein the subject has hemophilia.

75. The method of any one of claims 60-74, wherein the subject has hemophilic arthropathy.

76. The method of any one of claims 60-75, wherein the subject has hemarthrosis.

77. A composition comprising a microRNA 10a (miRlOa) for use in a method of treating a subject having hemophilic arthropathy or at risk of developing hemophilic arthropathy, the method comprising administering to the subject a therapeutically effective amount of the miRlOa.

78. The composition of claim 77, wherein the composition is a liquid solution.

79. The composition of claim 78, wherein the liquid solution comprises one or more of a buffer, a salt, a preservative, a stabilizer, a pH adjusting agent, and a solubilizing agent.

80. The composition of claim 77, further comprising a pharmaceutically acceptable excipient.Atty. Dkt. No. 4842-130WO181. The composition of claim 77, wherein the miRlOa is encapsulated in an extracellular vesicle (EV).

82. The composition of claim 81 wherein the EV is produced by a method comprising:(a) culturing a cell in a culture medium comprising one or more growth factors: and (b) isolating the EV from a supernatant of the culture medium from step a).

83. The composition of claim 81 or 82, wherein the EV is derived from an endothelial cell (eEV).

84. The composition of claim 83, wherein the endothelial cell is a human umbilical vein endothelial cell (HUVEC).

85. The composition of any one of claims 77-84, wherein the method comprises:(i) reducing or preventing macrophage infiltration into a joint of the subject; (ii) reducing or preventing neovascularization in a joint of the subject; (iii) reducing or preventing synovial hyperplasia in a joint of the subject; (iv) reducing or preventing cartilage degeneration in a joint of the subject;(v) reducing or preventing chondrocyte apoptosis in the subject;(vi) reducing expression of IL-6 in the subject’s synovial fluid(vii) administering a clotting factor; or(viii) a combination of i) - vii).

86. The composition of any one of claims 77-85, wherein the subject has hemophilia.

87. The composition of any one of claims 77-86, wherein the subject has hemophilic arthropathy.

88. The composition of any one of claims 77-87, wherein the subject has hemarthrosisAtty. Dkt. No. 4842-130WO189. A composition comprising a microRNA 10a (miRlOa) in combination with or encapsulated in an extracellular vesicle (EV).

90. The composition of claim 89, wherein the EV is an endothelial cell extracellular vesicle (eEV).

91. The composition of claim 90, wherein the endothelial cell is a human umbilical vein endothelial cell (HUVEC).

92. The composition of claim 90 or 91, wherein the endothelial cell is a FVIIa-released eEV.

93. A composition comprising a microRNA 10a (miRlOa) encapsulated in a nanoparticle.

94. The composition of claim 93, wherein the nanoparticle is a LNP.

95. A composition comprising a microRNA 10a (miRlOa) encapsulated in a liposome.

96. A liquid composition comprising a microRNA 10a (miRlOa) and one or more of a buffer, a salt, a preservative, a stabilizer, a pH adjusting agent, and a solubilizing agent.

97. A pharmaceutical composition comprising a microRNA 10a (miRlOa) and a pharmaceutically acceptable excipient.

98. A method of treating a subject having an inflammatory disorder, comprising administering the composition of any one of claims 77-97 to the subject.

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