Methods for treating muscle wasting diseases using MBV

Abstract

A method for treating muscle-wasting diseases, such as spinal muscular atrophy or muscular dystrophy, using matrix-bound vesicles (MBVs) is disclosed. A composition for use in treating muscle-wasting diseases is also disclosed. This composition is a) exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or CD63 lo CD81 lo The MBV comprises: a) exogenous MBV derived from the extracellular matrix that does not contain alkaline phosphatase; b) bone marrow progenitor cells or bone marrow-derived cells treated with the exogenous MBV; and / or c) an effective amount of conditioned medium or a fraction thereof obtained from macrophages cultured in the presence of the exogenous MBV.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 502,751, filed May 17, 2023, which is incorporated herein by reference in its entirety.

[0002] Areas of disclosure This disclosure relates to the field of treatment of muscle wasting conditions, specifically the use of extracellular matrix (ECM)-bound nanovesicles (MBVs) for treating muscle wasting conditions, such as muscular dystrophy or spinal muscular atrophy.

[0003] Sequence List The contents of the sequence listing (sequence listing.xml, size: 3,760 bytes, created: May 1, 2024) are thus incorporated herein by reference in their entirety. [Background technology]

[0004] background Muscle weakness and tissue decay affect a large number of individuals. In particular, atrophy involves the weakening or decay of organs, tissues, or body parts, typically caused by disease, injury, or lack of use. Many different types of atrophy are known. Primary diseases include muscular atrophy, which is the wasting or loss of muscle tissue; multiple system atrophy, which is a degenerative neurological disorder associated with the degeneration of nerve cells in the brain; and spinal muscular atrophy, which affects the spinal cord and nerves. Typically, diseases that cause or result in atrophy affect one or more muscles in the human body. Similarly, dystrophic disorders, also typically caused by disease, have substantially the same effect on muscles as atrophy.

[0005] Muscular dystrophy is a diverse group of hereditary neuromuscular disorders exhibiting a destructive set of neuromuscular diseases characterized by primary or secondary skeletal muscle involvement. Duchenne muscular dystrophy (DMD) is an X-linked disorder and the most common form of muscular dystrophy. DMD affects approximately 1 in 3,500 male births, with patients suffering from chronic muscle degeneration and weakness. Clinical symptoms are first detected between the ages of 2 and 5, and by the time the patient reaches adolescence, the ability to walk independently is lost. Death typically occurs in patients before the age of 30, due to cardiopulmonary failure. There is still a need for methods to treat muscular dystrophy in the population.

[0006] Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary muscular disorder that primarily causes progressive degeneration of the muscles of the face, scapula, and upper arm, but also results in weakness in other muscles. FSHD is the third most common type of muscular dystrophy, with an estimated prevalence of approximately 3 cases per 100,000 individuals. In most individuals diagnosed with FSHD, symptoms begin before the age of 20. FSHD is caused by mutations that result in inappropriate expression of the double homeobox protein 4 gene (DUX4). Currently, there are no pharmaceutical treatments for FSHD; therefore, patients must cope through surgical and mechanical aids, such as surgical procedures to stabilize the scapula, through the use of orthopedic devices, such as back supports, girdles, and braces, and through low-intensity exercise. Thus, novel therapies targeting muscle degeneration are needed to improve the quality of life for these individuals.

[0007] Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by genetic mutations in the survival motor neuron 1 (SMN1) gene. In SMA patients, affected motor neurons have a reduced ability to produce sustained firing, which worsens over time and can lead to motor neuron (MN) death. Severity of SMA ranges from respiratory failure in the neonatal period (types 1-2) to mild muscle weakness observed in adulthood (type 4). Novel gene therapies have successfully prevented the need for permanent respiratory support and death in neonatal patients (types 1-2). However, these gene therapies have not been able to help type 3-4 patients with severe locomotion deficits in adulthood. Furthermore, type 1-2 patients treated with gene therapy may develop severe locomotion deficits. Therefore, novel therapies that target motor impairments in SMA patients and improve the effectiveness of current treatments are needed to improve the quality of life for these individuals. [Overview of the Initiative] [Means for solving the problem]

[0008] Summary of Disclosure Methods for treating subjects with muscle wasting are disclosed. These methods include a) exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or CD63 lo CD81 lo The present invention comprises the step of administering to a subject a composition comprising an effective amount of: a) exogenous MBV derived from the extracellular matrix that does not contain alkaline phosphatase; b) myeloid progenitor cells or myeloid-derived cells treated with exogenous MBV; and / or c) a conditioned medium or fraction thereof obtained from a culture of macrophages cultured in the presence of exogenous MBV.

[0009] Methods for promoting muscle regeneration or repair in subjects requiring it are also disclosed. These methods include the step of administering to a subject a composition comprising an effective amount of exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or CD63 lo CD81 lo Therefore, MBV does not contain alkaline phosphatase, and thereby promotes muscle regeneration in the subject.

[0010] The above-mentioned and other features and advantages of the present invention will become more apparent from the following detailed description of several embodiments, which proceed with reference to the accompanying drawings. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a schematic diagram showing the procedure used to determine the direct (left) and indirect (right) effects of MBV administration on the transcriptome of FSHD myoblasts described in Examples 1-4. [Figure 2] Metabolic activity (%) of healthy and FSHD2 myoblasts from patients treated with different doses of MBV over 24 hours. [Figure 3] Effects of macrophage-derived secretomes (MBV, LPS(M1), and IL4(M2)) on the metabolic activity (%) of HMEC-1 cells after 24 hours (left) and 72 hours (right). [Figure 4] Results of a scratch assay showing the percentage migration area of ​​HMEC-1 cells after treatment with secretomes derived from M1, M2, and M-MBV macrophages. [Figure 5] Effects of macrophage-derived secretomes (MBV, LPS(M1), and IL4(M2)) on the metabolic activity (%) of N1E-155 cells after 24 hours (left) and 72 hours (right). [Figure 6A]The effects of macrophage-derived secretomes (MBV, LPS(M1), and IL4(M2)) on N1E-155 cell differentiation after 24 and 72 hours, as measured by morphological features such as roundness (Figure 5A upper panel), eccentricity (Figure 5A lower panel), and ferret diameter (Figure 5B). [Figure 6B] The effects of macrophage-derived secretomes (MBV, LPS(M1), and IL4(M2)) on N1E-155 cell differentiation after 24 and 72 hours, as measured by morphological features such as roundness (Figure 5A upper panel), eccentricity (Figure 5A lower panel), and ferret diameter (Figure 5B). [Figure 7] Classification of bone marrow cells that take up MBV after 3 and 24 hours. [Figure 8] Figure 8 shows the distribution of accessible sequences under different in vitro and in vivo treatments from ATAC sequence analysis. [Figure 9] Figure 9 shows the distribution of statistically significant regulated genes at the epigenetic level in different cells treated in vitro. The dots (corresponding to the larger circles in the Venn diagram below the figure) are shown on the right side of each graph, and the dots (corresponding to the smaller circles in the Venn diagram) are shown on the left side of each graph. The dot in the center of each graph corresponds to a gene in the overlapping region of the Venn diagram; the central section indicates the overlapping portion of the Venn diagram. Similarly, in the Venn diagram, the larger circles are the dots on the right, and the smaller circles are the dots on the left. [Figure 10A] Schematic experimental design for MBV accumulation in bone marrow cells (Figure 10A). Bone marrow and blood fluorescence imaging 24 hours after intraperitoneal injection of different MBV-tagged concentrates (Figure 10B) and their corresponding readings (Figure 10A). This accumulation was associated with changes in response to the M1 challenge (Figure 10B). [Figure 10B] Schematic experimental design for MBV accumulation in bone marrow cells (Figure 10A). Bone marrow and blood fluorescence imaging 24 hours after intraperitoneal injection of different MBV-tagged concentrates (Figure 10B) and their corresponding readings (Figure 10A). This accumulation was associated with changes in response to the M1 challenge (Figure 10B). [Figure 11]Schematic diagram of the study in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). Top: DUX4 expression was induced in 4-week-old female iDUX4pA-HSA mice by feeding them dox diet on day 0 and maintained throughout the duration of the study to simulate a "moderate" Dux4 environment (iDUX4pA-HSA mouse model). Bottom: DUX4 expression was induced in 14-week-old male mice by TMX injection on day 0 to simulate the FSHD phenotype in adults. The second arm of the study evaluated the effect of MBV treatment on the cumulative phenotype caused by low chronic DUX4 expression in aging FSHD mouse models (no TMX induction, 7-month-old mice; FLExDUX4(+TMX) mouse model). [Figure 12] Figure 12 is a bar graph showing the FSHD severity scores in normal (control) mice, MBV-treated FSHD mice, and saline-treated FSHD mice, as described in Example 8. A score of zero (0) was given to tissue sections indicating no injury (i.e., normal skeletal muscle tissue). A score of 1 indicated mild injury. A score of 2 indicated moderate injury with the presence of increased inflammatory cells around dead or dying muscle fibers. A score of 3 indicated severe diffuse injury with the presence of widespread inflammatory cells, activated satellite cells with scattered myoblasts, and multifocal tissue necrosis. [Figure 13] Figure 13 shows photographs of exemplary muscle tissue samples from control (PBS-treated; left) and MBV-treated (right) FSHD mice in the FLExDUX4(+TMX) model. Hematoxylin and eosin staining at approximately 400× magnification. [Figure 14] Figure 14 shows photographs of exemplary muscle tissue samples from control (PBS-treated; left) and MBV-treated (right) SMA mice. Hematoxylin and eosin staining at approximately 400× magnification. These images show longitudinal sections of skeletal muscle. Control-treated animals show amyotrophy gravis, separation of muscle fibers, the presence of increased inflammatory cells between fibers, and areas of tissue necrosis. MBV-treated animals also show amyotrophy, but to a lesser degree, with less inflammation and less severe muscle injury than PBS-treated controls. [Figure 15]Figure 15 is a schematic diagram of a study in a mouse model of spinal muscular atrophy (SMA). Mice received intramuscular injections of MBV on day 3 and were sacrificed on day 7. [Figure 16] Figures 16A–16B provide a comparison of surface markers for exosomes, bone microvesicles (MVs), and MBVs. The figures show the results of the EXO-CHECK® exosome antibody array (System Biosciences) comparing the levels of various markers of interest in mouse exosomes, mouse bone matrix vesicles (bone MVs), and mouse matrix-bound nanovesicles (MBVs). Figure 16A provides a digital image of the array, and Figure 16B is a graph showing the relative expression of each of the markers of interest in exosomes vs. bone MVs vs. MBVs. This data shows that MBVs differ from exosomes and bone microvesicles (MVs) based on the surface marker profiles. MBVs do not express or have low expression of CD63, EpCAM, ANXA5, TSG101, GM130, FLOT1, ICAM1, ALIX, and CD81 compared to bone MV or exosome levels of these markers, as shown in the bar graph in the lower panel. [Figure 17] Figure 17 is a Western blot showing that bone MV markers annexin V and tissue-nonspecific alkaline phosphatase (TNAP) are expressed by bone MVs. Lysates prepared from 1711A cells were used as a positive control. The results of this experiment show that matrix-bound nanovesicles (MBVs) lack expression of both bone microvesicle markers: TNAP and annexin V. Plasma exosomes express annexin V but not TNAP. These results clearly distinguish MBVs from both exosomes and bone microvesicles. In particular, the MBVs used were isolated from muscle tissue. [Figure 18]Figure 18 is a bar graph showing the different effects of activated gene expression in macrophages on exosomes, MVs, and MBVs. MBVs exhibit differential immunomodulatory effects; that is, they increase M2 macrophages compared to exosomes or bone MVs, which do not exhibit this effect. Bone marrow-derived macrophages (BMDMs) recovered from mice were either left untreated (M0) or treated for 24 hours with the following test materials: IFNγ+LPS (M1) to induce the M1 phenotype, IL-4 (M2) to induce the M2-like phenotype, plasma-derived exosomes, bone MVs derived from 17A cells, or MBVs isolated from muscle. After treatment, the change multipliers in the expression of the indicated genes (Arg, CD206, Fixx, IL-6, INOS, and TNF) were evaluated by qPCR. The pro-inflammatory markers IL-6 and TNF-α were downregulated by MBVs, which is clearly distinct from the downregulation of the same two inflammatory mediators by exosomes and bone MVs. MBV had a strong anti-inflammatory effect; on the other hand, exosomes and bone MV did not have this effect. [Figure 19] Figure 19 is a schematic diagram of the D2.mdx mouse study. D2.mdx mice (6-8 weeks old) were randomly assigned to the following groups: 1) saline treatment, n=6, and 2) MBV treatment, n=6. 100 μl containing 4.2 × 10^9 MBV cells was administered intraperitoneally on days 1, 3, and 5, and then weekly thereafter. Body weight was recorded weekly. Animals were sacrificed at 8 weeks, tissues were explanted, weighed, and processed for histological examination. [Figure 20] Figure 20 provides graphs showing body weight and organ weight from the D2.mdx mouse study. Animals (n=6 per group) were weighed at weeks 1, 3, 5, 7, and 8. The data show a statistically significant difference in body weight between MBV-treated animals and control animals. At week 8, the animals were sacrificed, and the gastrocnemius (GC), tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOLL), liver, and spleen were explanted and weighed. The results showed a significant difference in gastrocnemius (GC) between groups. Other muscles tested demonstrated a tendency toward higher weights in the MBV-treated group compared to the control group. [Figure 21] Figure 21 shows the results from muscle function tests. Functional analysis was performed by measuring isometric torque generation of the gastrocnemius muscle at weeks 1 and 7. The results showed a statistically significant increase in torque generation in animals treated with MBV compared to saline controls. [Figure 22] Figure 22 shows representative digital images of muscle structure (assessed by H&E) and fibrosis (assessed by Masson's trichrome). The saline-treated group showed widespread muscle degeneration, inflammation, thick bands of dense fibrous tissue replacing lost muscle tissue, and a moderate attempt at muscle cell regeneration. In contrast, the MBV-treated group showed a much less severe muscle degeneration, widely scattered areas of sparse muscle degeneration, fewer scattered mononuclear inflammatory cells, and a clearly robust muscle regeneration response indicated by satellite cell activation and the presence of myoblasts. [Modes for carrying out the invention]

[0012] Detailed description of several aspects Methods for treating subjects in a state of muscle wasting are disclosed herein. Various states of muscle wasting suitable for treatment by these methods are disclosed herein. These methods include the use of MBV, which may be prepared from an extracellular matrix source as disclosed herein. The method may include the steps of selecting a subject in a state of muscle wasting, and then treating the subject.

[0013] In some embodiments, a method for treating a subject having a state of muscle wasting, comprising: (1) exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or CD63 lo CD81 loA method is disclosed for treating muscle wasting in a subject, comprising the step of administering to a subject a composition having an effective amount of: (2) exogenous MBV derived from the extracellular matrix that does not contain alkaline phosphatase; (2) bone marrow progenitor cells or bone marrow-derived cells treated with exogenous MBV; and / or (3) a conditioned medium or fraction thereof obtained from a culture of macrophages cultured in the presence of exogenous MBV. The subject may be a human subject. The method may comprise the steps of selecting a subject having muscle wasting, and then treating the subject.

[0014] In some embodiments, methods are disclosed for promoting muscle regeneration or repair in subjects requiring it. These methods include the step of administering to a subject a composition comprising an effective amount of exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or express CD63 lo CD81 lo MBV does not contain alkaline phosphatase, thereby promoting muscle regeneration in the subject.

[0015] MBV can originate from the extracellular matrix of the bladder, small intestine, heart, dermis, liver, kidney, uterus, brain, blood vessels, lungs, bone, muscle, pancreas, placenta, stomach, spleen, colon, adipose tissue, or esophagus. In some cases, MBV does not originate from the bone ECM or cardiac ECM. For example, MBV originates from the extracellular matrix of the bladder, small intestine, dermis, liver, kidney, uterus, brain, blood vessels, lungs, muscle, pancreas, placenta, stomach, spleen, colon, adipose tissue, or esophagus. For example, MBV can originate from the urinary bladder matrix (UBM), small intestinal submucosa (SIS), or bladder submucosa (UBS). For example, MBV can originate from the dermis. For example, MBV originates from the extracellular matrix of a mammalian vertebrate selected from humans, monkeys, pigs, cattle, or sheep.

[0016] For example, in one aspect, a subject having a muscle wasting disease is administered an effective amount of a composition comprising exogenous matrix-binding nanovesicles (MBVs) derived from the extracellular matrix, and the MBVs do not express CD63 and CD81, or CD63 lo CD81 lo and the MBVs do not contain alkaline phosphatase.

[0017] In another aspect, a subject requiring muscle regeneration or repair is administered an effective amount of a composition comprising exogenous matrix-binding nanovesicles (MBVs) derived from the extracellular matrix, and the MBVs do not express CD63 and CD81, or CD63 lo CD81 lo and the MBVs do not contain alkaline phosphatase.

[0018] The MBVs can be administered to the subject by systemic administration, such as intravenous administration.

[0019] The exogenous MBVs can be administered in a physiologically acceptable solution, such as saline at physiological pH. The exogenous MBVs can be contained within an extracellular matrix (ECM) hydrogel or pregel prepared from the extracellular matrix (ECM), and the hydrogel or pregel is administered to the subject. The ECM hydrogel or pregel contains solubilized ECM at a concentration of 1 mg / mL to 500 mg / mL, such as 1 mg / mL to 50 mg / mL. For example, the extracellular matrix hydrogel or pregel can be an enzymatic ECM hydrogel or pregel and contains inactivated proteases, such as trypsin and / or pepsin. For example, the enzymatic hydrogel or pregel has a pH of about 7.0 to about 7.8. For example, the enzymatic pregel forms a gel at a temperature higher than about 25°C. In some examples, the ECM hydrogel has a storage modulus (G’) of about 50 Pa to about 200 Pa, a loss modulus (G’’) of about 5 Pa to about 20 Pa, and a ratio of G’ to G’’ of about 4:1 to about 15:1 at 37°C and is an acoustically treated hydrogel. In some examples, the ECM in the hydrogel or pregel is not dialyzed.

[0020] ECM hydrogels can be prepared from the extracellular matrix of the bladder, small intestine, heart, dermis, liver, kidney, uterus, brain, blood vessels, lungs, bone, muscle, pancreas, placenta, stomach, spleen, colon, adipose tissue, or esophagus. In some cases, ECM hydrogels can be prepared from bladder matrix (UBM), small intestinal submucosa (SIS), or bladder submucosa (UBS). ECM hydrogels can be prepared from the dermis. ECM hydrogels can be prepared from the extracellular matrix of mammalian vertebrates selected from humans, monkeys, pigs, cattle, or sheep.

[0021] For example, in another embodiment, a subject with muscle wasting disease is administered an effective amount of a composition containing myeloid progenitor cells or myeloid-derived cells treated with exogenous MBV. The myeloid progenitor cells or myeloid-derived cells may be autologous to the subject. The myeloid progenitor cells or myeloid-derived cells may be treated in vitro with exogenous MBV, for example, in a cell culture. The myeloid progenitor cells or myeloid-derived cells may then be isolated from the cell culture for administration to the subject. The myeloid-derived cells may be macrophages, monocytes, granule cells, or myeloid progenitor cells. The myeloid progenitor cells or myeloid-derived cells may be administered to the subject by systemic administration. For example, the myeloid progenitor cells or myeloid-derived cells may be administered by intravenous administration. The myeloid progenitor cells or myeloid-derived cells may be administered directly into the bone marrow cavity. The subject may be a human subject.

[0022] For example, in another embodiment, subjects with muscle wasting disease are administered an effective amount of a composition containing a conditioned medium or fraction thereof obtained from a culture of macrophages derived from the subject, cultured in the presence of exogenous MBV. The macrophages may be autologous to the subject. The conditioned medium may be purified by dialysis, size fractionation, and / or centrifugation before being administered to the subject. The subject may be a human subject.

[0023] In some manifestations, muscle wasting is a muscular dystrophy. For example, muscular dystrophy includes Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy (DMD), distal muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), limb-girdle muscular dystrophy, myotonic muscular dystrophy, oculopharyngeal muscular dystrophy, Bethlem myopathy, or Ulrich congenital muscular dystrophy. For example, muscle wasting is FSHD.

[0024] In some manifestations, muscle wasting is a condition of spinal muscular atrophy (SMA). For example, SMA may be infantile progressive spinal muscular atrophy (Type I SMA), intermediate spinal muscular atrophy (Type II SMA), juvenile spinal muscular atrophy (Type III SMA), or adult-onset spinal muscular atrophy (Type IV SMA).

[0025] In other embodiments, the state of muscle wasting is sarcopenia or cachexia.

[0026] In some embodiments, the methods disclosed herein for treating subjects having a state of muscle wasting increase myotubation in the subject, maintain neuronal progenitor cells in an undifferentiated state in the subject, and / or increase the migratory ability of endothelial cells in the subject. The methods disclosed herein may also increase muscle tissue growth in the subject.

[0027] In other embodiments, compositions for use in methods of treatment disclosed herein, (a) exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or CD63 lo CD81 loA composition is provided that provides an effective amount of MBV, which is (b) exogenous MBV derived from the extracellular matrix that does not contain alkaline phosphatase; (c) bone marrow progenitor cells or bone marrow-derived cells treated with exogenous MBV; and / or (d) a conditioned medium or fraction thereof obtained from macrophages cultured in the presence of exogenous MBV.

[0028] In some embodiments, the methods disclosed herein for promoting muscle regeneration or repair increase the number of satellite cells in the target muscle that are in need of regeneration or repair compared to the number before MBV administration. In some embodiments, the methods disclosed herein for promoting muscle regeneration or repair increase the number of myoblasts in the target muscle that are in need of regeneration or repair compared to the number before MBV administration.

[0029] term Unless otherwise specified, technical terms are used in accordance with their conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's genes XII, Jones & Bartlett Learning, 2017. The following explanations of terms and methods are provided to better describe this disclosure and to guide those skilled in the art in practicing this disclosure. The singular forms “a,” “an,” and “the” refer to one or more unless the context explicitly indicates otherwise. For example, the term “containing a (a) MBV” refers to one or more MBVs and is considered equivalent to the phrase “containing at least one MBV.” The term “or” refers to a single element or a combination of two or more elements from the alternative elements presented, unless the context explicitly indicates otherwise. As used herein, “comprises” means “includes.” Therefore, “comprising A or B” means “including A, B, or A and B” without excluding further elements. It is further understood that any and all molecular weight or molecular mass values ​​or percentages given for a composition are approximations and, unless otherwise indicated, are provided for descriptive purposes only. The dates of the GENBANK® accession numbers referred to herein are in sequence that were available as early as April 5, 2021. All references, patent applications and publications, and GENBANK® accession numbers cited herein are incorporated by reference. Unless otherwise indicated, “about” indicates within 5 percent. Where the term “about” is used before a quantitative value, this disclosure also includes that specific quantitative value itself. In case of any conflict, this specification, including the definitions of terms, shall prevail. To facilitate a review of the various aspects of this disclosure, the following definitions of specific terms are provided.

[0030] Acid proteases are enzymes that cleave peptide bonds, and these enzymes have increased activity in cleaving peptide bonds at acidic pH. For example, without limitation, acid proteases may include pepsin and trypsin.

[0031] Administration: Introduction of the composition (e.g., MBV, or a pharmaceutical preparation containing MBV) into the subject via the selected route. The route may be local or systemic. For example, if the selected route is intravenous, the composition is administered by introducing the composition into the vein of the subject. If the selected route is local, the composition may be administered by directly introducing the composition into the tissue of the subject.

[0032] Animals: A category of living, multicellular vertebrate organisms, including, for example, mammals and birds. The term "mammal" includes both human and non-human mammals. Similarly, the term "subject" includes both human subjects and veterinary subjects.

[0033] Biocompatibility: Any material that, when implanted in a mammalian subject, does not induce an adverse response in the subject. Biocompatible materials, once introduced into an organism, can perform their intended function, are neither toxic nor harmful to the organism, and do not induce immunological rejection of the material in the subject.

[0034] Cachexia: A disease-related muscle wasting syndrome that causes progressive muscle loss, often accompanied by loss of body fat, and which cannot be fully reversed by nutritional supplementation. A range of diseases, most commonly cancer, congestive heart failure, chronic obstructive pulmonary disease, chronic kidney disease, and AIDS, can cause cachexia. Key features of cachexia include progressive depletion of muscle and body fat mass, reduced food intake, abnormal metabolism of carbohydrates, proteins, and fats, reduced quality of life, and increased physical disability. Cachexia is distinct from weight loss due to malnutrition resulting from malabsorption, anorexia nervosa, or major depressive disorder. Weight loss from insufficient calorie intake generally causes fat loss before muscle loss, whereas cachexia predominantly causes muscle wasting. Cachexia is also distinct from sarcopenia.

[0035] Centrifugation: A process in which centrifugal force is applied to a mixture, causing more concentrated components of the mixture to move away from the axis of the centrifuge compared to other less concentrated components in the mixture. The force applied to the mixture is a function of the velocity of the centrifuge rotor and the radius of spin. In most applications, the force of spin causes the precipitate (pellet) to be pushed to the bottom of the centrifuge tube, and the remaining solution is appropriately called the “supernatant” or “clearance.” In other similar applications, density-based separation or “gradient centrifugation” techniques are used to isolate specific species from mixtures that contain both more concentrated and less concentrated components than the desired component.

[0036] During the circular motion of a centrifugal rotor, the force applied is the product of the spin radius and angular velocity, and this force is traditionally expressed as acceleration compared to "g," the standard acceleration due to gravity at the Earth's surface. The applied centrifugal force is called the "relative centrifugal force" (RCF) and is expressed as a multiple of "g."

[0037] Grinding (grinding and pulverizing): The process of breaking down larger particles into smaller particles, without limitation, including crushing, blending, tearing, slicing, milling, or cutting. ECM can be ground, however, in any form including but not limited to hydrated, frozen, air-dried, freeze-dried, powdered, or sheet form. "Ground ECM" contains intact collagen. In some cases, ground ECM has not been subjected to ultrasound or enzymatic digestion, for example, by proteases, such as acidic proteases.

[0038] Conditioning medium(single or multiple): A medium containing a basic medium and a cell secretome. The basic medium is suitable for culturing cells. Conditioning medium is generally produced by culturing cells of interest (e.g., macrophages) in a basic tissue culture medium so that the cells secrete active components (e.g., proteins and other factors) into the basic medium, thereby producing the conditioning medium. Cells are typically removed from the conditioning medium before use, for example, by centrifugation and / or other appropriate methods. The conditioning medium may be substantially cell-free (e.g., less than 5% of the volume of the conditioning medium) or completely cell-free, so as not to contain any viable cells.

[0039] Contact: To place in a state of direct physical association, which may be in solid or liquid form.

[0040] Cytokines: The term "cytokines" is used as a general term for a diverse group of soluble proteins and peptides that act as humoral regulators at nanomolar to picomolar concentrations, modulating the functional activity of individual cells and tissues under either normal or pathological conditions. These proteins also directly mediate intercellular interactions and regulate processes occurring in the extracellular environment. Examples of cytokines include, but are not limited to, tumor necrosis factor-α, interleukin (IL)-6, IL-10, IL-12, transforming growth factors, and interferon-γ.

[0041] Diagnosis: The process of identifying a disease based on its signs, symptoms, and the results of various tests. The conclusion reached through this process is also called a "diagnosis." Common forms of diagnostic tests include, without limitation, blood tests, medical imaging, and biopsies.

[0042] Enrichment: A process in which a desired component present in a mixture, such as nanovesicles, has an increased ratio of the amount of that component to the amount of other components in the mixture after the enrichment process, compared to before the enrichment process.

[0043] Extracellular matrix (ECM): A complex mixture of structural and functional biomolecules and / or biomacromolecules, including but not limited to structural proteins, specialized proteins, proteoglycans, glycosaminoglycans, and growth factors, that surround, support, and, unless otherwise indicated, are cell-free within a tissue. ECM preparations may be considered “decellularized” or “cell-free,” meaning that cells have been removed from the source tissue via processes described herein and known in the art. “ECM-derived material,” e.g., “ECM-derived nanovesicles,” “matrix-bound nanovesicles,” “MBV,” or “ECM-derived nanovesicles” means nanovesicles prepared from natural ECM or from an in vitro source in which ECM is produced by cultured cells. "Intact extracellular matrix" and "intact ECM" refer to an extracellular matrix that retains the activity of its structural and non-structural biomolecules, including but not limited to collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobial agents, chemoattractants, cytokines, and growth factors, e.g., pulverized ECM as described herein without limitation. The activity of biomolecules within the ECM can be removed chemically or mechanically, for example, by crosslinking and / or dialysis of the ECM. Intact ECM is essentially not crosslinked and / or dialysis, meaning that the ECM has not been subjected to dialysis and / or crosslinking processes or conditions other than those that occur naturally during storage and handling of the ECM prior to solubilization when preparing an enzymatic ECM hydrogel. Therefore, substantially crosslinked and / or dialysis-enhanced ECM (to a degree far removed from trivial forms that substantially do not affect the gelation and functional characteristics of the ECM in its use as described herein) is not considered "intact".

[0044] Exogenous: Originating from a different source. Exogenous MBV is produced separately, for example, extracted from an ECM source and added to an ECM hydrogel which may or may not have endogenous MBV present in the ECM hydrogel. Exogenous MBV may originate from the same or different tissue as the ECM used to prepare the ECM hydrogel. Exogenous MBV may originate from the same or different species as the ECM used to prepare the ECM hydrogel.

[0045] A gel is a state of matter between liquid and solid, generally defined as a cross-linked polymer network that swells in a liquid medium. Typically, a gel is a two-phase colloidal dispersion containing both solid and liquid, with the amount of solid being greater than the amount in the two-phase colloidal dispersion called a "sol." Thus, a "gel" possesses some of the properties of a liquid (i.e., its shape is elastic and deformable) and some of the properties of a solid (e.g., its shape is sufficiently discrete to maintain three dimensions on a two-dimensional surface). "Gelization time," also called "gel time," refers to the time it takes for a composition to become non-flowable under moderate stress.

[0046] Gelation: Formation of a gel from a sol.

[0047] Hydrogels: Networks of polymer chains that are hydrophilic and sometimes found as colloidal gels with water as the dispersion medium. Hydrogels are naturally occurring or synthetic polymeric networks that are highly absorbent. Hydrogels also possess a degree of flexibility similar to that of natural tissues. "Acoustically treated" hydrogels, e.g., acoustically treated ECM hydrogels, are produced using ultrasonic energy to solubilize ECM, for example. The characteristics of these hydrogels are disclosed herein. For hydrogels, G' (storage modulus) is typically about an order of magnitude greater than G'' (loss modulus). "Enzymatic" ECM hydrogels are produced by enzymatically digested ECM. The viscosity of enzymatic hydrogels increases when heated to a physiological temperature close to about 37°C. For example, enzymatic hydrogels are formed from injectable solutions that form a gel at a physiological temperature of 37°C, but at temperatures below 37°C. "Pre-gel" refers to an enzymatic hydrogel that is in a sol state due to not yet reaching the appropriate temperature for gelation, for example.

[0048] Improving muscle health: Improvement in muscle health compared to the existing condition or to a condition that would occur in the absence of treatment. For example, improving muscle health may include enhancing muscle regeneration, maintenance, or repair, or reducing or reversing muscle tissue degeneration. Improving muscle health may also include proactively treating the subject to prevent or reduce muscle damage, degeneration, or injury.

[0049] Isolated: “Isolated” biological components (e.g., nucleic acids, proteins, cells, or nanovesicles) are substantially separated or purified from cells of organisms in which they naturally occur or from other biological components in the ECM. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Isolated MBVs are extracted from fibrous material of the ECM. This term also includes nucleic acids and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0050] Isotonic buffer solution: A solution that is buffered to a pH between 7.0 and 7.8 and has an equilibrium concentration of salt to promote an isotonic environment.

[0051] Lysyl oxidase (Lox): A copper-dependent enzyme that catalyzes the formation of aldehydes from lysine residues in collagen and elastin precursors. These aldehydes are highly reactive and undergo spontaneous chemical reactions with other lysyl oxidase-derived aldehyde residues or with unmodified lysine residues. In vivo, this results in cross-linking of collagen and elastin, which plays a role in stabilizing collagen fibrils and for the integrity and elasticity of mature elastin. Complex cross-linking is formed in structurally different collagen (pyridinoline derived from three lysine residues) and elastin (desmosine derived from four lysine residues). Genes encoding the Lox enzyme have been cloned from various organisms (Hamalainen et al., Genomics 11:508, 1991; Trackman et al., Biochemistry 29:4863, 1990; thereby incorporated herein by reference). Residues 153–417 and 201–417 of the human lysyl oxidase sequence have been shown to be important for catalytic function. There are four Lox-like isoforms called LoxL1, LoxL2, LoxL3, and LoxL4.

[0052] Macrophages: A type of white blood cell that phagocytoses and breaks down cellular debris, foreign substances, microorganisms, and cancer cells. In addition to their role in phagocytosis, these cells play a crucial role in development, tissue maintenance and repair, as well as in both innate and adaptive immunity by mobilizing and influencing other cells, including immune cells such as lymphocytes. Macrophages can be present in many phenotypes, including those called M1 and M2. Macrophages that primarily perform pro-inflammatory functions are M1 macrophages (CD86) + / CD68 +) are called macrophages, but on the other hand, macrophages that reduce inflammation and promote and regulate tissue repair are called M2 macrophages (CD206) + / CD68 + ) is called. Markers for identifying macrophages of various phenotypes vary between species. It should be noted that macrophage phenotypes are indicated by a spectrum ranging between the M1 and M2 extremes. F4 / 80 (encoded by the adherent G protein-coupled receptor E1 (ADGRE1) gene) is a macrophage marker. See GENBANK® accession numbers NP_001243181.1, April 6, 2018 and NP_001965, March 5, 2018, both of which are incorporated herein by reference. While we do not wish to be bound by theory, MBV is thought to have the ability to modulate macrophage phenotypes, resulting in increases in M2-like, regulatory, or pro-remodeling macrophages. The effects of MBV on macrophages are further characterized in PCT publication number WO2017 / 151862A1, which is entirely incorporated herein by reference. In some embodiments, MBV may be used to induce the M2 phenotype in macrophages and inhibit M1 macrophages in a subject.

[0053] MicroRNAs are small, non-coding RNAs, typically 17 to 25 nucleotides long, that post-transcriptionally regulate gene expression, usually by repressing the translation of target mRNA. MicroRNAs ("miRNAs" or "miRs") can function as negative regulators, with larger amounts of specific miRNAs correlating to lower levels of target gene expression. There are three forms of miRNA: primary miRNA (pri-miRNA), immature miRNA (pre-miRNA), and mature miRNA. Primary miRNA (pri-miRNA) is expressed as stem-loop transcripts ranging from about several hundred nucleotides to over 1 kb. Pri-miRNA transcripts are cleaved in the nucleus by an RNase II endonuclease called Drosha, which cleaves both strands of the stem near the base of the stem-loop. Drosha cleaves the RNA double helix with alternating cuts, leaving a 5' phosphate and a 2-nucleotide overhang at the 3' end. The cleavage product, immature miRNA (pre-miRNA), is approximately 60–110 nucleotides long and has a hairpin structure formed in a folded manner. Pre-miRNA is transported from the nucleus to the cytoplasm by Ran-GTP and exportin-5. In the cytoplasm, pre-miRNA is further processed by another RNase II endonuclease called Dicer. Dicer recognizes the 5' phosphate and 3' overhang and cleaves the loop at the stem-loop branching point to form a miRNA double helix. The miRNA double helix binds to the RNA-induced silencing complex (RISC), where the antisense strand is preferentially degraded, and the sense strand mature miRNA directs the RISC to its target site. Mature miRNA is the biologically active form of miRNA and is approximately 17–25 nucleotides long.

[0054] Muscular dystrophy: A term used to refer to a group of genetic disorders that result in progressive muscle weakness. Muscular dystrophy can lead to skeletal muscle weakness and deficiencies in skeletal muscle proteins, resulting in a variety of impaired physiological functions. There is no satisfactory treatment for muscular dystrophy. Existing treatments typically focus on alleviating the effects of the disease and improving the patient's quality of life, for example, through physiotherapy, surgical intervention, or the provision of orthopedic devices.

[0055] Mutant genes associated with muscular dystrophy are responsible for encoding several proteins involved in the costamere protein network. These proteins include laminin-2, collagen, dystroglycan, integrin, caveolin-3, ankyrin, dystrophin, α-dystroblevin, vinculin, plectin, BPAG1b, muscle LIM protein, desmin, actinin-related LIM protein, α-actin, titin, telethonin, cypher, myotilin, and sarcoglycan / sarcospan complexes.

[0056] The most common form of muscular dystrophy is Duchenne muscular dystrophy (DMD), affecting 1 in 3,500 male live births. DMD is an X-linked recessive disorder characterized by a mutation in the gene encoding dystrophin. Dystrophin is a cytoskeletal protein approximately 430 kDa in size. This protein functions to connect the cytoskeleton and extracellular matrix of a cell. Loss of dystrophin in DMD patients results in loss of muscle fiber attachment to the extracellular matrix during contraction, which ultimately leads to progressive fiber damage, membrane leakage, and loss of muscle function. Most patients die before the age of 30, due to respiratory or heart failure.

[0057] Becker muscular dystrophy (also known as benign pseudohypertrophic muscular dystrophy) is related to Duchenne muscular dystrophy in that both arise from mutations in the dystrophin gene, but in Duchenne muscular dystrophy, functional dystrophin is not produced, which makes DMD far more severe than BMD. BMD is an X-linked recessive genetic disorder characterized by slowly progressing muscle weakness of the legs and pelvis. BMD is one type of dystrophin disorder that encompasses a spectrum of muscle diseases in which insufficient dystrophin is produced in muscle cells, leading to instability in the structure of the muscle cell membrane. This is caused by mutations in the dystrophin gene. The pattern of symptom onset in BMD is similar to that of DMD, but with later progression at a considerably slower rate.

[0058] Congenital muscular dystrophy is caused by gene mutations that affect the production of other costamere proteins. Merosin-deficient congenital muscular dystrophy (MDC1A) is a congenital muscular dystrophy caused by a genetic mutation in the LAMA2 gene that results in the absence or complete loss of the laminin-α2 protein. This loss of laminin-α2 leads to the absence of laminin-211 / 221. Laminin-211 / 221 are major components of the extracellular matrix and play a crucial role in muscle cell development. During muscle cell differentiation, laminin binds to α7β1 integrin. In the absence of laminin-α2, muscle fibers cannot adhere to the basement membrane, and myotubes undergo apoptosis. Muscle regeneration also fails, resulting in loss of muscle repair, as well as increased myofibrosis and inflammation. This chronic tissue injury is the main cause of morbidity and mortality in MDC1A.

[0059] Congenital muscular dystrophy (CMD) and limb-girdle muscular dystrophy (LGMD) are common forms of highly heterogeneous muscular dystrophy that can be distinguished by the age of onset. In CMD, the onset of symptoms is at birth or within the first six months of life; in LGMD, the onset of symptoms is in late childhood, adolescence, or even adulthood. Inheritance in LGMD can be autosomal dominant (type 1 LGMD) or autosomal recessive (type 2 LGMD), while CMD is recessive. CMD and LGMD can overlap both clinically and genetically.

[0060] MDC1A is a progressive muscle-wasting disease that causes children to be wheelchair-bound, require mechanical ventilation to breathe, and result in premature death. Symptoms are detected at birth by low muscle tone and "floppy" baby syndrome. DMD, BMD, and LGMD are progressive muscle-degenerative diseases that are usually diagnosed at age 3-5 when children exhibit developmental delays, including the ability to walk and climb stairs. The disease is progressive, and children are usually wheelchair-bound and require mechanical ventilation by their teens.

[0061] Facioscapulohumeral muscular dystrophy (FSHD) is a form of muscular dystrophy associated with progressive muscle weakness and loss of muscle tissue. Unlike DMD and BMD, which primarily affect the lower body, FSHD affects the upper body, mainly the muscles of the face, shoulders, and upper arms. However, it can also affect the muscles around the pelvis, buttocks, and lower legs. Symptoms of FSHD often do not appear until between the ages of 10 and 26, but it is not uncommon for symptoms to appear much later. In some cases, symptoms never develop. Symptoms are usually mild and worsen very slowly. Facial muscle weakness is common and may include ptosis, inability to whistle, decreased facial expression, depressed or angry facial expressions, difficulty pronouncing words, shoulder muscle weakness (leading to deformities, e.g., prominent scapulae (scapular winging) and sloping shoulders), weakness in the lower extremities, hearing loss, and possible cardiac conditions. FSHD is caused by mutations that result in the abnormal expression of the double homeobox protein 4 gene (DUX4), which leads to the expression of proteins toxic to muscle cells.

[0062] Myeloid cells: "Myeloid cells" or "cells derived from bone marrow" refer to cells derived from myeloid progenitor cells, which include, for example, granule cells (e.g., basophils, neutrophils, eosinophils) and monocytes. Since monocytes can differentiate into macrophages or dendritic cells, macrophages and dendritic cells are also considered myeloid cells or cells derived from bone marrow.

[0063] Myoblasts: Proliferating satellite cells and their offspring are called myoblasts, also known as myogenic progenitor cells. Myoblasts can fuse together to create new muscle fibers, which can then fuse with existing muscle fibers in skeletal muscle tissue, resulting in skeletal muscle regeneration.

[0064] Muscle cells: Muscle cells are mature, contractile cells found in the muscles of animals. Three types of muscle exist: skeletal muscle, smooth muscle, and cardiac muscle. Skeletal muscle cells are long, thread-like structures with many nuclei, called muscle fibers, and they arise from myoblasts. Skeletal muscle cells are formed by the fusion of myoblasts in a process known as myogenesis, giving rise to multinucleated cells (synthia). Skeletal muscle cells contain myofibrils, which are formed from filaments and sarcomeres, and form striated muscle tissue. Myofilaments, which are filaments of myofibrils, consist of three types: thick, thin, and elastic filaments. Thin filaments are mainly composed of actin, a protein that spirals together with nebulin filaments. Thick filaments are mainly composed of myosin, a protein responsible for force generation. Elastic filaments are composed of a protein called titin, which holds the thick filaments in place. Actin and myosin filaments each have a specific, constant length on the order of a few micrometers, which is much shorter than the length of an elongated muscle cell (several millimeters in the case of human skeletal muscle cells). These filaments are organized into repetitive subunits along the length of the myofibrils. Muscle cells are almost entirely filled with myofibrils that run parallel to each other along the long axis of the cell.

[0065] Nanovesicles: Extracellular vesicles that are nanoparticles with a diameter of approximately 10 to 1,000 nm. Nanovesicles are lipid membrane-bound particles that carry biologically active signaling molecules (e.g., microRNAs, proteins) among other molecules. Generally, nanovesicles are confined by a lipid bilayer, and biological molecules can be encapsulated and / or embedded within the bilayer. Thus, nanovesicles contain a lumen surrounded by a plasma membrane. Different types of vesicles can be distinguished based on diameter, intracellular origin, density, shape, sedimentation rate, lipid composition, protein markers, nucleic acid content, and origin, e.g., whether they are extracellular matrix-derived or secreted. Nanovesicles can be identified by their origin, e.g., matrix-bound nanovesicles derived from the ECM (see above), protein content, and / or miR content.

[0066] Exosomes, or liquid-phase extracellular vesicles (EVs), are membranous vesicles secreted by cells, ranging in diameter from 10 to 150 nm. Generally, late endosomes or polyvesicles contain intraluminal vesicles formed by inward budding and cleavage of vesicles from the restricted endosomal membrane into these enclosed vesicles. These intraluminal vesicles are then released from the polyvesicle lumen into the extracellular environment, typically into body fluids such as blood, cerebrospinal fluid, or saliva, during exocytosis upon fusion with the plasma membrane. Exosomes are created within a cell when a segment of the membrane invaginates and is invaginated into the plasma membrane. The internalized segment, which is broken down into smaller vesicles and eventually expelled from the cell, contains proteins as well as RNA molecules, such as mRNA and miRNA. Plasma-derived exosomes are largely deficient in ribosomal RNA. Extracellular matrix-derived exosomes contain specific miRNAs and protein components and have been shown to be present in virtually all body fluids, such as blood, urine, saliva, semen, and cerebrospinal fluid. Exosomes can express CD11c, CD63, CD81, and / or CD9, and therefore CD11c + and / or CD63 + and / or C81 + and / or CD9 + This is possible. Exosomes do not have high levels of lysyl oxidase on their surface.

[0067] "ECM-derived nanovesicles," "matrix-bound nanovesicles," "MBVs," or "ECM-derived nanovesicles" all refer to the same membrane-bound particles that are in the size range of 10 nm to 1000 nm, reside in the extracellular matrix, and contain biologically active signaling molecules, such as proteins, lipids, nucleic acids, growth factors, and cytokines, that influence cellular behavior. These terms are interchangeable and refer to the same vesicles. These nanovesicles are embedded within the ECM, bound to the ECM, and are not simply bound to the surface or freely circulating in body fluids. These nanovesicles are resistant to harsh isolation conditions, such as freeze-thaw, as well as digestion by proteases, such as pepsin, elastase, hyaluronidase, proteinase K, and collagenase, and digestion by detergents. MBVs are distinct from other extracellular vesicles containing exosomes and have a phospholipid composition distinct from exosomes. MBVs do not express alkaline phosphatase, and therefore are distinct from bone matrix vesicles that express alkaline phosphatase. In certain circumstances, MBVs may also be distinguished from exosomes based on the absence of certain markers generally attributed to exosomes.

[0068] In some embodiments, MBVs are characterized by one or more of the following features of protein expression or lipid content: (i) MBV may not express one or more of CD63 and / or CD81 and / or CD9, or may have low or barely detectable levels of CD63 and / or CD81 and / or CD9 compared to other vesicles, e.g., exosomes (CD63 lo and / or CD81 lo and / or CD9 lo(See, for example, Example 1). Various methods, such as antibody-based methods, e.g., Western blotting or flow cytometry, may be used to distinguish between low expression, barely detectable expression, or absence of expression of CD63 and / or CD81 and / or CD9 in MBV (see, for example, Bashashati and Brinkman, Adv Bioinformatics, 2009: 584603). In some embodiments, MBV expression of CD63 and / or CD81 and / or CD9 is considered low or barely detectable compared to other vesicles, in which case the expression of CD63 and / or CD81 and / or CD9 in MBV is at least one standard deviation or at least two standard deviations lower than the mean expression of other vesicles, e.g., exosomes; (ii) MBV has a phospholipid content in which at least 55% of the total phospholipids constitute a combination of phosphatidylcholine (PC) and phosphatidylinositol (PI); (iii) MBV has a phospholipid content in which 10% or less of the total phospholipids constitute sphingomyelin (SM); (iv) MBV has a phospholipid content in which 20% or less of the total phospholipids constitute phosphatidylethanolamine (PE); (v) MBV has a phospholipid content in which phosphatidylinositol (PI) constitutes 15% or more of the total phospholipid content, and the percentage indicates the percentage of lipid concentration.

[0069] In some embodiments, MBV is characterized by all of the following features: (i) Not expressing one or more of CD63 and / or CD81 and / or CD9, or having low or barely detectable levels of CD63 and / or CD81 and / or CD9 (CD63 lo and / or CD81 lo and / or CD9 lo (As further described above); (ii) Phospholipid content in which at least 55% of the total phospholipids constitutes combined phosphatidylcholine (PC) and phosphatidylinositol (PI); (iii) Phospholipid content in which sphingomyelin (SM) constitutes 10% or less of the total phospholipids; (iv) Phospholipid content in which 20% or less of the total phospholipids constitute phosphatidylethanolamine (PE); and (v) Phospholipid content in which phosphatidylinositol (PI) accounts for 15% or more of the total phospholipid content.

[0070] In some embodiments, MBV is characterized by all of the following features: (i) Phospholipid content in which at least 55% of the total phospholipids constitutes combined phosphatidylcholine (PC) and phosphatidylinositol (PI); (ii) Phospholipid content in which sphingomyelin (SM) constitutes 10% or less of the total phospholipids; (iii) Phospholipid content in which 20% or less of the total phospholipids constitute phosphatidylethanolamine (PE); and (iv) Phospholipid content in which phosphatidylinositol (PI) accounts for 15% or more of the total phospholipid content.

[0071] In some embodiments, MBV is characterized by one or more of the following features: (i) Phospholipid content in which at least 55% of the total phospholipids constitutes combined phosphatidylcholine (PC) and phosphatidylinositol (PI); (ii) Phospholipid content in which sphingomyelin (SM) constitutes 10% or less of the total phospholipids; (iii) Phospholipid content in which 20% or less of the total phospholipids constitute phosphatidylethanolamine (PE); and (iv) Phospholipid content in which phosphatidylinositol (PI) accounts for 15% or more of the total phospholipid content.

[0072] In some embodiments, MBV is characterized by one or more of the following features: (i) Does not contain detectable levels of alkaline phosphatase; (ii) Does not contain detectable levels of osteopontin; (iii) Does not contain detectable levels of osteoprogeterin; (iv) Does not contain detectable levels of complement C5; and / or (v) Does not contain detectable levels of c-reactive protein.

[0073] In some embodiments, MBV contains IL33, and IL33 + That is the case.

[0074] The ECM from which MBV is isolated may be a tissue-derived ECM, may be produced from cells in a culture, or may be purchased from a commercial source.

[0075] Pharmacopoecitable carriers: Pharmacopoecitable carriers useful in the claimed pharmaceutical preparations are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975) describes suitable compositions and formulations for the delivery of the fusion proteins disclosed herein.

[0076] Generally, the properties of the carrier depend on the specific mode of administration used. For example, parenteral formulations typically contain an injectable fluid as a vehicle, such as a pharmaceutically and physiologically acceptable fluid, e.g., water, physiological saline, equilibrium salt solution, aqueous dextrose, glycerol, etc. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical preparation may contain trace amounts of non-toxic adjuncts, such as humectants or emulsifiers, preservatives, and pH buffers, e.g., sodium acetate or sorbitan monolaurate.

[0077] Pharmaceutical agent: A chemical compound or composition that, when administered appropriately to a target or cell, can induce a desired therapeutic or prophylactic effect.

[0078] Phospholipids are a class of lipids having a structure consisting of two hydrophobic fatty acid tails and a hydrophilic head consisting of a phosphate group. Major classes of phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), sphingomyelin (SM), cardiolipin (CL), phosphatidic acid (PA), and bis-monoacylglycerol (BMP). Phospholipids can be measured by various methods. For example, liquid chromatography-mass spectrometry (LC-MS) based global lipidomics and redox lipidomics may be used. In some embodiments, the specific phospholipid content is expressed as a percentage concentration of total phospholipids (e.g., total phospholipids in MBV), and the percentage concentration is in w / w (weight / weight) units.

[0079] Polynucleotides are nucleic acid sequences of any length (e.g., linear sequences). Therefore, polynucleotides include oligonucleotides, as well as gene sequences found in chromosomes. An "oligonucleotide" is a group of conjugated nucleotides joined by native phosphodiester bonds. Oligonucleotides are polynucleotides between 6 and 300 nucleotides in length. Oligonucleotide analogs refer to parts that function similarly to oligonucleotides but have parts that do not exist naturally. For example, oligonucleotide analogs, such as phosphorothioate oligodeoxynucleotides, may contain parts that do not exist naturally, such as modified sugar moieties or intersugar links. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and these include peptide nucleic acid (PNA) molecules.

[0080] Prophylactic: As used herein, refers to a drug therapy or treatment designed and used to prevent the occurrence of a disease or disorder. As used herein, the terms “prophylactic” and “prevention” are interchangeable.

[0081] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Therefore, for example, a purified nucleic acid molecular preparation is a preparation in which the nucleic acid referred to is purer than the nucleic acid present in its natural environment within the cell. For example, a nucleic acid preparation is purified so that the nucleic acid accounts for at least 50% of the total protein content of the preparation. Similarly, a purified MBV preparation is a preparation in which the exosomes are purer than those present in the environment containing the cell in which the microvesicles and exosomes reside. Purified populations of nucleic acids or MBVs are higher than approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure, or free from other nucleic acids or cellular components, respectively.

[0082] Preventing or treating a disease: “Preventing” a disease refers to, for example, inhibiting the onset of a disease in a person known to be predisposed to the disease. Examples of people with a known predisposition include those with a family history of the disease or those exposed to factors that make the subject susceptible to the condition. “Treatment” refers to therapeutic interventions that alleviate the signs or symptoms of a disease or pathological condition after it has begun to develop.

[0083] Regeneration: When referring to skeletal muscle, this refers to the formation of new skeletal muscle tissue. Regeneration is a natural process that occurs in response to injury or stress, or other events that cause the loss of existing skeletal muscle in an object. Skeletal muscle regeneration occurs through a process initiated by muscle stem cells (satellite cells) that results in the production of new muscle fibers and the repair of existing muscle fibers. The presence of satellite cells and myoblasts in muscle tissue indicates that the muscle tissue is in a state of regeneration.

[0084] Sarcopenia: Age-related progressive loss of muscle mass and strength. The primary symptom of this condition is muscle weakness. Sarcopenia affects both males and females equally; the incidence increases with age. In humans, sarcopenia generally affects individuals aged 60 and older. This disease affects both sexes equally. The condition is characterized by a degenerative loss of the quantity, quality, and strength of skeletal muscle. The rate of muscle loss depends on exercise level, comorbidities, nutrition, and other factors. Pathological changes in sarcopenia include a decrease in the quality of muscle tissue, reflected in the replacement of muscle fibers with fat; an increase in fibrosis; changes in muscle metabolism; oxidative stress; and degeneration of the neuromuscular junction. Sarcopenia is diagnosed when a patient has muscle mass at least two standard deviations below the patient mean and has a slow walking speed. Subjects with this condition have the presence of low muscle mass and either low muscle strength or low physical ability.

[0085] Satellite cells: Satellite cells are myogenic (muscle) stem cells that play a crucial role in the maintenance, remodeling, and repair of muscle fibers. These precursors to skeletal muscle cells are mononuclear and are found between the basement membrane and plasma membrane of muscle fibers. While normally quiescent in adult muscle, they can proliferate in response to injury or stress, giving rise to regenerated muscle and more satellite cells.

[0086] Solubilized ECM: ECM that has been treated, for example, by ultrasonic cavitation or enzymatic digestion, which involves the physical disruption or digestion of protein aggregates, thereby causing microstructural changes.

[0087] Spinal muscular atrophy (SMA) is a hereditary and acquired central nervous system (CNS) neurodegenerative disorder characterized by progressive motor neuron loss in the spinal cord and brainstem, leading to muscle weakness and muscular atrophy. The most common form of SMA is caused by mutations in the survival motor neuron (SMN) gene and manifests in a wide range of severity, affecting individuals from infancy to adulthood (Crawford and Pardo, Neurobiol. Dis., 1996, 3:97).

[0088] Specifically, SMA is caused by a genetic mutation in the survival motor neuron 1 (SMN1) gene, which prevents the normal expression of the SMN protein. In most cases, SMA is diagnosed based on clinical symptoms and the presence of at least one copy of the SMN1 gene test. However, in approximately 5% of cases, SMA is caused by mutations in the gene other than SMN1 inactivation. In some cases, if the SMN1 gene test is not feasible or shows no abnormalities, other tests, such as electromyography (EMG) or muscle biopsy, may be indicated.

[0089] Remarkably, despite the ubiquitous expression of the SMN1 gene in all motor neurons (MNs), not all muscles are affected. SMA particularly affects the lower extremities, and in more severe cases, respiratory function is impaired. Affected motor neurons in SMA patients are less able to produce sustained firing and deteriorate over time, potentially leading to motor neuron (MN) death. Experiments in mouse models have shown that insufficient expression of the SMN protein initially leads to dysfunction and MN death in the later stages of the disease. MN dysfunction in SMA patients is caused by non-cellular autonomous mechanisms and is unrelated to MN death. Therefore, MN dysfunction and their death in SMA patients are two independent processes.

[0090] The severity of SMA ranges from respiratory failure in the neonatal period (types 1-2) to mild muscle weakness observed in adulthood (type 4). Infant SMA is the most severe form of this neurodegenerative disorder. Symptoms include muscle weakness, low muscle tone, weak crying, a tendency to limp or flop, difficulty sucking or swallowing, accumulation of secretions in the lungs or throat, difficulty feeding, and increased susceptibility to respiratory infections. The legs tend to be weaker than the arms, and developmental milestones such as lifting the head or sitting up may not be reached. Generally, the earlier symptoms appear, the shorter the life expectancy. Symptoms appear soon after motor neuron cell decay. Severe forms of the disease are fatal. The course of SMA is directly related to the rate of motor neuron cell decay and the severity of the resulting weakness. Infants with severe forms of SMA have a high mortality rate from respiratory illness due to weakness in the muscles that support breathing. Children with milder forms of SMA live longer, but they, especially those in the final stages of the more severe spectrum, may require extensive medical support. The clinical spectrum of SMA disorders is divided into the following five groups: 1) Type 0 SMA (In utero SMA) is the most severe form of the disease and begins before birth. Typically, the first symptom of type 0 SMA is reduced fetal movement, which can first be observed between the 30th and 36th weeks of gestation. After birth, these newborns have little movement and experience difficulty swallowing and breathing. 2) Type 1 SMA (infantile SMA or Werdnig-Hoffmann disease) presents with symptoms between 0 and 6 months of age. This form of SMA is also very severe. Patients never acquire the ability to sit, and death usually occurs within the first two years if ventilatory support is not provided. 3) Type 2 SMA (intermediate SMA) has an age of onset between 7 and 18 months. Patients acquire the ability to sit without support but never stand or walk without assistance. The prognosis in this group largely depends on the degree of respiratory involvement. 4) Type 3 SMA (juvenile SMA or Kugelberg-Welander disease) is generally diagnosed after 18 months of age. Individuals with type 3 SMA may be able to walk independently at some point during their disease course, but often become wheelchair-bound during adolescence or adulthood. 5) Type 4 SMA (adult-onset SMA). Weakness usually begins in late adolescence in the tongue, hands, or feet and then progresses to other areas of the body. The course of adult-onset SMA is fairly slow and has little to no impact on life expectancy.

[0091] Subjects: Humans, and all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, mice, rabbits, sheep, dogs, cats, horses, cattle, chickens, amphibians and reptiles. In some aspects of the methods described, the subjects are humans. The term "subject" is used interchangeably with the term "patient." A subject may be an individual diagnosed with a disease or disorder, e.g., an infectious disease or disorder (e.g., an immunocompromised individual, a healthcare professional), an individual diagnosed with a disease or disorder, e.g., an infectious disease or disorder, an individual who has previously suffered from a disease or disorder, e.g., an infectious disease or disorder, or an individual evaluated for symptoms or signs of a disease or disorder, e.g., an infectious disease or disorder.

[0092] Therapeutic effective dose: The amount of a particular substance, e.g., MBV, sufficient to achieve the desired effect in the treated subject. When administered to a subject, the dose used is generally the dose that achieves the target tissue concentration (e.g., in the lungs) that has been shown to achieve the desired in vitro effect.

[0093] Thermoreversible hydrogels: Hydrogels formed due to entanglement of polymer chains, where viscosity changes at a temperature characteristic of gelation. The disclosed acoustically treated ECM hydrogels are thermoreversible hydrogels that exhibit gelation (transition from sol to gel) upon cooling.

[0094] Topical application: Topical medications are applied only to specific areas of the body, not to the entire body. In specific cases, the composition is applied to the skin or eyes in areas where hemostasis is desired. For example, a pharmaceutical composition may be applied in a topical preparation to wounds, such as epithelial wounds or defects, such as traumatic or surgical wounds, such as skin or corneal abrasions or surgical incisions.

[0095] Total phospholipid content: When used herein in relation to MBV, "total phospholipids" or "total phospholipid content" refers to the sum of all phospholipids present in a given amount of isolated MBV, i.e., MBV isolated from ECM. MBV can be isolated, for example, by enzymatic digestion of decellularized ECM and differential centrifugation. Total phospholipid content can be determined by methods such as LC-MS-based global lipidomics and redox lipidomics. Total phospholipid content is measured by weight. The percentage of total phospholipid content refers to the percentage concentration on a weight / weight basis.

[0096] Transplantation: Placement of a biocompatible substrate, such as MBV, into the target organism that requires it.

[0097] Treatment, treatment and therapy: any success or signs of success in reducing or relieving an injury, pathology or condition, e.g., relief, improvement, reduction of symptoms, or making the condition more tolerable for the patient, slowing the rate of degeneration or decline, making the final stage of degeneration less debilitating, or improving the physical or mental well-being of the subject, including any objective or subjective parameters. Treatment may be evaluated by objective or subjective parameters, including the results of a physical examination, neurological examination or psychiatric evaluation.

[0098] Ultrasonic treatment: A process of exposing an object to ultrasound with a frequency higher than 20 kHz.

[0099] Matrix-bound nanovesicles (MBVs) derived from the extracellular matrix (ECM) Nanovesicles derived from ECM (also known as matrix-bound nanovesicles, or "MBVs") are generally described in PCT publication numbers WO2017 / 151862, WO2018 / 204848 and WO2019 / 213482, all of which are incorporated herein by reference. It has been disclosed that MBVs are embedded in the extracellular matrix. These MBVs can be isolated and are biologically active. MBVs either do not express CD63 and CD81, or CD63 lo CD81 lo MBV is free of alkaline phosphatase. MBV may contain IL-33. These MBVs may be used for therapeutic purposes. In some embodiments, MBV does not contain alkaline phosphatase, osteopontin, osteoprotegerin, complement C5 and / or c-reactive proteins.

[0100] The extracellular matrix is ​​a complex mixture of structural and functional biomolecules and / or biomacromolecules that surround and support cells within mammalian tissues and, unless otherwise indicated, are cell-free. These include, but are not limited to, structural proteins, specialized proteins, proteoglycans, glycosaminoglycans, and growth factors. Generally, the MBVs disclosed are embedded within any type of extracellular matrix (ECM) and can be isolated from this site. Therefore, MBVs are not detectably present on the surface of the ECM and are not exosomes (also known as extracellular vesicles or EVs).

[0101] The extracellular matrix, for example, without limitation, is incorporated by reference in its entirety, as per U.S. Patent Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463; 5,372,821; 5,554,389; 5,573,784; 5,645,860; 5,771,969; 5,753,267; 5,762,966; 5,866,414; and 6 Disclosed in Patent Nos. 099,567; Nos. 6,485,723; Nos. 6,576,265; Nos. 6,579,538; Nos. 6,696,270; Nos. 6,783,776; Nos. 6,793,939; Nos. 6,849,273; Nos. 6,852,339; Nos. 6,861,074; Nos. 6,887,495; Nos. 6,890,562; Nos. 6,890,563; Nos. 6,890,564; and Nos. 6,893,666. However, ECM can be produced from any tissue, or from any in vitro source in which ECM is produced by cultured cells and contains one or more polymeric components (components) of native ECM. ECM preparations may be considered "decellularized" or "cell-free," meaning that cells have been removed from the source tissue or culture.

[0102] In some embodiments, the extracellular matrix (ECM) is isolated from vertebrates, including but not limited to mammalian vertebrates such as humans, monkeys, pigs, cattle, and sheep. The ECM may originate from any organ or tissue, including, without limitation, the bladder, intestines (e.g., small or large intestine), heart, dermis, liver, kidneys, uterus, brain, blood vessels, lungs, bones, muscles, pancreas, placenta, stomach, spleen, colon, adipose tissue, or esophagus. In specific, non-limiting examples, the extracellular matrix is ​​isolated from esophageal tissue, bladder (e.g., bladder matrix (UBM) or bladder submucosa (UBS)), small intestinal submucosa (SIS), dermis, umbilical cord, pericardium, cardiac tissue, or skeletal muscle. For example, the ECM may be UBM, or the ECM may be SIS, or the ECM may be UBS, or the ECM may be dermis. The ECM may include, for example, any part or tissue obtained from an organ, including, without limitation, submucosa, epithelial basement membrane, lamina propria, etc. In one non-restrictive embodiment, the ECM is isolated from the bladder. In some embodiments, the ECM is derived from human subjects. In other embodiments, the ECM is derived from porcine subjects. In some embodiments, the ECM is not porcine ECM. In some embodiments, the ECM is not porcine bladder-derived ECM.

[0103] The ECM may or may not include the basement membrane. In another non-limiting embodiment, the ECM includes at least a portion of the basement membrane. The ECM material may or may not retain some of the cellular elements that make up the original tissue, such as capillary endothelial cells or fibrous cells. In some embodiments, the ECM includes both basement membrane surfaces and non-basement membrane surfaces.

[0104] In some embodiments, the extracellular matrix (ECM) is recovered from pig bladder (also known as bladder matrix or UBM). Briefly, the ECM is prepared by removing bladder tissue from a mammal, e.g., a pig, and trimming off the residual external connective tissue, which includes adipose tissue. All residual urine is removed by repeated washing with tap water. The tissue is delaminated by initially immersing it in a deepithelializing solution, e.g., hypertonic saline (e.g., 1.0 N saline), for a period ranging from 10 minutes to 4 hours. Exposure to hypertonic saline removes epithelial cells from the underlying basement membrane. Calcium chelating agents may be added to the saline solution as needed. The tissue remaining after the initial delamination procedure includes the epithelial basement membrane and the tissue layers anti-luminal (abluminal) relative to the epithelial basement membrane. The relatively brittle epithelial basement membrane is always damaged and removed by any mechanical delamination against the luminal surface. This tissue is then subjected to further processing that removes most of the anti-luminal tissue while preserving the epithelial basement membrane and lamina propria. The outer serosal, adventitia, muscularis mucosa, submucosa, and most of the muscularis mucosa are removed from the remaining deepithelialized tissue by mechanical dissection or by a combination of enzymatic treatment (e.g., using trypsin or collagenase) followed by hydration and dissection. Mechanical removal of these tissues is achieved, for example, without limitation, by removal of mesenteric tissue with Adson-Brown forceps and Metzenbaum scissors, and by wiping the muscularis and submucosa using longitudinal wiping motions with a scalpel handle wrapped in moistened gauze or other hard object. Automated robotic procedures involving cutting blades, lasers, and other methods of tissue separation are also contemplated. After these tissues have been removed, the resulting ECM consists mainly of the epithelial basement membrane and the underlying lamina propria.

[0105] In another embodiment, the ECM is prepared by exfoliating the porcine bladder tissue to remove the outer layers, including both the serous (tunica serosa) and muscular (muscular) layers, using a longitudinal wiping motion with a scalpel handle and moistened gauze. After turning the tissue segments inside out, the luminal portion of the mucosal layer (tunica mucosa) is interlaminated from the underlying tissue using the same wiping motion. Care is taken to avoid perforating the submucosa. After these tissues have been removed, the resulting ECM will consist mainly of the submucosa layer (see Figure 2 of U.S. Patent No. 9,277,999, incorporated herein by reference).

[0106] ECM can also be prepared as a powder. Such a powder can be prepared according to the method of Gilbert et al., Biomaterials 26 (2005) 1431-1435, which is thus incorporated herein by reference in whole. For example, a UBM sheet can be freeze-dried and then cut into smaller sheets for immersion in liquid nitrogen. The flash-frozen material can then be pulverized so that the particles are small enough to be placed in a rotary knife mill where the ECM is pulverized. Similarly, by precipitating the NaCl in the ECM structure, the material can be broken down into uniformly sized particles that can be flash-frozen, freeze-dried, and pulverized.

[0107] In one non-limiting embodiment, the ECM is derived from the submucosa of the small intestine or SIS. Commercial preparations include, but are not limited to, SURGISIS®, SURGISIS-ES®, STRATASIS® and STRATASIS-ES® (Cook Urological Inc.; Indianapolis, Ind.) and GRAFTPATCH® (Organogenesis Inc.; Canton Mass.). In another non-limiting embodiment, the ECM is derived from the dermis. Commercial preparations include, but are not limited to, PELVICOL® (marketed as PERMACOL® in Europe; Bard, Covington, Ga.), REPLIFORM® (Microvasive; Boston, Mass.) and ALLODERM® (LifeCell; Branchburg, NJ). In yet another embodiment, the ECM is derived from the bladder. Commercial preparations include, but are not limited to, UBM (ACell Corporation; Jessup, Md.).

[0108] MBV may be induced from (and released from) the extracellular matrix using the methods disclosed below. For example, MBV may be obtained from the extracellular matrix according to the method disclosed in U.S. Patent Application Publication No. 2019 / 0117837, the contents of which are incorporated herein by reference for all purposes. In some embodiments, the ECM is digested with enzymes, e.g., pepsin, collagenase, elastase, hyaluronidase and / or proteinase K, and MBV is isolated. In other embodiments, MBV is released from and separated from the ECM by altering the pH with a solution, e.g., glycine HCl, citrate, ammonium hydroxide; by the use of chelating agents, e.g., EDTA, EGTA, etc.; by ionic strength and / or chaotropic effect with the use of salts, e.g., potassium chloride (KCl), sodium chloride, magnesium chloride, sodium iodide, sodium thiocyanate, etc.; or by exposing the ECM to denaturing conditions such as guanidine HCl or urea.

[0109] MBV may originate from the extracellular matrix of the bladder, small intestine, heart, dermis, liver, kidney, uterus, brain, blood vessels, lungs, bone, muscle, pancreas, placenta, stomach, spleen, colon, adipose tissue, and / or esophagus. In specific, non-limiting examples, MBV originates from the bladder matrix (UBM), small intestinal submucosa (SIS), or bladder submucosa (UBS). In one embodiment, MBV originates from the dermis. In another embodiment, MBV originates from the UBM. In a further embodiment, MBV originates from the extracellular matrix of a mammalian vertebrate selected from humans, monkeys, pigs, cattle, or sheep. In specific, non-limiting examples, MBV originates from a non-human mammal. In some embodiments, MBV does not originate from the bone ECM. In some embodiments, MBV does not originate from the cardiac ECM. In some embodiments, MBV does not originate from either the cardiac ECM or the bone ECM. In certain embodiments, MBV is prepared after digestion of ECM with enzymes, such as pepsin, elastase, hyaluronidase, proteinase K, saline solution and / or collagenase, or a combination thereof. ECM may be frozen-thawed or subjected to mechanical degradation.

[0110] In some embodiments, the expression of CD63, CD81, and / or CD9 is undetectable on MBV. Therefore, in some embodiments, MBV does not express CD63, and / or CD81, and / or CD9. In one specific example, CD63, CD81, and CD9 are undetectable on nanovesicles. In other embodiments, MBV has barely detectable levels of CD63, CD81, and CD9, such that they are detectable by Western blotting. These MBVs express CD63 lo CD81 lo CD9 lo In other embodiments, MBV does not express one or more of CD63, CD81, or CD9 at a detectable level. In other embodiments, MBV expresses one or more of CD63, CD81, or CD9 at a barely detectable level. Those skilled in the art can, for example, use antibodies that specifically bind to CD63, CD81, and CD9 to detect CD63lo and / or CD81 lo and / or CD9 lo MBVs can be easily identified. Low levels of these markers can be established using procedures, e.g., fluorescence-activated cell sorting (FACS) and fluorescently labeled antibodies, to determine thresholds for low and high levels of CD63, CD81, and CD9. In some cases, the disclosed MBVs do not contain detectable alkaline phosphatase, osteopontin, osteoprotegerin, complement C5, and / or c-reactive proteins. Since MBVs bind to the ECM in vivo and are not found in biological fluids, the disclosed MBVs are distinct from nanovesicles, e.g., exosomes, which may transiently bind to the surface of the ECM due to their presence in biological fluids.

[0111] MBVs have a unique phospholipid content compared to, for example, exosomes. In some embodiments, the total phospholipid content of MBVs is at least 50%, 55%, 60%, 65%, 70%, 75%, 85%, or 90%, or about 50%-90%, 50%-65%, 50%-60%, 50%-70%, 60%-70%, 60%-90%, or 70%-90%, of combined phosphatidylcholine (PC) and phosphatidylinositol (PI). In specific embodiments, the total phospholipid content of MBVs is at least 55% of combined phosphatidylcholine (PC) and phosphatidylinositol (PI). In specific embodiments, the total phospholipid content of MBVs is at least 60% of combined phosphatidylcholine (PC) and phosphatidylinositol (PI). In some embodiments, the phospholipid content of MBV includes a ratio of phosphatidylcholine (PC) to phosphatidylinositol (PI) of less than 8:1 (e.g., less than 7:1, less than 6:1, less than 5:1, less than 4:1, less than 3:1, or less than 2:1). In some embodiments, the phospholipid content of MBV includes a ratio of phosphatidylcholine (PC) to phosphatidylinositol (PI) in the range of 0.5 to 1:1, or 1:0.5 to 1, or 0.5 to 1:2, or 2:0.5 to 1, or 0.8 to 1:1, or 1:0.8 to 1. In one embodiment, the phospholipid content of MBV includes a ratio of phosphatidylcholine (PC) to phosphatidylinositol (PI) of approximately 1:1. In a specific embodiment, the phospholipid content of MBV includes a ratio of phosphatidylcholine (PC) to phosphatidylinositol (PI) of approximately 0.9:1.

[0112] In some embodiments, the total phospholipid content of MBV is 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or less, or approximately 5%-10%, 5%-15%, 10%-15%, or 8%-12% sphingomyelin (SM). In specific embodiments, the total phospholipid content of MBV is 10% or less sphingomyelin (SM). In some embodiments, the total phospholipid content is 15% or less sphingomyelin (SM), 14% or less sphingomyelin, 13% or less sphingomyelin, 12% or less sphingomyelin, 11% or less sphingomyelin, 10% or less sphingomyelin, 9% or less sphingomyelin, 8% or less sphingomyelin, 7% or less sphingomyelin, 6% or less sphingomyelin, 5% or less sphingomyelin, or 4% or less sphingomyelin.

[0113] In some embodiments, the total phospholipid content of MBV is 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or less than 10%, or approximately 10%-20%, 15%-20%, 14%-18%, or 12%-16% phosphatidylethanolamine (PE). In specific embodiments, the total phospholipid content of MBV is 20% or less phosphatidylethanolamine (PE).

[0114] In some embodiments, the total phospholipid content of MBV is 5%, 10%, 12%, 15%, 18%, 20%, 25%, or 30% or more, or approximately 5%–30%, 10%–20%, 10–25%, 15%–25%, or 12%–18% phosphatidylinositol (PI). In specific embodiments, MBV contains 15% or more phosphatidylinositol (PI) as its phospholipid content.

[0115] In specific embodiments, the total phospholipid content of MBV includes 15% or more phosphatidylinositol, less than 20% phosphatidylethanolamine, and less than 10% sphingomyelin. In specific embodiments, the total phospholipid content of MBV is 15% or more phosphatidylinositol and less than 20% phosphatidylethanolamine. In specific embodiments, the total phospholipid content of MBV is 15% or more phosphatidylinositol and less than 10% sphingomyelin. In specific embodiments, the total phospholipid content of MBV includes 20% or less phosphatidylethanolamine and less than 10% sphingomyelin. In specific embodiments, the total phospholipid content of MBV is more than 15% phosphatidylinositol, less than 20% phosphatidylethanolamine, less than 10% sphingomyelin, and at least 55% combined phosphatidylinositol and phosphatidylcholine. In one embodiment, the total phospholipid content of MBV is at least 55% combined phosphatidylcholine (PC) and phosphatidylinositol (PI), and less than 10% sphingomyelin (SM). In specific embodiments, the total phospholipid content of MBV is at least 55% combined phosphatidylinositol and phosphatidylcholine, and more than 15% phosphatidylinositol. In specific embodiments, the total phospholipid content of MBV is 55% combined phosphatidylinositol and phosphatidylcholine, and less than 20% phosphatidylethanolamine.

[0116] MBVs may also contain lysyl oxidase (Lox). Generally, nanovesicles derived from the extracellular matrix (ECM) have a higher Lox content than exosomes. Lox is expressed on the surface of MBVs. Nano-LC MS / MS proteomics analysis can be used to detect Lox proteins. Quantification of Lox can be performed (see, for example, Hill RC, et al., Mol Cell Proteomics. 2015;14(4):961-73, which is thus incorporated herein by reference in its entirety).

[0117] In some embodiments, MBV is characterized by one or more of the following features: (i) Does not contain detectable levels of alkaline phosphatase; (ii) Does not contain detectable levels of osteopontin; (iii) Does not contain detectable levels of osteoprotegerin; (iv) Does not contain detectable levels of complement C5; and / or (v) Does not contain detectable levels of c-reactive protein.

[0118] In some embodiments, MBV is characterized by one or more of the following features: (i) Contains low levels of EpCAM or does not contain detectable levels of EpCAM. (ii) Contains low levels of ANXA5 or does not contain detectable levels of ANXA5. (iii) Contains low levels of TSG101 or does not contain detectable levels of TSG101; (iv) Contains low levels of FLOT1 or does not contain detectable levels of FLOT1; (v) Contains low levels of ICAM1 or does not contain detectable levels of ICAM1; (vi) Contains low levels of GM130 or does not contain detectable levels of GM130; and / or (vii) Contains low levels of ALIX or does not contain detectable levels of ALIX.

[0119] In one embodiment, MBV is characterized by low or undetectable levels of ANXA5, TSG101, and ICAM1.

[0120] In one embodiment, the MBV is characterized by low or undetectable levels of CD81, CD63, ANXA5, TSG101, and ICAM1.

[0121] In certain embodiments, MBV comprises one or more miRNAs. In specific, non-limiting examples, MBV comprises one, two, or all three of miR-143, miR-145, and miR-181. miR-143, miR-145, and miR-181 are known in the art.

[0122] The miR-145 nucleic acid sequence is provided by MiRbase accession number MI0000461, which is thus incorporated herein by reference. [ka] The miR-181 nucleic acid sequence is provided by the miRbase accession number MI0000269, which is thus incorporated herein by reference. [ka] The miR-143 nucleic acid sequence is provided by NCBI accession number NR_029684.1, dated March 30, 2018, which is thus incorporated herein by reference. The DNA encoding the miR-143 nucleic acid sequence is [ka] That is the case.

[0123] Following administration, MBV maintains the expression of F4 / 80 (macrophage marker) and CD-11b on macrophages in the subjects. Nanovesicle-treated macrophages are predominantly F4 / 80+Fizz1+ and exhibit the M2 phenotype.

[0124] The MBV disclosed herein may be formulated into compositions for drug delivery. The MBV is further disclosed and described in PCT Publication No. WO2017 / 151862, which is thus incorporated herein by reference.

[0125] Isolation of MBV from ECM MBV can be produced from ECM produced by any cell of interest, or isolated from a commercial source of ECM as described above. MBV can be produced from the same species as the subject being treated, or from a different species. In some embodiments, these methods include a step of enzymatically digesting the ECM to produce digested ECM. In specific embodiments, the ECM is digested by one or more of pepsin, elastase, hyaluronidase, collagenase, metalloproteinase, and / or proteinase K, or a combination thereof. In specific non-limiting embodiments, the ECM is digested by elastase and / or metalloproteinase alone. In another non-limiting embodiment, the ECM is not digested by collagenase and / or trypsin and / or proteinase K. In other embodiments, the ECM is treated with detergent. In further embodiments, the method does not involve the use of enzymes. In specific non-limiting embodiments, the method utilizes chaotropic agents or ionic strengths, e.g., salts, e.g., potassium chloride, to isolate MBV. In a further embodiment, the ECM may be manipulated to increase the MBV content before isolation of MBV. Techniques for isolating MBV from the ECM are described, for example, in U.S. Patent Application Publication 2019 / 0117837, the contents of which are incorporated herein by reference for all purposes. Techniques for isolating MBV are also disclosed in Quijano et al., Tissue Eng Part C Methods. 2020 Oct;26(10):528-540, which are also incorporated herein by reference.

[0126] In some embodiments, ECM is digested enzymatically. ECM may be digested enzymatically for about 12 to about 48 hours, for example, about 12 to about 36 hours. ECM may be digested enzymatically for about 12, about 24, about 36, or about 48 hours. In a specific, non-limiting example, ECM is digested enzymatically at room temperature. However, digestion may occur at about 4°C, or at any temperature between about 4°C and 25°C. Generally, ECM is digested enzymatically for any length of time sufficient to remove collagen fibrils, and at any such temperature. The digestion process may vary depending on the tissue source. If necessary, ECM may be treated by freezing and thawing either before or after enzymatic digestion. ECM may be treated with detergents containing ionic and / or nonionic detergents.

[0127] Next, the digested ECM is treated, for example, by centrifugation to isolate the non-fibrous supernatant. In some embodiments, the digested ECM is centrifuged at about 300 to about 1000 g for the first step, for example. Thus, the digested ECM may be centrifuged at about 400 g to about 750 g, for example, about 400 g, about 450 g, about 500 g, or about 600 g. This centrifugation may be carried out over about 10 to about 15 minutes, for example, about 10 to about 12 minutes, for example, about 10, about 11, about 12, about 14, about 14, or about 15 minutes. The supernatant containing the digested ECM is collected.

[0128] In some embodiments, MBV comprises Lox. In some embodiments, a method for isolating such MBV includes the steps of: digesting the extracellular matrix with elastase and / or metalloproteinase to produce digested extracellular matrix; centrifuging the digested extracellular matrix to remove collagen fibril residues, thereby producing fibril-free supernatant; centrifuging the fibril-free supernatant to isolate solid material; and suspending the solid material in a carrier.

[0129] In some embodiments, the digested ECM may also be centrifuged at approximately 2000g to approximately 3000g for a second step. Thus, the digested ECM may be centrifuged at approximately 2,500g to approximately 3,000g, for example, approximately 2,000g, 2,500g, 2,750g, or 3,000g. This centrifugation may be carried out over approximately 20 to approximately 30 minutes, for example, approximately 20 to approximately 25 minutes, for example, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29, or approximately 30 minutes. The supernatant containing the digested ECM is collected.

[0130] In a further embodiment, the digested ECM may be centrifuged at approximately 10,000 to approximately 15,000 g for a third step. Thus, the digested ECM may be centrifuged at approximately 10,000 g to approximately 12,500 g, for example, approximately 10,000 g, 11,000 g, or 12,000 g. This centrifugation may be carried out over approximately 25 to approximately 40 minutes, for example, approximately 25 to approximately 30 minutes, for example, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29, approximately 30, approximately 31, approximately 32, approximately 33, approximately 34, approximately 35, approximately 36, approximately 37, approximately 38, approximately 39, or approximately 40 minutes. The supernatant containing the digested ECM is collected. One, two, or all three of these centrifugation steps may be used independently. In some embodiments, all three centrifugation steps are used. The centrifugal separation step can be repeated, for example, two, three, four, or five times. In one embodiment, all three centrifugal separation steps are repeated three times.

[0131] In some embodiments, the digested ECM is centrifuged at approximately 500 g for approximately 10 minutes, at approximately 2,500 g for approximately 20 minutes, and / or at approximately 10,000 g for approximately 30 minutes. These steps(s), for example, all three steps, are repeated two, three, four, or five times, for example, three times. Thus, in a non-limiting example, the digested ECM is centrifuged at approximately 500 g for approximately 10 minutes, at approximately 2,500 g for approximately 20 minutes, and at approximately 10,000 g for approximately 30 minutes. These three steps are repeated three times. Thus, a non-filamentous supernatant is produced. The non-filamentous supernatant is then centrifuged to isolate MBV. In some embodiments, the non-filamentous supernatant is centrifuged at approximately 100,000 g to approximately 150,000 g. Therefore, the non-fiber supernatant is centrifuged at approximately 100,000 g to approximately 125,000 g, for example, approximately 100,000 g, approximately 105,000 g, approximately 110,000 g, approximately 115,000 g, or approximately 120,000 g. This centrifugation may be carried out over approximately 60 to approximately 90 minutes, for example, approximately 70 to approximately 80 minutes, for example, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, or approximately 90 minutes. In a non-limiting example, the non-fiber supernatant is centrifuged at approximately 100,000 g for approximately 70 minutes. The solid material, which is MBV, is collected. These MBVs can then be resuspended in any carrier of interest, for example, a buffer, but not limited to this.

[0132] In further embodiments, the ECM is not digested by enzymes. In these methods, the ECM is suspended in an isotonic saline solution, such as phosphate-buffered saline. Then, salt is added to the suspension so that the final concentration of salt is greater than about 0.1 M. The concentration can be, for example, up to about 3 M, e.g., about 0.1 M of salt to about 3 M or about 0.1 M to about 2 M. The salt can be, for example, about 0.1 M, 0.15 M, 0.2 M, 0.3 M, 0.4 M, 0.7 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M or 2 M. In some non-limiting examples, the salt is potassium chloride, sodium chloride, or magnesium chloride. In other embodiments, the salt is sodium chloride, magnesium chloride, sodium iodide, sodium thiocyanate, sodium salt, lithium salt, cesium salt, or calcium salt.

[0133] In some embodiments, the ECM is suspended in a salt solution for about 10 minutes to about 2 hours, for example, about 15 minutes to about 1 hour, about 30 minutes to about 1 hour, or about 45 minutes to about 1 hour. The ECM may be suspended in a salt solution for about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes. The ECM may be suspended in a salt solution at temperatures of, for example, about 4°C to about 25°C or about 4°C to about 37°C, but not limited to these, or between 4°C and about 50°C. In a specific non-limiting example, the ECM is suspended in a salt solution at about 4°C. In another specific non-limiting example, the ECM is suspended in a salt solution at about 22°C or about 25°C (room temperature). In a further non-limiting example, the ECM is suspended in a salt solution at approximately 37°C.

[0134] In some embodiments, the method comprises the steps of: incubating the extracellular matrix at a salt concentration higher than about 0.4 M; centrifuging the digested extracellular matrix to remove collagen fibril residues and isolating the supernatant; centrifuging the supernatant to isolate the solid material; and suspending the solid material in a carrier, thereby isolating MBV from the extracellular matrix.

[0135] After incubation in a salt solution, the ECM is centrifuged to remove collagen fibrils. In some embodiments, the digested ECM may also be centrifuged at about 2000g to about 5000g. Thus, the digested ECM may be centrifuged at about 2,500g to about 4,500g, for example, about 2,500g, about 3,000g, 3,500g, about 4,000g, or about 4,500g. In a specific non-limiting example, the centrifugation is at about 3,500g. This centrifugation may be carried out over about 20 to about 40 minutes, for example, about 25 to about 35 minutes, for example, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 minutes, about 31, about 32, about 33, about 34, or about 35 minutes. The supernatant is then collected.

[0136] In a further embodiment, the supernatant may then be centrifuged at approximately 100,000 to approximately 150,000 g for the third step. Thus, the digested ECM may be centrifuged at approximately 100,000 g to approximately 125,000 g, for example, approximately 100,000 g, 110,000 g, or 120,000 g. This centrifugation may be carried out over approximately 30 minutes to approximately 2.5 hours, for example, approximately 1 hour to approximately 3 hours, for example, approximately 30 minutes, approximately 45 minutes, approximately 60 minutes, approximately 90 minutes, or approximately 120 minutes (2 hours). The solid material is collected and suspended in a solution, for example, buffered saline, thereby isolating the MBV.

[0137] In yet another embodiment, the ECM is suspended in an isotonic buffer solution, for example, phosphate-buffered saline, but not limited to this. Centrifugation or other methods may be used to remove larger particles (see below). Ultrafiltration is then used to isolate particles from the ECM that are between MBV, between about 10 nm and about 10,000 nm, for example, between about 10 nm and about 1,000 nm, for example, between about 10 nm and about 300 nm.

[0138] In specific, non-limiting examples, isotonic buffered saline solution has a total salt concentration of about 0.164 mM and a pH of about 7.2 to about 7.4. In some embodiments, the isotonic buffered saline solution contains 0.002 M to about 0.164 M KCl, for example, about 0.0027 M KCl (concentration of KCl in phosphate-buffered saline). This suspension is then treated by ultracentrifugation.

[0139] After incubation in an isotonic buffer solution, the ECM is centrifuged to remove collagen fibrils. In some embodiments, the digested ECM may also be centrifuged at about 2000 g to about 5000 g. Thus, the digested ECM may be centrifuged at about 2,500 g to about 4,500 g, for example, about 2,500 g, about 3,000 g, 3,500, about 4,000 g, or about 4,500 g. In a specific non-limiting example, the centrifugation is at about 3,500 g. This centrifugation may be carried out over about 20 to about 40 minutes, for example, about 25 to about 35 minutes, for example, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 minutes, about 31, about 32, about 33, about 34, or about 35 minutes.

[0140] Microfiltration and centrifugation may be used and combined to remove large molecular weight materials from a suspension. In one embodiment, for example, large molecular weight materials larger than 200 nm are removed using microfiltration. In another embodiment, large molecular weight materials are removed by the use of centrifugation. In a third embodiment, both microfiltration and ultracentrifugation are used to remove large molecular weight materials. Large molecular weight materials, for example, materials larger than about 10,000 nm, larger than about 1,000 nm, larger than about 500 nm, or larger than about 300 nm, are removed from the suspended ECM.

[0141] Next, the eluent or supernatant from the microfiltration is subjected to ultrafiltration. Thus, eluents containing particles smaller than approximately 10,000 nm, 1,000 nm, 500 nm, or 300 nm are collected and used. These eluents are then subjected to ultrafiltration using a membrane with a molecular weight cutoff (MWCO) of 3,000 to 100,000.

[0142] Preparation of extracellular matrix (ECM) and hydrogels Any type of extracellular matrix can be used to produce mammalian ECM hydrogels (see U.S. Patent Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463; 5,372,821; 5,554,389; 5,573,784; 5,645,860; 5,771,969; 5,753,267; 5,762,966; 5,866,414 related to ECM). See also Nos. 6,099,567; 6,485,723; 6,576,265; 6,579,538; 6,696,270; 6,783,776; 6,793,939; 6,849,273; 6,852,339; 6,861,074; 6,887,495; 6,890,562; 6,890,563; 6,890,564; and 6,893,666). In certain embodiments, ECM is isolated from vertebrates, from mammals including, but not limited to, humans, monkeys, horses, pigs, cattle, and sheep. In a specific, non-specific example, ECM is a pig.

[0143] ECM may originate from any organ or tissue, without limitation, including the bladder, intestines (e.g., small or large intestine), heart, kidneys, uterus, brain, blood vessels, lungs, bones, muscles, pancreas, stomach, spleen, adipose tissue, liver, esophagus, and dermis. ECM may be obtained from cell cultures. In one embodiment, ECM is isolated from the bladder. In another embodiment, ECM is derived from the esophagus. In another embodiment, ECM is derived from the dermis. In another embodiment, ECM is derived from the submucosa (SIS) of the small intestine. ECM may or may not include a basement membrane portion of the ECM. In certain embodiments, ECM includes at least a portion of the basement membrane. Tissues may be decellularized to produce ECM, for example, by removing cells and cellular material from the source tissue or organ. For example, when ECM is implanted in a subject, for example, as a component of a hydrogel disclosed herein, it is desirable to use decellularized material to prevent an immune response. Removal of cellular material prevents such immune responses, for example, when forming a hydrogel using ECM.

[0144] U.S. Patent No. 8,361,503 (which is incorporated herein by reference in its entirety for all purposes) discloses that a preparation of bladder ECM, for example, a preparation of porcine bladder ECM, is prepared by exfoliating the bladder tissue to remove the outer layers, including both the serosal and muscular layers, using a longitudinal wiping motion with a scalpel handle and moistened gauze. After inversion of the tissue segments, the luminal portion of the mucosal layer is interlaminated from the underlying tissue using the same wiping motion. In some embodiments, perforation of the submucosa is prevented. After these tissues have been removed, the resulting ECM consists mainly of the submucosa layer.

[0145] The production of hydrogels from dermal ECM is disclosed in Wolf et al., Biomaterials 33: 7028-7038, 2012, which is thereby incorporated herein by reference. The production of ECM from esophageal tissue is disclosed, for example, in Badylak et al., J Pediatr Surg. 35(7):1097-103, 2000 and Badylak et al., J Surg Res. 2005 September; 128(1):87-97, 2005, both of which are thereby incorporated herein by reference. U.S. Patent No. 6,893,666, which is thereby incorporated herein by reference, discloses the production of ECM from the bladder, skin, esophagus, and small intestine. ECM may be produced from any of these tissues.

[0146] Commercially available ECM preparations may also be used. In one embodiment, the ECM is derived from the submucosa of the small intestine or SIS. Commercially available preparations include, but are not limited to, SURGISIS®, SURGISIS-ES®, STRATASIS® and STRATASIS-ES® (Cook Urological Inc.; Indianapolis, Ind.) and GRAFTPATCH® (Organogenesis Inc.; Canton Mass.). In another embodiment, the ECM is derived from the dermis. Commercially available preparations include, but are not limited to, PELVICOL® (sold as PERMACOL® in Europe; Bard, Covington, Ga.), REPLIFORM® (Microvasive; Boston, Mass.) and ALLODERM® (LifeCell; Branchburg, NJ). In yet another embodiment, the ECM is derived from the bladder. Commercially available preparations include, but are not limited to, UBM (Acell Corporation; Jessup, Md.).

[0147] Tissue for the preparation of ECM can be recovered in a wide variety of ways, and once recovered, various parts of the recovered tissue can be used. ECM has also been prepared from the esophagus and small intestine. See, for example, Keane et al., Tissue Eng. Part A, 21(17-18): 2293-2300, 2015, which is incorporated herein by reference. Esophageal ECM can be prepared by mechanically separating the mucosa and submucosa from the muscularis exostata, digesting the mucosa in a trypsin-containing buffer, and then exposing it to sucrose, TRITON-X100®, deoxycholic acid, peracetic acid, and DNAse. Small intestinal submucosa (SIS) can be prepared by mechanically removing the superficial layers of the mucosa, serosal layer, and muscularis exostata from the intact small intestine, leaving the submucosa, muscularis mucosa, and basal stratum compactum intact. Next, the SIS is treated with peracetic acid. An exemplary protocol is provided in Keane et al. Dermal hydrogels can be produced, for example, as disclosed in Wolf et al, J Biomed Mater Res A. 2013. 35(25):6838-49. PMID: 23873846. PMCID: 3808505, which is thus incorporated herein by reference.

[0148] In one embodiment, the extracellular matrix (ECM) is isolated from a recovered pig bladder to prepare the bladder matrix (UBM). Excess connective tissue and residual urine are removed from the bladder. Most of the serosal layer, muscular layer, submucosal layer, and muscularis mucosa can be removed by mechanical exfoliation or by a combination of enzymatic treatment, hydration, and exfoliation. Mechanical removal of these tissues can be achieved by exfoliation using longitudinal wiping motions to remove the outer layers (particularly the anti-luminal smooth muscle layer) and even the luminal portion of the mucosal layer (epithelial layer). Mechanical removal of these tissues can be achieved, for example, by removing the mesenteric tissue with Adson-Brown forceps and Metzenbaum scissors, and by wiping the muscular and submucosal layers using longitudinal wiping motions with a scalpel handle wrapped in moistened gauze or other hard object. Epithelial cells of the mucosal layer can also be dissociated by immersing the tissue in an epithelializing solution, e.g., hypertonic saline, without limitation. The resulting UBM comprises the basement membrane and adjacent lamina propria of the mucosal layer, which is further treated with peracetic acid, freeze-dried, and powdered. See U.S. Patent No. 8,361,503 incorporated herein by reference.

[0149] Dermal sections may be used for the preparation of ECM hydrogels. See PCT application number 2015 / 15164728 incorporated herein by reference. In a specific, non-limiting example, the dermis can be decellularized using 0.25% trypsin / 1% TRITON-X(registered trademark)-100 (i.e., without SDS) in the following solutions at room temperature in a vortex shaker at 300 RPM: 0.25% trypsin for 6 hours, 1×; deionized water, 15 minutes, 3×; 70% ethanol, 10-12 hours, 1×; 3% H2O2, 15 minutes, 1×; deionized water, 15 minutes, 2×; 1% TRITON-X(registered trademark)-100 in 0.26% EDTA / 0.69% Tris for 6 hours, 1× and then overnight, 1×; deionized water, 15 minutes, 3×; 0.1% peracetic acid / 4% ethanol, 2 hours, 1×; PBS, 15 minutes, 2×; and finally deionized water, 15 minutes, 2×. Next, the dermal sheets are freeze-dried, and then processed into granular form using a Waring blender and Wiley Mill with a #20 mesh screen.

[0150] In some embodiments, epithelial cells may be initially delaminated by immersing the tissue in an epithelial delamination solution, such as hypertonic saline, for a period ranging from 10 minutes to 4 hours, without limitation. Exposure to hypertonic saline efficiently removes epithelial cells from the underlying basement membrane. The tissue remaining after the initial delamination procedure includes the epithelial basement membrane and the tissue layers antiluminal to the epithelial basement membrane. This tissue is then subjected to further processing that removes most of the antiluminal tissue but not the epithelial basement membrane. Most of the outer serosa, adventitia, smooth muscle tissue, submucosa, and muscularis mucosa are removed from the remaining deepithelialized tissue by mechanical delamination or by a combination of enzymatic treatment, hydration, and delamination.

[0151] In some embodiments, the ECM itself can be sterilized by several standard techniques, including but not limited to exposure to peracetic acid, low-dose gamma radiation, gas plasma sterilization, ethylene oxide treatment, or electron beam treatment. More typically, sterilization of the ECM is achieved by immersion in 0.1% (v / v) peracetic acid, 4% (v / v) ethanol, and 95.9% (v / v) sterile water for two hours. The peracetic acid residue is removed by washing twice with PBS (pH=7.4) for 15 minutes each, and then twice with sterile water for 15 minutes each. ECM materials can be sterilized by propylene oxide or ethylene oxide treatment, gamma irradiation (0.05–4 mRad), gas plasma sterilization, peracetic acid sterilization, or electron beam treatment. ECM can also be sterilized by treatment with glutaraldehyde, which causes crosslinking of protein materials, but this treatment substantially alters the material to drive a different type of host remodeling, which is more similar to scar tissue formation or encapsulation rather than constructive remodeling, where it is slowly reabsorbed or not reabsorbed at all. Crosslinking of protein materials can also be induced by carbodiimide, dehydrothermal, or photo-oxidation methods. As disclosed in U.S. Patent No. 8,361,503, ECM is disinfected by immersion for 2 hours in 0.1% (v / v) peracetic acid (a), 4% (v / v) ethanol, and 96% (v / v) sterile water. The ECM material is then washed twice for 15 minutes each with PBS (pH=7.4) and twice for 15 minutes each with deionized water.

[0152] Generally, after isolation of the tissue of interest, decellularization is carried out by various methods, for example, without limitation, exposure to hypertonic saline, peracetic acid, TRITON-X®, or other detergents. Sterilization and decellularization may occur simultaneously. For example, without limitation, sterilization with peracetic acid may also be used for decellularization. The ECM may then be dried by either lyophilization (freeze-drying) or air-drying. The dried ECM may be pulverized by methods including, but not limited to, tearing, milling, cutting, crushing, and shearing. The pulverized ECM may also be further processed into a powder form by methods, for example, without limitation, crushing or milling while frozen or freeze-dried.

[0153] Mammalian ECMs are also commercially available. These include AVITENE®, MICROMATRIX®, and XENMATRIX®. These commercially available products can also be used to produce mammalian acoustically treated ECM hydrogels.

[0154] Preparation of acoustically treated ECM hydrogels In some embodiments, the fragmented ECM, e.g., mammalian ECM, is diluted in a liquid for the preparation of an acoustically treated ECM hydrogel. The ECM may or may not be lyophilized prior to fragmentation. The ECM can be fragmented, for example, by crushing, mincing or cutting the ECM. The fragmented ECM should have pieces within the range of about 10 μm to about 5000 μm, about 10 μm to about 4000 μm, about 10 μm to about 3000 μm, about 10 μm to about 2000 μm, about 10 μm to about 1000 μm, about 10 μm to about 500 μm, about 30 μm to about 300 μm, about 40 to about 400 μm, about 25 μm to about 500 μm, about 50 μm to about 500 μm, about 100 μm to about 300 μm, about 10 μm to about 50 μm or about 10 μm to about 100 μm. In one embodiment, the ECM is provided as pieces having a range of about 10 μm to about 1000 μm. In another preferred embodiment, the ECM is provided as pieces having a range of about 10 μm to about 2000 μm. In a non-limiting example, the pieces are within the range of about 30 μm to about 300 μm. The liquid can be a buffer at neutral pH, e.g., about 7.0 to about 7.6, e.g., about 7.1 to about 7.5, e.g., about 7.2 to about 7.4, e.g., about 7.0 to 7.2, e.g., about 7.0 to 7.4, e.g., about 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6. The ECM can be diluted in an isotonic buffered saline solution such as, but not limited to, phosphate buffered saline (PBS) or Tris buffered saline. In some embodiments, the buffered saline solution has an osmolality of about 290 mOsm / L. The liquid can be water. In some embodiments, an isotonic buffer containing, without limitation, phosphate buffered saline (PBS) can be used to bring the solution to the target pH or to help maintain the pH and ionic strength of the gel at target levels, e.g., physiological pH and ionic conditions. This forms a liquid ECM solution.

[0155] Methods for preparing acoustic treatment hydrogels generally do not involve the use of acidic proteases, such as pepsin, trypsin or hyaluronidase, nor the enzymatic digestion of ECM tissue. See PCT Application No. WO2015 / 164728, which is hereby incorporated by reference in its entirety. Generally, solubilized ECM in a liquid is not contacted with acidic proteases.

[0156] Methods for preparing extracellular matrix hydrogels by acoustic treatment techniques, such as the application of ultrasonic frequencies, can be found in US Patent Application Publication No. 2022 / 0143265, the contents of which are hereby incorporated by reference for all purposes. In some embodiments, the ECM is utilized at a concentration higher than about 25 mg / ml in a liquid. The ECM can be utilized at a concentration of about 25 mg / ml to about 600 mg / ml in a liquid, such as a buffer. Suitable concentrations also include about 25 mg / ml to about 300 mg / ml, about 25 mg / ml to about 200 mg / ml and about 25 mg / ml to about 150 mg / ml. The ECM can be utilized at a concentration of about 50 mg / ml to 600 mg / ml in a liquid, such as a buffer. Suitable concentrations also include about 50 mg / ml to about 300 mg / ml, about 50 mg / ml to about 200 mg / ml and about 50 mg / ml to about 150 mg / ml. Suitable concentrations include about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 and 200 mg / ml. Exemplary concentrations include about 25 mg / ml, 100 mg / ml and 150 mg / ml. In one non-limiting example, the ECM is in a liquid at a concentration of about 25 mg / ml to about 150 mg / ml. In one non-limiting example, the ECM is in a liquid at a concentration of 100 mg / ml.

[0157] ECM in a liquid, such as a buffered saline solution, is treated with ultrasonic frequencies. In one embodiment, the ultrasonic frequencies are approximately 20 kHz to approximately 100 kHz. ECM in a liquid can be treated with ultrasonic frequencies of approximately 20 kHz to approximately 30 kHz, approximately 20 kHz to approximately 40 kHz, approximately 20 kHz to approximately 50 kHz, approximately 20 kHz to approximately 60 kHz, approximately 20 kHz to approximately 70 kHz, approximately 20 kHz to approximately 80 kHz, or approximately 20 kHz to approximately 90 kHz. ECM in a liquid can be treated with ultrasonic frequencies of approximately 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, 90 kHz, or 100 kHz. In a non-limiting example, ECM in a liquid can be treated with ultrasonic frequencies of approximately 20 kHz.

[0158] ECM in a liquid, for example, a buffered saline solution, is treated with ultrasound for at least 20 seconds, for example, at least 30 seconds. ECM in a liquid, for example, a buffered saline solution, is treated with ultrasound for at least 60 seconds. In some embodiments, ECM in a liquid is treated with ultrasound for at least 60 seconds to about 1 hour. In further embodiments, ECM in a liquid is treated with ultrasound for at least 60 seconds to about 30 minutes. In further embodiments, ECM in a liquid is treated with ultrasound for at least 30 seconds to about 30 minutes. In additional embodiments, ECM in a liquid is treated with ultrasound for at least 60 seconds to about 15 minutes. In additional embodiments, ECM in a liquid is treated with ultrasound for at least 30 seconds to about 15 minutes. In some embodiments, ECM in a liquid is treated with ultrasound for at least 60 seconds to about 10 minutes. In some embodiments, ECM in a liquid is treated with ultrasound for at least 30 seconds to about 10 minutes. In some embodiments, the ECM in liquid is treated with ultrasound for at least 60 seconds to about 5 minutes. In some embodiments, the ECM in liquid is treated with ultrasound for at least 30 seconds to about 5 minutes. The ECM in liquid may be treated with ultrasound for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 minutes. In some embodiments, the ECM in a liquid is treated with pulsed ultrasound for a total time enumerated herein. Thus, in some embodiments, the ECM in a liquid, for example, a buffered saline solution, is treated with pulses of, for example, a length of at least about 30 seconds, for example, a length of about 30, about 40, or about 60 seconds. The ECM in a liquid, for example, a buffered saline solution, may be treated with ultrasound 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, such that the total treatment time is 60 seconds to 1 hour, or any of the total times enumerated.ECM in a liquid, such as saline solution, may be treated for 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 seconds. ECM in a liquid, such as saline solution, may be treated for at least 30 seconds. Generally, when multiple treatments are used, they are performed over a period of less than one hour. An exemplary method is a 30-second pulse of ultrasound followed by 30-45 seconds of no treatment, and then another treatment. This treatment may be applied 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more. One exemplary, non-limiting method is to use six repetitions of, for example, six 30-second pulses of ultrasound at approximately 20 kHz, followed by a 45-second off period, resulting in a total of three minutes of ultrasound treatment.

[0159] Ultrasound can have an amplitude of approximately 20 μm to approximately 320 μm. Generally, amplitude is measured from the center of the probe used to generate the ultrasound. The amplitude of the vibrating surface of the probe is the distance, measured in microns (μm), between its position when the probe is fully extended and its position when it is fully retracted. In some embodiments, the amplitude is approximately 30 μm to approximately 200 μm. In further embodiments, the amplitude is approximately 36 μm to approximately 180 μm. The amplitude may be approximately 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 150, 160, 70, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 μm. In some embodiments, the amplitudes are approximately 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, 100-110 μm, 110-120 μm, 120-130 μm, 130-140 μm, 140-150 μm, 150-160 μm, 160-170 μm, 170-180 μm, 180-190 μm, 190-200 μm, 200-210 μm, and 210-220 μm. The amplitude can be 220-230 μm, 230-240 μm, 240-250 μm, 250-260 μm, 260-270 μm, 270-280 μm, 280-290 μm, or 290-300 μm. In a specific, non-limiting example, the ultrasound has a frequency of approximately 20 kHz and an amplitude of approximately 36 μm to approximately 180 μm. In a further non-limiting example, the ultrasound has a frequency of approximately 20 kHz and an amplitude of approximately 36 μm to approximately 180 μm, and the treatment lasts for a total of approximately 1, 2, 3, 4, or 5 minutes, for example, approximately 3 minutes. The ultrasonic treatment may last for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes. The ultrasonic treatment may last from about 30 seconds to about 5 minutes. The ultrasonic treatment may, for example, last from about 1 to about 5 minutes. The ultrasonic treatment may last from about 1 to about 10 minutes. The ultrasonic treatment may, for example, last from 1 to about 20 minutes. In additional embodiments, the ultrasonic treatment may last for less than about 1 hour, less than about 30 minutes, less than about 20 minutes, or less than about 10 minutes. In some embodiments, the ultrasonic treatment may last for at least 30 seconds.In other embodiments, the ultrasonic treatment may last from about 10 minutes to about 24 hours, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the ultrasonic treatment may last up to 48 hours.

[0160] In some embodiments, the ECM in liquid is ultrasonically treated at a temperature in the range of approximately 30°C to approximately 43°C. In one embodiment, the ECM in liquid is ultrasonically treated at a temperature in the range of approximately 35°C to approximately 40°C. In another embodiment, the ECM in liquid is ultrasonically treated at a temperature in the range of approximately 36°C to approximately 38°C. In yet another embodiment, the ECM in liquid is ultrasonically treated at a temperature in the range of approximately 37°C or higher, for example, approximately 37°C to approximately 55°C, for example, approximately 37°C to approximately 50°C, for example, approximately 37°C to approximately 45°C, for example, approximately 37°C to approximately 40°C. The ECM in liquid is ultrasonically treated at a temperature of approximately 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55°C. In a further embodiment, the ECM in liquid is treated ultrasonically at a temperature higher than about 38°C, for example, about 38°C to about 50°C, for example, about 38°C to about 45°C, for example, about 38°C to about 40°C.

[0161] In some embodiments, ultrasonic treatment produces an acoustically treated ECM hydrogel. The acoustically treated ECM hydrogel generally undergoes a phase transition from sol to gel at around 37°C, and therefore transitions to a liquid phase at temperatures above 37°C and to a gel phase at temperatures below 37°C. At 37°C, the acoustically treated ECM hydrogel is sufficiently viscous to resemble a gel; however, if the temperature is increased above 37°C, the gel transitions to a sol. The acoustically treated ECM hydrogel forms a gel (sol-to-gel transition) when the temperature decreases below 37°C. Therefore, in some embodiments, after ultrasonic treatment, the acoustically treated ECM hydrogel is cooled to a temperature below 37°C, for example, about 4°C to about 36°C. The acoustically treated ECM hydrogel can generally be cooled to room temperature, which is about 25°C. In some embodiments, the acoustically treated ECM hydrogel is cooled to about 15°C to about 25°C. Acoustically treated ECM hydrogels can be cooled to approximately 23°C to approximately 27°C. Acoustically treated ECM hydrogels can be cooled to approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30°C to induce the gel phase.

[0162] In some embodiments, exogenous MBV may be added to the ECM solution before sonication. In yet other embodiments, exogenous MBV may be added to the acoustically treated ECM hydrogel after sonication. Exogenous MBV may be added to the ECM hydrogel before the hydrogel transitions to a gel (e.g., while it is in the liquid phase); therefore, in one embodiment, exogenous MBV is added to the acoustically treated ECM hydrogel at a temperature higher than 37°C to produce a composition comprising the acoustically treated hydrogel disclosed herein containing exogenous MBV. In another embodiment, exogenous MBV is added to the acoustically treated ECM hydrogel during its gel phase, for example, at a temperature below 37°C. For example, an acoustically treated ECM hydrogel containing exogenous MBV is disclosed herein.

[0163] In some embodiments, acoustically treated mammalian ECM hydrogels are disclosed, which are thermoreversible and exist in a solid (gel) phase at temperatures below about 37°C and in a liquid (sol) phase at temperatures above 37°C. Acoustically treated hydrogels can be produced using any of the methods disclosed herein. In some embodiments, the storage modulus (G') for the acoustically treated ECM hydrogel is about an order of magnitude greater than the loss modulus (G''). In further embodiments, the viscosity of the acoustically treated ECM hydrogel decreases with increasing stress at temperatures of about 15 to about 37°C, e.g., about 15, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and / or 36°C. In a further embodiment, the viscosity of the acoustically treated ECM hydrogel decreases with increasing stress at room temperature and / or about 23°C to about 27°C and / or about 15°C to about 25°C. In one embodiment, the gel-to-sol transition of the acoustically treated ECM hydrogel occurs at about 37°C, so that the hydrogel can be used as a submucosal cushion, because the hydrogel is sufficiently viscous at body temperature.

[0164] These acoustically treated ECM hydrogels may be prepared from any mammalian ECM disclosed above. In specific non-limiting examples, the ECM is human ECM. In other non-limiting examples, the ECM is bladder ECM, small intestinal submucosal ECM, esophageal ECM, or dermal ECM. In one embodiment, the ECM is bladder ECM. In another embodiment, the ECM is dermal ECM. In yet another embodiment, the ECM is esophageal ECM. The source of the ECM may be, for example, pig, cattle, or sheep.

[0165] In some embodiments, the acoustically treated ECM hydrogel contains ECM at concentrations of about 25 mg / ml to about 600 mg / ml. In further embodiments, the acoustically treated ECM hydrogel contains ECM at concentrations of about 20 mg / ml to about 600 mg / ml, about 25 mg / ml to about 300 mg / ml, about 25 mg / ml to about 200 mg / ml, and about 25 mg / ml to about 150 mg / ml. In additional embodiments, the acoustically treated ECM hydrogel contains ECM at concentrations of about 50 mg / ml to 600 mg / ml in a liquid, for example, in a buffer. The acoustically treated ECM hydrogel may also have ECM concentrations of about 50 mg / ml to about 300 mg / ml, about 50 mg / ml to about 200 mg / ml, about 50 mg / ml to about 150 mg / ml, about 50 to 100 mg / ml, or about 100 to 150 mg / ml. In some non-limiting examples, acoustically treated ECM hydrogels contain ECM at concentrations of approximately 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 mg / ml. In some non-limiting cases, acoustically treated ECM hydrogels are approximately 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-105, 105-110, 110-115, and 11 This includes ECM in concentrations of 5-120, 120-125, 125-130, 130-135, 135-140, 140-145, 145-150, 150-155, 155-160, 160-165, 165-170, 170-175, 175-180, 180-185, 185-190, 190-195, and 195-200 mg / ml. Exemplary, non-limiting concentrations of ECM also include approximately 25 mg / ml, 100 mg / ml, and 150 mg / ml. In one non-limiting example, the acoustically treated ECM hydrogel contains ECM in concentrations of approximately 25 mg / ml to approximately 150 mg / ml. In one embodiment, the ECM concentration is approximately 100 mg / ml.

[0166] In some embodiments, when the ECM concentration is approximately 150 mg / mL, the acoustically treated ECM hydrogel is at approximately 1400 Pa at 15°C. * It has a viscosity of s and a viscosity of approximately 400 Pa at a temperature of 25°C. * It has a viscosity of s. In another embodiment, when the concentration of ECM is about 150 mg / mL, the acoustically treated ECM hydrogel has a viscosity of about 2700 Pa at 15°C. * At 25°C, the pressure is approximately 800 Pa. * s, and 600 Pa at 37°C * It has a storage modulus of s.

[0167] The acoustically treated ECM hydrogel in liquid phase can be placed in a three-dimensional mold before cooling, or spread onto a TEFLON® sheet to form a film. The high concentration of ECM (50-600 mg / ml) in the acoustically treated ECM hydrogel allows for the formation of very thin sheets, e.g., sheets as thin as 4 microns. The acoustically treated ECM hydrogel can be configured to any size greater than 4 microns in any two-dimensional or three-dimensional shape. In some embodiments, sheets with a thickness of about 4 to about 10 microns are formed, e.g., sheets with a thickness of about 4, 5, 6, 7, 8, 9, or 10 microns. The acoustically treated ECM hydrogel can be formed into any three-dimensional shape, without limitation, including cylinders, spheres, ellipsoids, disks, sheets, cubes, cuboids, cones, triangular prisms or prisms with rectangular bases, as well as hollow spheres, hollow ellipsoids and open-end hollow cylinders, etc. The acoustically treated ECM hydrogel can also be used as an injectable by placing it in a syringe and extruding it from the syringe in either the gel or sol phase.

[0168] In some embodiments, the mammalian acoustically treated ECM hydrogel contains solubilized ECM at a concentration higher than approximately 0.1 mg / ml. The mammalian acoustically treated ECM hydrogel may contain solubilized ECM at a concentration ranging from approximately 0.1 mg / ml to approximately 1,000 mg / ml. Appropriate concentrations also include solubilized ECM at concentrations of approximately 1 mg / ml to 1,000 mg / ml, 1 mg / ml to 500 mg / ml, 1 mg / ml to 300 mg / ml, 1 mg / ml to 200 mg / ml, 1 mg / ml to 100 mg / ml, 10 mg / ml to 100 mg / ml, 10 mg / ml to 200 mg / ml, 100 mg / ml to 500 mg / ml, 50 mg / ml to 150 mg / ml, 20 mg / ml to 70 mg / ml, 4 mg / ml to 20 mg / ml, or 40 mg / ml to 66 mg / ml. Mammalian acoustically treated ECM hydrogels may contain solubilized ECM at concentrations of approximately 10 mg / ml to 500 mg / ml in a liquid, for example, a buffer solution. Mammalian acoustically treated ECM hydrogels may contain solubilized ECM in concentrations of 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 mg / ml. Exemplary concentrations include solubilized ECM in concentrations of approximately 20 mg / ml, 40 mg / ml, 66 mg / ml, 70 mg / ml, and 150 mg / ml. In a non-limiting example, mammalian acoustically treated ECM hydrogels may contain solubilized ECM in concentrations ranging from approximately 20 mg / ml to approximately 70 mg / ml. In a non-limiting example, mammalian acoustically treated ECM hydrogels contain solubilized ECM at concentrations of approximately 40 mg / ml or approximately 66 mg / ml. In a non-limiting example, mammalian acoustically treated ECM hydrogels contain solubilized ECM at concentrations of approximately 10 mg / ml to approximately 100 mg / ml. In a non-limiting example, mammalian acoustically treated ECM hydrogels contain solubilized ECM at concentrations of approximately 50 mg / ml to approximately 150 mg / ml. In a non-limiting example, mammalian acoustically treated ECM hydrogels contain solubilized ECM at concentrations of approximately 10 mg / ml to approximately 200 mg / ml.In a non-limiting example, mammalian acoustically treated ECM hydrogels contain solubilized ECM at concentrations ranging from approximately 10 mg / ml to approximately 500 mg / ml.

[0169] Exemplary concentrations include solubilized ECM at approximately 20 mg / ml, 40 mg / ml, 66 mg / ml, 70 mg / ml, and 150 mg / ml. In a non-limiting example, mammalian acoustic-treated ECM hydrogels contain approximately 20 mg / ml to approximately 70 mg / ml of solubilized ECM. In a non-limiting example, mammalian acoustic-treated ECM hydrogels contain approximately 40 mg / ml or approximately 66 mg / ml of solubilized ECM.

[0170] In some embodiments, the mammalian acoustically treated ECM hydrogel contains solubilized ECM at concentrations ranging from about 25 mg / ml to about 600 mg / ml. In further embodiments, the mammalian acoustically treated ECM hydrogel contains solubilized ECM at concentrations ranging from about 20 mg / ml to about 600 mg / ml, about 25 to about 500 mg / ml, about 25 to about 400 mg / ml, about 25 mg / ml to about 300 mg / ml, about 25 mg / ml to about 200 mg / ml, and about 25 mg / ml to about 150 mg / ml. In additional embodiments, the mammalian acoustically treated ECM hydrogel contains solubilized ECM at concentrations ranging from about 50 mg / ml to about 600 mg / ml. Mammalian acoustically treated ECM hydrogels may also contain solubilized ECM at concentrations of approximately 50 mg / ml to 300 mg / ml, 50 mg / ml to 200 mg / ml, 50 mg / ml to 150 mg / ml, 50 to 100 mg / ml, or 100 to 150 mg / ml. In some non-limiting examples, mammalian acoustically treated ECM hydrogels may contain approximately 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-105, 105-110, 110-115, and 115. Contains solubilized ECM at concentrations of ~120, 120~125, 125~130, 130~135, 135~140, 140~145, 145~150, 150~155, 155~160, 160~165, 165~170, 170~175, 175~180, 180~185, 185~190, 190~195, and 195~200 mg / ml.

[0171] In some embodiments, a composition comprising mammalian acoustic treatment ECM hydrogel and trehalose is produced. In additional embodiments, a composition comprising about 0.1 mg / ml to about 700 mg / ml of trehalose is used. In some embodiments, the composition comprises about 1 mg / ml to about 700 mg / ml of trehalose. In further embodiments, the composition comprises 50 mg / ml to about 500 mg / ml of trehalose. In other embodiments, the composition comprises about 10 mg / ml to about 600 mg / ml of trehalose, about 10 mg / ml to about 500 mg / ml, about 10 mg / ml to about 400 mg / ml, about 10 mg / ml to about 300 mg / ml, about 10 mg / ml to about 200 mg / ml, or about 10 mg / ml to about 100 mg / ml of trehalose. In further embodiments, the composition may contain about 0.1 to about 100 mg / ml of trehalose, about 0.1 to about 10 mg / ml of trehalose, or about 0.1 to about 1 mg / ml of trehalose. In additional embodiments, the composition may contain about 50 mg / ml to about 400 mg / ml of trehalose, about 50 mg / ml to about 300 mg / ml of trehalose, about 50 mg / ml to about 200 mg / ml of trehalose, or about 50 mg / ml to about 100 mg / ml of trehalose. In some embodiments, the composition contains about 20 mg / ml to about 70 mg / ml of trehalose. In some embodiments, the composition contains about 10 mg / ml to about 100 mg / ml of trehalose. In some embodiments, the composition contains 15 to 30 mg / ml of trehalose. In some embodiments, the composition contains 60 to 70 mg / ml of trehalose. In some embodiments, the composition contains 20 mg / ml of trehalose. In some embodiments, the composition contains 66 mg / ml of trehalose. In other embodiments, the composition may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 66, 70, 80, 90, 100, 200, 300, 400, 500, or 600 mg / ml of trehalose. In other embodiments, the composition contains about 100 mg / ml to about 700 mg / ml of trehalose, for example, about 100, 150, 20, 250, 300, 350, 400, 450, 500, 550, or 600 mg / ml of trehalose.In additional embodiments, the composition may contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / ml of trehalose.

[0172] In additional embodiments, the composition comprises a mammalian acoustically treated ECM hydrogel, comprising solubilized ECM, further pulverized mammalian ECM, and optionally trehalose. The pulverized ECM is not ultrasonically treated and is not solubilized into a hydrogel. The pulverized ECM is a separate additive to the composition, which also comprises a mammalian ECM hydrogel. The composition may contain about 1 to about 30 wt / volume (w / v)% of unsolubilized pulverized ECM in the acoustically treated ECM hydrogel. While not bound by theory, pulverized ECM generally has intact collagen particles, whereas the acoustically treated ECM hydrogel has ultrasonically disrupted collagen, resulting in an increase in soluble collagen content (the whole of which is incorporated herein by reference Hussey et al., Ultrasonic cavitation to prepare ECM hydrogels Acta Biomater. 2020 May;108:77-86). Thus, the acoustically treated ECM hydrogel composition containing further pulverized mammalian ECM includes both intact collagen and disrupted collagen.

[0173] The composition may contain approximately 5% to approximately 30% w / v, approximately 10% to approximately 30%, approximately 15% to approximately 30%, approximately 20% to approximately 30%, approximately 25% to approximately 30%, approximately 1% to approximately 20%, approximately 5% to approximately 20%, approximately 10% to approximately 20%, approximately 15% to approximately 20%, approximately 10% to approximately 20%, or approximately 15% to approximately 20% of ground ECM (w / v). The composition may contain approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30% of ground ECM (w / v). The composition may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30% or less of ground ECM (w / v). The composition may contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30% of ground ECM (w / v).

[0174] The pulverized ECM may originate from the same species as the mammalian acoustically treated ECM hydrogel. In one specific, non-limiting example, both the mammalian acoustically treated ECM hydrogel and the pulverized ECM may be from pigs. In another non-limiting example, both the mammalian acoustically treated ECM hydrogel and the pulverized ECM may be from humans.

[0175] The pulverized ECM may be derived from the same or a different tissue as the mammalian acoustically treated ECM hydrogel. In one embodiment, the mammalian acoustically treated ECM hydrogel and the pulverized ECM are derived from the same tissue. In one embodiment, the mammalian acoustically treated ECM hydrogel and the pulverized ECM are dermal ECM. In one embodiment, the mammalian acoustically treated ECM hydrogel and the pulverized ECM are porcine dermal ECM.

[0176] The composition may be sterilized before application to the target. The composition may be sterilized using any method known to those skilled in the art, including filtration and radiation. In some embodiments, the composition is sterilized using ionizing radiation, e.g., e-beam or gamma radiation. The composition may be sterilized using gamma radiation, for example, the composition is sterilized using irradiation of 10–50 kGy, e.g., 15–45 kGy, 20–40 kGy, or 10–30 kGy. In some non-limiting examples, the composition is sterilized using irradiation of 10, 15, 20, 25, 30, 35, 40, 45, or 50 kGy. Generally, the composition is sterilized for a time sufficient to achieve the absence of detectable viable pathogens, e.g., viruses and bacteria, but not limited to these.

[0177] Preparation of enzymatic ECM hydrogels Methods for preparing ECM hydrogels are disclosed above in this specification and are also disclosed in, for example, U.S. Patent No. 8,361,503, the contents of which are hereby incorporated by reference herein for all purposes. Any type of extracellular matrix tissue can be used to produce hydrogels that can be used in the methods disclosed herein (see U.S. Patents related to ECM: No. 4,902,508; No. 4,956,178; No. 5,281,422; No. 5,352,463; No. 5,372,821; No. 5,554,389; No. 5,573,784; No. 5,645,860; No. 5,771,969; No. 5,753,267; No. 5,762,966; No. 5,866,414; No. 6,099,567; No. 6,485,723; No. 6,576,265; No. 6,579,538; No. 6,696,270; No. 6,783,776; No. 6,793,939; No. 6,849,273; No. 6,852,339; No. 6,861,074; No. 6,887,495; No. 6,890,562; No. 6,890,563; No. 6,890,564; and No. 6,893,666). In certain embodiments, the ECM is isolated from vertebrates, such as, without limitation, warm-blooded mammalian vertebrates including, but not limited to, humans, monkeys, horses, pigs, cows, and sheep. In specific non-limiting examples, the ECM is from a pig or a human.

[0178] The ECM can be derived from any organ or tissue including, without limitation, the bladder, intestine, liver, esophagus, and dermis. For example, the ECM can be derived from the bladder, small intestine, heart, dermis, liver, kidney, uterus, brain, blood vessels, lung, bone, muscle, pancreas, placenta, stomach, spleen, colon, adipose tissue, and / or esophagus. The ECM can be obtained from cell cultures. In one embodiment, the ECM is isolated from the bladder. In another embodiment, the ECM is of esophageal origin. The ECM may or may not contain the basement membrane portion of the ECM. In certain embodiments, the ECM contains at least a portion of the basement membrane.

[0179] In some embodiments, as in U.S. Patent No. 8,361,503 (thereby incorporated herein by reference), bladder ECM, for example, porcine bladder ECM, is prepared by exfoliating the bladder tissue to remove the outer layers, including both the serosal and muscular layers, using a longitudinal wiping motion with a scalpel handle and moistened gauze. After inversion of the tissue segments, the luminal portion of the mucosal layer is exfoliated from the underlying tissue using the same wiping motion. In some embodiments, perforation of the submucosa is prevented. After these tissues are removed, the resulting ECM consists mainly of the submucosa layer. The production of hydrogels from decellularized dermal ECM is disclosed in Wolf et al., Biomaterials 33: 7028-7038, 2012 (thereby incorporated herein by reference). The production of ECM from esophageal tissue is disclosed, for example, in Badylak et al. J Pediatr Surg. 35(7):1097-103, 2000 and Badylak et al., J Surg Res. 2005 September; 128(1):87-97, 2005, both of which are incorporated herein by reference. U.S. Patent No. 6,893,666, also incorporated herein by reference, discloses the production of ECM from the bladder, skin, esophagus, and small intestine.

[0180] Commercially available ECM preparations may also be used in the methods, devices, and compositions described herein. In one embodiment, the ECM is derived from the submucosa of the small intestine or SIS. Commercially available preparations include, but are not limited to, SURGISIS®, SURGISIS-ES®, STRATASIS® and STRATASIS-ES® (Cook Urological Inc.; Indianapolis, Ind.) and GRAFTPATCH® (Organogenesis Inc.; Canton Mass.). In another embodiment, the ECM is derived from the dermis. Commercially available preparations include, but are not limited to, PELVICOL® (marketed as PERMACOL® in Europe; Bard, Covington, Ga.), REPLIFORM® (Microvasive; Boston, Mass.) and ALLODERM® (LifeCell; Branchburg, NJ). In yet another embodiment, the ECM is derived from the bladder. Commercial preparations include, but are not limited to, UBM (Acell Corporation; Jessup, Md.).

[0181] Tissue for the preparation of ECM can be recovered in a wide variety of ways, and once recovered, various parts of the recovered tissue can be used. ECM has also been prepared from the esophagus and small intestine, and hydrogels have been prepared from this ECM. See, for example, Keane et al., Tissue Eng. Part A, 21(17-18): 2293-2300, 2015, which is incorporated herein by reference. Esophageal ECM can be prepared by mechanically separating the mucosa and submucosa from the muscularis exostata, digesting the mucosa in a trypsin-containing buffer, and then exposing it to sucrose, TRITON-X100®, deoxycholic acid, peracetic acid, and DNAse. Small intestinal submucosa (SIS) can be prepared by mechanically removing the superficial layers of the mucosa, serosal layer, and muscularis exostata from the intact small intestine, leaving the submucosa, muscularis mucosa, and basal compacta intact. Next, the SIS is treated with peracetic acid. An exemplary protocol is provided in Keane et al. Dermal hydrogels can be produced, for example, as disclosed in Wolf et al, J Biomed Mater Res A. 2013. 35(25):6838-49. PMID: 23873846. PMCID: 3808505, which is thus incorporated herein by reference.

[0182] In one embodiment, the extracellular matrix (ECM) is isolated from a recovered pig bladder to prepare the bladder matrix (UBM). Excess connective tissue and residual urine are removed from the bladder. Most of the serosal layer, muscular layer, submucosal layer, and muscularis mucosa can be removed by mechanical exfoliation or by a combination of enzymatic treatment, hydration, and exfoliation. Mechanical removal of these tissues can be achieved by exfoliation using longitudinal wiping motions to remove the outer layers (particularly the anti-luminal smooth muscle layer) and even the luminal portion of the mucosal layer (epithelial layer). Mechanical removal of these tissues can be achieved, for example, by removing the mesenteric tissue with Adson-Brown forceps and Metzenbaum scissors, and by wiping the muscular and submucosal layers using longitudinal wiping motions with a scalpel handle wrapped in moistened gauze or other hard object. Epithelial cells of the mucosal layer can also be dissociated by immersing the tissue in an epithelializing solution, e.g., hypertonic saline, without limitation. The resulting UBM comprises the basement membrane and adjacent lamina propria of the mucosal layer, which is further treated with peracetic acid, freeze-dried, and powdered. See U.S. Patent No. 8,361,503 incorporated herein by reference.

[0183] Dermal sections may be used for the preparation of an enzymatic ECM hydrogel. See PCT application number 2015 / 15164728 incorporated herein by reference. In a specific, non-limiting example, the dermis can be decellularized using 0.25% trypsin / 1% Triton® X-100 (i.e., without SDS) in the following solutions at room temperature in a vortex shaker at 300 RPM: 0.25% trypsin for 6 hours, 1×; deionized water, 15 minutes, 3×; 70% ethanol, 10-12 hours, 1×; 3% H2O2, 15 minutes, 1×; deionized water, 15 minutes, 2×; 1% Triton X-100 in 0.26% EDTA / 0.69% Triton for 6 hours, 1× and then overnight, 1×; deionized water, 15 minutes, 3×; 0.1% peracetic acid / 4% ethanol, 2 hours, 1×; PBS, 15 minutes, 2×; and finally deionized water, 15 minutes, 2×. Next, the dermal sheets are freeze-dried, and then processed into granular form using a Waring blender and Wiley Mill with a #20 mesh screen.

[0184] In some embodiments, epithelial cells may be initially delaminated by immersing the tissue in an epithelial delamination solution, such as hypertonic saline, for a period ranging from 10 minutes to 4 hours, without limitation. Exposure to hypertonic saline efficiently removes epithelial cells from the underlying basement membrane. The tissue remaining after the initial delamination procedure includes the epithelial basement membrane and the tissue layers antiluminal to the epithelial basement membrane. This tissue is then subjected to further processing that removes most of the antiluminal tissue but not the epithelial basement membrane. Most of the outer serosa, adventitia, smooth muscle tissue, submucosa, and muscularis mucosa are removed from the remaining deepithelialized tissue by mechanical delamination or by a combination of enzymatic treatment, hydration, and delamination.

[0185] ECM can be sterilized by several standard techniques, including but not limited to exposure to peracetic acid, low-dose gamma radiation, gas plasma sterilization, ethylene oxide treatment, or electron beam treatment. More typically, ECM sterilization is achieved by immersion in 0.1% (v / v) peracetic acid, 4% (v / v) ethanol, and 95.9% (v / v) sterile water for two hours. The peracetic acid residue is removed by washing twice for 15 minutes each with PBS (pH=7.4) and twice for 15 minutes each with sterile water. ECM materials can be sterilized by propylene oxide or ethylene oxide treatment, gamma irradiation (0.05–4 mRad), gas plasma sterilization, peracetic acid sterilization, or electron beam treatment. ECM can also be sterilized by treatment with glutaraldehyde, which causes crosslinking of protein materials, but this treatment substantially alters the material to drive a different type of host remodeling, which is more similar to scar tissue formation or encapsulation rather than constructive remodeling, where it is slowly reabsorbed or not reabsorbed at all. Crosslinking of protein materials can also be induced by carbodiimide, thermal dehydration, or photooxidation. As disclosed in U.S. Patent No. 8,361,503, ECM is disinfected by immersion for 2 hours in 0.1% (v / v) peracetic acid (a), 4% (v / v) ethanol, and 96% (v / v) sterile water. The ECM material is then washed twice for 15 minutes each with PBS (pH=7.4) and twice for 15 minutes each with deionized water.

[0186] Following the isolation of the target tissue, decellularization is carried out by various methods, for example, by exposure to hypertonic saline, peracetic acid, TRITON-X®, or other detergents, without limitation. Sterilization and decellularization may occur simultaneously. For example, sterilization with peracetic acid, without limitation, may also function to decellularize the ECM. The decellularized ECM may then be dried by either lyophilization (freeze-drying) or air-drying. The dried ECM may be pulverized by methods including, but not limited to, tearing, milling, cutting, crushing, and shearing. The pulverized ECM may also be further processed into a powder form by methods, for example, by crushing or milling while frozen or freeze-dried, without limitation. To prepare a solubilized ECM tissue, the pulverized ECM is digested with an acidic protease in an acidic solution to form a digest solution. The acidic protease may be, for example, trypsin and / or pepsin, or a combination thereof.

[0187] In one embodiment, the decellularized ECM material is partially digested by an acidic protease. In one example, the decellularized ECM material is not as completely digested compared to the digestion of 1 mg / mL lyophilized powdered ECM material over 48 hours with 1 mg / mL pepsin in 0.01 M HCl. In another example, the decellularized ECM material is not as completely digested compared to the digestion of 10 mg / mL lyophilized powdered ECM material over 48 hours with 1 mg / mL pepsin in 0.01 M HCl. In a further embodiment, hyaluronic acid in the ECM material is digested by 50%, 40%, 30%, 25%, 20%, or less than 10% compared to undigested ECM material. See PCT application number WO2015 / 164728 incorporated herein by reference.

[0188] ECM digest solutions are typically maintained at room temperature with constant stirring for a certain amount of time. ECM digests can be used immediately, stored at -20°C, or frozen at -20°C or -80°C, for example, without limitation. Thus, ECM digests can be maintained in a solubilized form. Methods for maintaining hydrogels in a solubilized form are disclosed, for example, in PCT application no. PCT / US16 / 52261, filed September 10, 2016, which is thus incorporated herein by reference.

[0189] When ECM is solubilized (typically substantially completely), the pH of the solution is raised to between 7.2 and 7.8, and according to one embodiment, to pH 7.4. The pH can be raised to approximately 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8. Bases, such as those containing hydroxyl ions, including NaOH, can be used to raise the pH of the solution. Similarly, buffers, such as isotonic buffers, including without limitation phosphate-buffered saline (PBS), can be used to bring the solution to a target pH or to help maintain the pH and ionic strength of the gel at target levels, e.g., physiological pH and ionic conditions. This forms a “pregel” solution, which is a solubilized ECM hydrogel. The neutralized digest solution (pregel, solubilized ECM hydrogel) can gel at the lower critical solution temperature. See PCT publication number 2015 / 164728 incorporated herein by reference.

[0190] ECM hydrogels form a gel (sol-to-gel transition) with increasing temperature. The lower critical solution temperature (LCST) in a reverse gel is the temperature below which the reverse-gelling polymer is soluble in its solvent (e.g., water or aqueous solvent). When the temperature rises above the LCST in a reverse gel, a hydrogel is formed. The general concepts of reverse gelation of polymers and their relationship to the LCST are widely known in the field of chemistry. The ECM gels described herein are prepared, for example, from decellularized intact ECM as described below, by digestion of the ECM material with an acidic protease, neutralization of the material to form a pregel, and raising the temperature of the pregel to a temperature above the LCST of the pregel to gel the pregel, for example, to form a hydrogel. With respect to acidic protease-digested materials, the transition temperature from solution to gel is typically in the range of 10°C to 40°C and any increment or range between them, e.g., 20°C to 35°C. For example, Pregel can be heated to 37°C to form a hydrogel. For example, Pregel.

[0191] Therefore, the extracellular matrix (ECM) can typically be derived from mammalian tissue, for example, without limitation, one of the bladder, esophagus, or small intestine. In a specific, non-limiting example, the ECM is derived from the bladder. According to one embodiment, the decellularized ECM material prepared from the tissue is not dialyzed before partial or complete digestion by an acidic protease, and / or after digestion by an acidic protease and before gelation of the neutralized digested ECM material.

[0192] In one non-limiting embodiment, the ECM is freeze-dried and pulverized. The ECM is then solubilized with an acidic protease in an acidic solution to produce digested ECM, e.g., bladder ECM. The acidic protease may be pepsin, trypsin, or a combination thereof, without limitation. The ECM can then be solubilized in a 0.01 M HCl solution, for example, at an acid pH suitable or optimal for the protease, e.g., a pH higher than about pH 2, or between pH 2 and 4. The ECM is typically solubilized over about 12 to about 48 hours using mixing (stirring, agitation, blending, mixing, rotating, tilting, etc.), depending on the tissue type (see, for example, the following examples). ECM hydrogels are prepared by (i) pulverizing the extracellular matrix, (ii) solubilizing intact non-dialysis or non-crosslinked extracellular matrix by digestion with an acidic protease in an acidic solution to produce a digested solution, (iii) raising the pH of the digested solution to between 7.2 and 7.8 to produce a neutralized digested solution (pregel solution), and (iv) gelling the solution.

[0193] Accordingly, a composition containing exogenous MBV of ECM enzymatically digested by an acidic protease in an acidic solution is disclosed. For example, when neutralized to pH 7.0-7.8 and warmed to about 37°C, the composition forms a gel and the protease is inactivated. In one embodiment, the exogenous MBV does not originate from bone tissue or heart tissue. In a further embodiment, the concentration of exogenous MBV in the composition is higher than 5 mg / mL.

[0194] For example, a composition of enzymatically digested ECM in a neutral solution of pH 7.0-7.8 is also disclosed, the solution containing an inactivated acid protease, e.g., inactivated pepsin and / or trypsin, or another inactivated acid protease suitable in its active form for digesting ECM; the composition also contains exogenous MBV. When the solution is heated to about 37°C, it forms a gel. In one embodiment, the exogenous MBV is not derived from bone tissue or heart tissue. In a further embodiment, the concentration of exogenous MBV in the composition is higher than 5 mg / mL. The acid protease may be inactivated or deactivated, for example, due to a change in pH.

[0195] In a further embodiment, the ECM hydrogel may be centrifuged to collect the soluble fraction. An exemplary method for fractionating an ECM hydrogel is disclosed, for example, in PCT publication number WO2015 / 164728, which is thus incorporated herein by reference. The method disclosed in this PCT publication comprises the steps of: partially or completely digesting a decellularized ECM material prepared from tissue with an acidic protease, such as pepsin; neutralizing the digested ECM material to a pH of 7.0–8.0, 7.2–7.8, or 7.4; gelling the neutralized digested ECM material at a temperature above its lower critical solution temperature; centrifuging the gelled ECM material to produce a pellet and a supernatant; and separating the supernatant and pellet to separate the structural and soluble fractions of the ECM material.

[0196] ECM hydrogels form a gel when exposed to temperatures above the lower critical solution temperature, for example, about 37°C. ECM hydrogels in “pre-gel” form (solubilized ECM hydrogels) can be frozen and stored at, for example, -20°C or -80°C, without limitation. ECM hydrogels in “pre-gel” form can be stored at room temperature, for example, about 25°C. In some non-limiting examples, ECM hydrogels are in pre-gel form at temperatures below 37°C, for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, and 4°C. ECM hydrogels can be frozen for storage and therefore stored at temperatures below 0°C. As used herein, the terms “pre-gel form” or “pre-gel” refer to an ECM hydrogel whose pH is increasing but which has not gelled. For example, without limitation, ECM hydrogels in pregel form have a pH between 7.2 and 7.8. In some embodiments, solubilized ECM hydrogels are used in the methods disclosed herein. Methods for maintaining the hydrogel in a solubilized form are disclosed, for example, in PCT application number PCT / US16 / 52261, filed September 10, 2016, which is thus incorporated herein by reference. In some embodiments, ECM compositions prepared by any of the methods described herein are absorbed into a biocompatible substrate, adsorbed onto a biocompatible substrate, or dispersed on or in a biocompatible substrate by other means. Non-limiting examples of biocompatible substrates include: meshes, nonwovens, decellularized tissues, polymer compositions, polymeric structures, cell growth scaffolds, implants, orthopedic implants, and intraocular lenses, sutures, intravascular implants, stents, and grafts. The compositions described herein may be applied to or incorporated into nonwoven materials, such as bandages, sutures, implants, such as ceramic, metallic, or polymeric implants, such as orthotics, artificial or otherwise modified tubes, valves, intraocular lenses, or tissue implants, by any suitable method.As used herein, the term “coating” and related cognates, such as “coated” and “to coat,” refer to a process that involves partially or entirely covering an inorganic structure with a composition described herein. For example, coating an inorganic structure with a solubilized fraction may include methods such as pouring, embedding, layering, dipping, and spraying. Ultrasonication may be used to assist in the coating of an inorganic structure.

[0197] The composition used comprises an ECM hydrogel, which is an "enzymatic" ECM hydrogel containing exogenous MBV. Exogenous MBV is added to enrich the bioactive properties of the ECM hydrogel and to improve its therapeutic efficacy in reducing inflammation and enhancing tissue growth and repair when administered to or implanted in a subject. The enzymatic ECM hydrogel is prepared from solubilized ECM. To prepare the solubilized ECM tissue, the pulverized ECM is digested with an acidic protease in an acidic solution to form a digested solution. As used herein, the term "acidic protease" refers to an enzyme that cleaves peptide bonds, which has increased activity in cleaving peptide bonds at an acidic pH. For example, without limitation, acidic proteases may include pepsin and trypsin. In one embodiment, the ECM is lyophilized before pulverization.

[0198] ECM digest solutions are typically maintained with constant agitation for a certain amount of time at room temperature. ECM digests can be used immediately, stored at -20°C, or frozen at -20°C or -80°C, for example, without limitation. To form a "pregel" solution, the pH of the digest solution is raised to between 7.2 and 7.8. The pH can be raised by adding one or more bases or isotonic buffer solutions, for example, without limitation, NaOH, or PBS at pH 7.4. This method typically does not involve a pre-gelling dialysis step and produces a more complete ECM-like matrix that typically gels more slowly than comparable collagen or dialyzed ECM preparations at 37°C. Therefore, the gel is more suitable for injection into the patient and also retains more of the attributes of native ECM due to the retention of many native soluble factors, for example, cytokines, without limitation.

[0199] As used herein, the term “isotonic buffer solution” refers to a solution that is buffered to a pH between 7.2 and 7.8 and has an equilibrium concentration of salt to promote an isotonic environment. As used herein, the term “base” refers to any compound or solution of a compound having a pH higher than 7. For example, without limitation, a base is an alkaline hydroxide or an aqueous solution of an alkaline hydroxide. In certain embodiments, a base is NaOH or NaOH in PBS.

[0200] The “Pregel” solution can be incubated at a moderately warm temperature, for example, about 37°C, without limitation, to gel at that point. Pregel can be frozen and stored at -20°C or -80°C, for example, without limitation. As used herein, the terms “Pregel solution” or “Pregel” refer to a digested solution with increased pH. For example, Pregel has a pH between 7.2 and 7.8. The ECM hydrogel composition may contain an inactivated acidic protease. The ECM hydrogel composition may have a pH between 7.2 and 7.8. “Pregel” may contain exogenous MBV. In one embodiment, the exogenous MBV does not originate from the cardiac ECM or the bone ECM.

[0201] ECM hydrogels, digest solutions, or pregels may contain solubilized ECM at concentrations between 1 mg / mL and 500 mg / mL. In some embodiments, the amount of solubilized ECM in the ECM hydrogel, digest solution, or pregel is between 1 mg / mL and 400 mg / mL, for example, 1 mg / mL to 350 mg / mL, or 1 mg / mL to 300 mg / mL, or 1 mg / mL to 250 mg / mL, or 1 mg / mL to 200 mg / mL, or 1 mg / mL to 150 mg / mL, or 1 mg / mL to 100 mg / mL, or 1 mg / mL to 50 mg / mL, or 5 mg / mL to 250 mg / mL, or 20 mg / mL to 200 mg / mL, or 5 mg / mL to 200 mg / mL, or 5 mg / mL to 100 mg / mL. In additional embodiments, the amount of solubilized ECM in the ECM hydrogel, digest solution, or pregel is between approximately 5 mg / ml and approximately 50 mg / ml, for example, approximately 10 mg / ml to approximately 50 mg / ml, approximately 20 mg / ml to approximately 50 mg / ml, for example, approximately 30 mg / ml to approximately 50 mg / ml, approximately 40 mg / ml to approximately 50 mg / ml, approximately 5 mg / ml to approximately 40 mg / ml, approximately 5 mg / ml to approximately 30 mg / ml, approximately 5 mg / ml to approximately 20 mg / ml, or approximately 5 mg / ml to approximately 10 mg / ml. For example, an ECM hydrogel, digest solution, or pregel may contain solubilized ECM at concentrations of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / ml. In one non-limiting example, the amount of solubilized ECM in the ECM hydrogel, digest solution, or pregel is between 10 mg / mL and 30 mg / mL. In another non-limiting example, the amount of solubilized ECM in the ECM hydrogel, digest solution, or pregel is between 1 mg / mL and 20 mg / mL. In yet another non-limiting example, the amount of solubilized ECM in the ECM hydrogel, digest solution, or pregel is between 4 mg / mL and 20 mg / mL. In another non-limiting example, the amount of solubilized ECM in the ECM hydrogel, digest solution, or pregel is between 1 mg / mL and 50 mg / mL.In some embodiments, the amount of solubilized ECM in the ECM hydrogel, digest solution, or pregel is about 5 to about 100 mg / mL, for example, 50 to 100 mg / mL, 25 to 75 mg / mL, 60 to 80 mg / mL, 40 to 60 mg / mL, 50 to 80 mg / mL, or about 30 to about 60 mg / mL.

[0202] Preparation of ECM hydrogel containing exogenous MBV Exogenous MBV may be added to ECM hydrogels (or pregels) disclosed herein, such as enzymatic hydrogels or acoustically treated hydrogels. Exogenous MBV may be present in ECM hydrogels at concentrations of less than 1 mg / mL. For example, MBV may be present at least about 1 × 10⁻⁶ 5 ~Approx. 1×10 20 It can be present in the ECM hydrogel at a concentration of particles / mL. In some embodiments, exogenous MBV is approximately 1 × 10⁶ 5 ~Approx. 1×10 20 particles / mL, for example, about 1 × 10⁶ 5 ~Approx. 1×10 18 particles / mL, for example, about 1 × 10⁶ 5 ~Approx. 1×10 16 particles / mL, for example, about 1 × 10⁶ 5 ~Approx. 1×10 14 particles / mL, or, for example, about 1 × 10⁶ 5 ~Approx. 1×10 12 It is present in the ECM hydrogel (or pregel) at a concentration of particles / mL. In some embodiments, exogenous MBV is approximately 1 × 10⁶ 6 ~Approx. 1×10 20 particles / mL, for example, about 1 × 10⁶ 6 ~Approx. 1×10 18 particles / mL, for example, about 1 × 10⁶ 6 ~Approx. 1×10 16 particles / mL, for example, about 1 × 10⁶ 6 ~Approx. 1×10 14 particles / mL, for example, about 1 × 10⁶ 6 ~Approx. 1×10 12 particles / mL, for example, about 1 × 10⁶ 7 ~Approx. 1×10 12 particles / mL, for example, about 1 × 10⁶ 7 ~Approx. 1×10 11particles / mL, for example, about 1×10 8 ~ about 1×10 12 particles / mL, for example, about 1×10 8 ~ about 1×10 11 particles / mL, for example, about 1×10 9 ~ about 1×10 12 particles / mL, for example, about 1×10 9 ~ about 1×10 11 particles / mL, for example, about 1×10 10 ~ about 1×10 12 particles / mL, for example, about 1×10 10 ~ about 1×10 11 particles / mL, for example, about 1×10 11 ~ about 1×10 12 It is present in the ECM hydrogel (or pregel) at a concentration of particles / mL. In a non-limiting example, exogenous MBV is about 1×10 8 ~ 1×10 11 particles / mL. In a non-limiting example, exogenous MBV is about 1×10 5 ~ 1×10 12 particles / mL. In a non-limiting example, exogenous MBV is about 1×10 6 ~ 1×10 12 It is present in the ECM hydrogel (or pregel) disclosed herein at a concentration of particles / mL. In some embodiments, exogenous MBV is about 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 14 , 1×10 16 , 1×10 18 [[ID=6º]]or about 1×10 20 particles / mL. In other embodiments, exogenous MBV is about 5×10 6 , 5×10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 or about 5×10 12It is present in the ECM hydrogel (or pregel) at a concentration of particles / mL. In a specific, non-limiting example, exogenous MBV is approximately 1 × 10⁶ 11 It is present in the ECM hydrogel (or pregel) at a concentration of particles / mL. In another non-limiting example, exogenous MBV is approximately 1 × 10⁶ 12 It is present in the ECM hydrogel (or pregel) at a concentration of particles / mL. In further non-limiting examples, exogenous MBV is approximately 1 × 10⁶ 10 particles / mL or approximately 1 × 10⁶ 9 It is present in the ECM hydrogel (or pregel) at a concentration of particles / mL. In some embodiments, exogenous MBV is approximately 1 × 10⁶ 6 ~1 × 10 18 particles / mL, for example, about 1 × 10⁶ 6 ~1 × 10 14 , about 1×10 10 ~1 × 10 14 , 1 x 10 12 ~1 × 10 18 , 1 x 10 14 ~1 × 10 18 or 1 × 10 10 ~1 × 10 18 In another non-limiting example, exogenous MBV is present in the ECM hydrogel (or pregel) at concentrations less than 1 mg / mL, e.g., ≤0.9 mg / mL, ≤0.8 mg / mL, ≤0.7 mg / mL, ≤0.6 mg / mL, ≤0.5 mg / mL, ≤0.4 mg / mL, ≤0.3 mg / mL, ≤0.2 mg / mL, ≤0.1 mg / mL, ≤90 μg / mL, ≤80 μg / mL, ≤70 μg / mL, ≤60 μg / mL, ≤50 μg / mL, ≤40 μg / mL, ≤30 μg / mL, ≤20 μg / mL, or ≤10 μg / mL, but higher than 0 μg / mL, e.g., higher than 0.1 μg / mL, higher than 0.5 μg / mL, or higher than 1 μg / mL, in the ECM hydrogel (or pregel).

[0203] MBV can be added to a hydrogel, for example, before the enzymatic gelation of the hydrogel. For example, MBV can be added to an enzymatic hydrogel before raising the temperature above 25°C, for example, to 37°C. For example, MBV can be added to an acoustically treated hydrogel at any point after sonication of the ECM to produce the hydrogel.

[0204] Treatment method Various methods for treating subjects with muscle wasting diseases are disclosed herein. Various muscle wasting diseases are disclosed herein, and these methods may be used to treat any of the disclosed disorders. These methods involve using MBV directly administered to subjects with muscle wasting diseases, or using MBV in combination with cells to provide cells for adoptive transfer to subjects or to prepare conditioned media for administration to subjects resulting from cell cultures with MBV. In some examples, human subjects with muscle wasting diseases are selected and treated according to the methods disclosed herein.

[0205] Muscle wasting diseases include small muscular atrophy and muscular dystrophy, as well as other conditions that cause muscular atrophy. For example, the treatment methods disclosed herein may be used to treat spinal muscular atrophy ("SMA"), such as infantile progressive spinal muscular atrophy (Type I SMA), intermediate spinal muscular atrophy (Type II SMA), juvenile spinal muscular atrophy (Type III SMA), or adult spinal muscular atrophy (Type IV SMA). The treatment methods disclosed herein may also be used to treat muscular atrophy occurring in cachexia or sarcopenia. The treatment methods disclosed herein may also be used to treat muscular dystrophy (MD), such as Becker MD, congenital MD, Duchenne MD, distal MD, Emery-Dreyfus MD, facioscapulohumeral MD (FSHD), limb-girdle MD, myotonic MD, oculopharyngeal MD, Bethlem myopathy, or Ulrich congenital muscular dystrophy. For example, the disclosed methods may be used to treat FSHD. For example, the disclosed methods may be used to treat SMA. Subjects having SMA may be selected for treatment using the disclosed methods.

[0206] A method for promoting muscle regeneration or repair in a subject requiring it, wherein the subject is administered an effective amount of a composition comprising exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or CD63 lo CD81 lo A method is also disclosed in which MBV does not contain alkaline phosphatase.

[0207] MBV administration In some embodiments, a method for treating a subject having a state of muscle wasting comprises the step of administering to the subject a composition comprising an effective amount of exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or CD63 lo CD81 loA method is disclosed in which MBV does not contain alkaline phosphatase. In an additional embodiment, a method for promoting muscle regeneration or repair in a subject requiring it is provided to the subject with an effective amount of a composition comprising exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or CD63 lo CD81 lo A method is disclosed in which MBV does not contain alkaline phosphatase. In all of these embodiments, MBV may be prepared as disclosed herein.

[0208] MBV may be administered in ECM hydrogels disclosed herein, such as enzymatic hydrogels or acoustically treated hydrogels. Enzymatic hydrogels may be in a pregel state. Exogenous MBV contained in the hydrogel may be present in the hydrogel in the amounts described herein. ECM hydrogels containing MBV may be administered systemically, for example, intravenously. For example, ECM hydrogels containing MBV may be administered topically to muscles affected by atrophy or dystrophy, as well as any of the muscle wasting disorders described herein. The amount of hydrogel administration depends on the concentration of MBV in the hydrogel and the desired outcome in the patient, including the severity of the disease.

[0209] MBV can also be suspended in a pharmaceutically acceptable carrier. For example, MBV may be supplied in a carrier that can be isotonic, hypotonic, or hypertonic, depending on the needs of the subject, such as an equilibrium salt solution or physiological saline. For example, the salt solution may be a magnesium chloride solution, a sodium chloride solution, a potassium chloride solution, or a calcium chloride solution. The carrier may be lactated Ringer's solution, or a dextrose or other sugar solution. The carrier may be buffered to maintain the physiological pH in the subject, where applicable. The carrier may contain glycerol. MBV may be provided in a carrier at a concentration less than 1 mg / mL, for example, ≤0.9 mg / mL, ≤0.8 mg / mL, ≤0.7 mg / mL, ≤0.6 mg / mL, ≤0.5 mg / mL, ≤0.4 mg / mL, ≤0.3 mg / mL, ≤0.2 mg / mL, ≤0.1 mg / mL, ≤90 μg / mL, ≤80 μg / mL, ≤70 μg / mL, ≤60 μg / mL, ≤50 μg / mL, ≤40 μg / mL, ≤30 μg / mL, ≤20 μg / mL, or ≤10 μg / mL, but higher than 0 μg / mL, for example, higher than 0.1 μg / mL, higher than 0.5 μg / mL, or higher than 1 μg / mL.

[0210] The amount of solution administered depends on the concentration of MBV in the solution and the desired dose, and may be, for example, 100 microliters to 10 mL.

[0211] For example, administration of MBV in a pharmaceutically acceptable carrier or in a hydrogel prepared as described herein may be administered to subjects suffering from muscle wasting disorders, such as human subjects, to treat muscle wasting disorders. Muscle wasting disorders may be any of those disclosed herein, for example. Administration may be, for example, intravenous, intramuscular, or subcutaneous.

[0212] MBV may be administered to subjects daily, weekly, every two weeks, monthly (e.g., every 28 days), every two months (e.g., every 56 days), or every three months (e.g., every 84 days). The amount of MBV administered and the frequency of administration may be adjusted depending on the desired outcome in the subject and the type and severity of muscle wasting.

[0213] For example, the subject has approximately 1 x 10 1 ~Approx. 1×10 20 A dose of 1 MBV / kg of body weight is administered. For example, a subject may receive approximately 1 x 10⁶ units of MBV. 6 ~Approx. 1×10 20 The MBV per 1 kg of body weight is, for example, approximately 1 × 10 6 ~Approx. 1×10 12 The dosage is one MBV / kg of body weight / administered. In some cases, the subjects were given approximately 1 × 10⁶ units. 6 ~Approx. 1×10 19 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 18 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 17 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 16 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 15 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 14 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 13 1 MBV or approximately 1 x 10 6 ~Approx. 1×10 12 In other cases, the subjects receive approximately 1 × 10⁶ MBV / dose. 7 ~Approx. 1×10 11 The dose is 1 MBV / kg of body weight / administered. In another example, the subject receives 1 × 10⁶ 7 ~1 × 10 8 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 8 ~1 × 10 10 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 9 ~1 × 10 10A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 6 ~1 × 10 8 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 7 ~1 × 10 9 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 8 ~1 × 10 11 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 9 ~1 × 10 11 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 10 ~1 × 10 11 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 11 ~1 × 10 12 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 6 ~1 × 10 14 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 12 ~1 × 10 14 The MBV dose is administered at a rate of 1 MBV / kg of body weight / administered dose. In one embodiment, the administration of MBV according to any of the above amounts is by systemic administration. For example, in one embodiment, the administration is intravenous. In another embodiment, the administration is by, for example, a bolus subcutaneous injection into the muscle.

[0214] Adoption In some embodiments, a method is disclosed for treating a subject having a state of muscle wasting, comprising the step of administering to the subject a composition comprising an effective amount of myeloid progenitor cells or their offspring ("myeloid-derived cells") treated with exogenous MBV. In some embodiments, the myeloid-derived cells are macrophages. Macrophages are commensal phagocytic cells in lymphoid and non-lymphoid tissues that have a highly diverse range of roles in maintaining the biological integrity of an organism, from development and homeostasis to repair and immune responses to pathogens. Macrophages exert these functions through the clearance of cellular debris, the production of growth factors, highly efficient phagocytosis (particularly of tumor cells), and the production of inflammatory cytokines. Macrophages are typically classified into classically activating, pro-inflammatory, or M1 macrophages, and alternatively activating, anti-inflammatory, or M2 macrophages. In some embodiments, the myeloid cells used in the disclosed method are anti-inflammatory M2 macrophages.

[0215] M2 or anti-inflammatory macrophages (also called surrogate activated macrophages) can be induced by IL-4 or IL-13 secreted by innate and adaptive immune cells, such as mast cells, basophils, and TH-2 lymphocytes. M2a or activated macrophages typically express surface markers including CD206, CD36, ILHRa, and CD163; transcription factors including STAT6, GATA3, SOCS1, and PPARy; and metabolic enzymes including ARG1 and CARKL, and may secrete cytokines including but not limited to IL10 and TGFp. M2b or regulatory macrophages are typically induced by stimulation with immune complexes and TLR ligands, or by IL-1R agonists. M2b macrophages typically express surface markers including CD86 and MHC-II molecules; transcription factors including STATS, IRF4, and p50 (NF-κB); and metabolic enzymes including ARG1 and CARKL. In some embodiments, bone marrow-derived cells are M2b macrophages. M2c macrophages are activated by glucocorticoids or IL-10 and exhibit a strong anti-inflammatory profile and phagocytotic activity of apoptotic bodies. M2c macrophages typically express surface markers including CD163, TLR1, and TLR8; transcription factors including STATS, STAT6, IRF4, and p50 (NF-κB); and metabolic enzymes including ARG1 and GS. M2c macrophages typically secrete cytokines including IL10 and TGF-β. The expression of surface markers including CD14, CD206, and CD163 on M2 macrophages has been reported to be associated with phagocytosis competence (Schulz et al. In-Depth Characterization of Monocyte-Derived Macrophages using a Mass Cytometry-Based Phagocytosis Assay. Sci Rep 9, 1925 (2019), doi.Org / 10.1038 / s41598-018-38127-9).The disclosed method may utilize M2 macrophages, which include M2a, M2b, and / or M2c macrophages.

[0216] In some embodiments, the cells are bone marrow progenitor cells or bone marrow-derived cells, e.g., those directly isolated from the subject (autologous) and / or those isolated from the subject and treated with MBV. In other embodiments, the cells are derived from a cell lineage and treated with MBV. The cells may also be obtained from a heterogeneous source, e.g., from a mouse, rat, non-human primate, or pig, and treated with MBV. In some embodiments, the cells are human cells. In some embodiments, bone marrow-derived cells, such as M2 macrophages, can be obtained from human induced pluripotent stem cells (IPSCs) (see Hansen et al., Stem cell research vol. 29 (2018): 232-244; Lachmann et al., Stem cell reports vol. 4,2 (2015): 282-96. doi:10.1016 / j.stemcr.2015.01 .005; Mukherjee et al., A Simple Multistep Protocol for Differentiating Human Induced Pluripotent Stem Cells into Functional Macrophages. In: Rousselet G. (eds) Macrophages. Methods in Molecular Biology, vol 1784. Humana Press, New York, NY.).

[0217] With respect to the subject being treated, the myeloid progenitor cells or myeloid-derived cells may be allogeneic and / or autologous. The cells and compositions are typically isolated from a sample, particularly a biological sample, e.g., obtained from or derived from a subject. Typically, the subject is one that requires and / or receives cell therapy (adoptive cell therapy). The subject is preferably a mammal, particularly a human.

[0218] In autologous adoptive cell transfer, myeloid progenitor cells or myeloid-derived cells are collected from a subject, treated with MBV, and returned to the subject. In allogeneic adoptive cell transfer, myeloid progenitor cells or myeloid-derived cells are collected from a healthy donor, not a patient, treated with MBV, and administered to a target subject with muscle wasting disorder who is being treated. In some embodiments, these allogeneic cells are HLA-matched to reduce the likelihood of rejection by the host. Therefore, myeloid-derived cells described herein may also include modifications such as the disruption or removal of HLA class I molecules. For example, Torikai et al., Blood. 2013; 122: 1341-1349 and Ren et al., Clin. Cancer Res. 2017; 23:2255-2266.

[0219] In certain embodiments, bone marrow progenitor cells or bone marrow-derived cells may be obtained from collected units of blood, or from bone marrow, or collected from subjects, using several techniques known to those skilled in the art, for example, Ficoll separation for blood cells. In one embodiment, cells from the circulating blood of an individual may be obtained by apheresis or leukocyte apheresis. Apheresis products typically contain lymphocytes, monocytes, granule cells, B cells, other nucleated leukocytes, erythrocytes, and platelets, including T cells. Cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium, for example, phosphate-buffered saline (PBS) or a washing solution which may be deficient in calcium and magnesium, or deficient in many, if not all, divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in various biocompatible buffers, for example, Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed, and the cells may be directly resuspended in the culture medium. In another embodiment, cells may be isolated from peripheral blood by lysing erythrocytes and depleting lymphocytes and erythrocytes, for example, by centrifugation via a PERCOLL® gradient. Alternatively, cells may be isolated from the umbilical cord. In any event, a specific subpopulation of myeloid cells, typically macrophages, may be further isolated by positive or negative selection techniques.

[0220] In one embodiment, cells or populations of cells, including bone marrow-derived cells such as macrophages, are cultured for expansion. In another embodiment, cells or populations of cells, including bone marrow progenitor cells, are cultured for differentiation and expansion into bone marrow-derived cells, such as macrophages. When expanding cells, bone marrow-derived cells, such as macrophages, can be enlarged by approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 100,000, 1,000,000, 10,000,000 or more, and any and all whole or partial integer multiples between them. In one embodiment, cells are enlarged within a range of approximately 20 to 50 times.

[0221] After culturing, the cells may be incubated in cell medium in a culture device for a certain period of time, or until the cells reach confluence or high cell density for optimal subculturing, before transferring the cells to another culture device. The culture device may be any culture device commonly used for culturing cells in vitro. In some embodiments, the confluence level is 70% or higher before transferring the cells to another culture device. More preferably, the confluence level is 90% or higher. The period may be any time appropriate for culturing cells in vitro. The culture medium may be replaced at any point during cell culture. In some embodiments, the culture medium is replaced approximately every 2-3 days. The cells are treated with an effective amount of MBV and harvested from the culture device, at which point the cells may be used immediately or stored for use at a later time, for example, by freezing.

[0222] The culturing steps described herein (including contact with MBV as described herein) may be very short, e.g., less than 24 hours, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours. The culturing steps described further herein (including contact with MBV as described herein) may be longer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or longer.

[0223] In one embodiment, bone marrow progenitor cells or bone marrow-derived cells may be cultured for several hours (about 3 hours) to about 14 days, or any integer value of time in between. Bone marrow-derived cells may be cultured for 1 to 10 days, for example, 2 to 9 days, for example, 3 to 8 days, for example, 3, 4, 5, 6, 7, 8 or 9 days. In some embodiments, bone marrow-derived cells are cultured for 7 days. Suitable conditions for cell culture include a suitable medium that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), L-glutamine, insulin, M-CSF, GM-CSF, IL-10, IL-12, IL-15, TGF-b and TNF-a, or any other additives for cell growth known to those skilled in the art (e.g., macrophage complete medium, DMEM / F12, DMEM / F12-10 (Invitrogen) or DMEM high glucose). Other additives for cell growth include, but are not limited to, surfactants, plasmamates, and reducing agents, such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, AEVI-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a prescribed set of hormones, and / or sufficient amounts of cytokines for cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in the cultures of cells injected into the subject. Cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0224] After this period, MBV is, for example, approximately 1 × 10⁻⁶ 6 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 20 The MBV / ml of each individual, for example, 1 × 10⁻⁶. 9 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 14A single MBV / ml, for example, about 1 × 10⁻⁶. 10 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 12 1 MBV / ml, or 1 × 10⁻⁶ 10 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 11 1 MBV / ml, or 1 × 10⁻⁶ 11 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 12 1 MBV / ml, or 1 × 10⁻⁶ 10 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 14 1 MBV / ml, or 1 × 10⁻⁶ 11 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 14 1 MBV / ml, or 1 × 10⁻⁶ 9 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 11 A single MBV / ml, for example, about 1 × 10⁻⁶. 9 MBV / ml, approximately 1 × 10⁻⁶ 10 MBV / ml, approximately 1 × 10⁻⁶ 11 MBV / ml, approximately 1 × 10⁻⁶ 12 MBV / ml, approximately 1 × 10⁻⁶ 13 1 MBV / ml, or 1 × 10⁻⁶ 14 It can be added to the culture medium at a concentration of 1 MBV / ml. In some embodiments, the MBV is approximately 1 × 10⁶. 6 ~Approx. 1×10 12 It can be added to the culture at a concentration of 1 × 10⁶ MBV / ml. In some cases, the MBV is approximately 1 × 10⁶. 6 ~Approx. 1×10 19 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 18 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 17 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 16 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 15 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 14 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 13 1 MBV / ml, or approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 12It can be added to the culture at a concentration of 1 MBV / ml. In other examples, the MBV is, for example, about 1 × 10⁶. 7 ~Approx. 1×10 11 It can be added to the culture medium at a concentration of 1 MBV / ml.

[0225] In some embodiments, bone marrow-derived cells are treated with MBV for approximately 2 to 72 hours, for example, approximately 12 to 48 hours, or for example, approximately 24 to 36 hours. Bone marrow progenitor cells or bone marrow-derived cells, for example, macrophages, for example, M2 macrophages, may be treated with MBV for approximately 12, approximately 24, approximately 36, or approximately 48 hours. Bone marrow-derived cells, for example, macrophages, for example, M2 macrophages, may be treated with MBV for approximately 24 hours.

[0226] The present invention relates to a method of treatment, particularly adoptive cell therapy, preferably adoptive bone marrow-derived cell therapy, which involves administering bone marrow progenitor cells or bone marrow-derived cells that have been in contact with MBV to a subject in need thereof. The administration may be carried out, for example, within about 2 to 48 hours after contact between bone marrow progenitor cells or bone marrow-derived cells and MBV, for example, within about 12 to 24 hours after contact between bone marrow progenitor cells or bone marrow-derived cells and MBV, for example, within about 2, 4, 6, 8, 10, 12, 14, 16, 19, 20, 22, or 24 hours. In other embodiments, bone marrow progenitor cells or bone marrow-derived cells are frozen after contact with MBV, as disclosed herein, and then used at a later date.

[0227] In some embodiments, an effective amount of myeloprogenitor cells or myelo-derived cells treated with MBV, or a composition containing such myelo-derived cells, is administered to a subject, for example, a subject having a muscle-wasting condition. In some embodiments, these methods thereby treat, for example, alleviate one or more symptoms of a muscle-wasting condition.

[0228] In some embodiments, adoptive cell therapy is carried out by autotransfer, in which bone marrow progenitor cells or bone marrow-derived cells are isolated from and / or otherwise prepared from a subject receiving bone marrow-derived cell therapy. Thus, in some embodiments, bone marrow progenitor cells or bone marrow-derived cells are produced from a subject with a muscle-wasting condition. After isolation and treatment with MBV, the bone marrow progenitor cells or bone marrow-derived cells are administered to the same subject. In some embodiments, adoptive cell therapy is carried out by allogeneic transfer, in which bone marrow progenitor cells or bone marrow-derived cells are isolated from and / or otherwise prepared from a different subject (first subject) other than the subject with a muscle-wasting condition (second subject). In such embodiments, bone marrow progenitor cells or bone marrow-derived cells are treated with MBV and then administered to a different subject of the same species, for example, that has a muscle-wasting condition.

[0229] In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject. In some embodiments, HLA matching is less important if the immune cells are modified to reduce the expression of endogenous TCRs and HLA class I molecules.

[0230] Administration of MBV-treated myeloid progenitor cells or myeloid-derived cells may be combined with one or more additional therapeutic agents, or in conjunction with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, myeloid-derived cells are co-administered with another treatment in sufficiently close temporal proximity so that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cell population is administered before one or more additional therapeutic agents. In some embodiments, the cell population is administered after one or more additional therapeutic agents.

[0231] Administration of myeloid progenitor cells or myeloid-derived cells prepared as described herein may be administered to subjects suffering from muscle wasting disorders, such as human subjects, to treat muscle wasting disorders. Muscle wasting disorders may be, for example, any of those disclosed herein. Myeloid progenitor cells or myeloid-derived cells may be administered systemically, for example, intravenously.

[0232] Myeloid progenitor cells or myeloid-derived cells treated with MBV may be administered to subjects weekly, every two weeks, monthly (e.g., every approximately 28 days), every two months (e.g., every approximately 56 days), or every three months (e.g., every approximately 84 days). The number of cells administered and the frequency of dosing may be adjusted depending on the desired outcome in the subject and the type and severity of muscle wasting disorder. conditioned medium

[0233] In some embodiments, a method for treating a subject having muscle wasting is disclosed, comprising the step of administering to the subject a composition containing an effective amount of a conditioned medium or fraction thereof obtained from a culture of macrophages cultured in the presence of exogenous MBV.

[0234] The isolation of bone marrow progenitor cells or bone marrow-derived cells, and their in vitro treatment with MBV, are disclosed above. In some embodiments, a medium adapted by culturing such cells may be used in the disclosed method.

[0235] Furthermore, macrophages, such as M2 macrophages, may be cultured with MBV in any medium suitable for mammalian cell culture, such as RPMI-1640 medium, which may contain human or fetal bovine serum as needed, or Dulbecco's Modified Eagle Medium / Nutrient Mixture F12 (DMEM-F12), in order to produce a conditioned medium for use in treating muscle wasting conditions.

[0236] MBV is approximately 1 × 10 6 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 20 A single MBV / ml, for example, about 1 × 10⁻⁶. 9Individual MBV / ml ~ approximately 1 × 10⁻⁶ 14 A single MBV / ml, for example, about 1 × 10⁻⁶. 10 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 12 1 MBV / ml, or 1 × 10⁻⁶ 10 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 11 1 MBV / ml, or 1 × 10⁻⁶ 11 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 12 1 MBV / ml, or 1 × 10⁻⁶ 10 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 14 1 MBV / ml, or 1 × 10⁻⁶ 11 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 14 1 MBV / ml, or 1 × 10⁻⁶ 9 Individual MBV / ml ~ approximately 1 × 10⁻⁶ 11 A single MBV / ml, for example, about 1 × 10⁻⁶. 9 MBV / ml, approximately 1 × 10⁻⁶ 10 MBV / ml, approximately 1 × 10⁻⁶ 11 MBV / ml, approximately 1 × 10⁻⁶ 12 MBV / ml, approximately 1 × 10⁻⁶ 13 1 MBV / ml, or 1 × 10⁻⁶ 14 MBV / ml can be added to acclimate culture media containing macrophages. In some embodiments, the MBV is approximately 1 × 10⁶. 6 ~Approx. 1×10 12 It can be added to the culture at a concentration of 1 × 10⁶ MBV / ml. In some cases, the MBV is approximately 1 × 10⁶. 6 ~Approx. 1×10 19 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 18 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 17 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 16 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 15 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 14 MBV / ml, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 13 1 MBV / ml, or approximately 1 × 10⁻⁶ 6~Approx. 1×10 12 It can be added to the culture at a concentration of 1 MBV / ml. In other examples, the MBV is, for example, about 1 × 10⁶. 7 ~Approx. 1×10 11 It can be added to the culture medium at a concentration of 1 MBV / ml.

[0237] In some embodiments, macrophages are cultured in a medium containing MBV for approximately 2 to 72 hours, for example, approximately 12 to 48 hours, or for example, approximately 24 to 36 hours. Macrophages, for example, M2 macrophages, may be treated with MBV for approximately 12, approximately 24, approximately 36, or approximately 48 hours. Macrophages, for example, M2 macrophages, may be treated with MBV for approximately 24 hours.

[0238] Once the desired period for preparing the conditioned medium has been reached, for example, when the desired concentration of macrophage secretome molecules is reached in the medium, the culture medium can be separated from the cells to isolate the conditioned medium. This can be achieved, for example, by centrifugation and other known techniques. In some cases, when centrifugation is used, the conditioned medium is the supernatant and the macrophages are in the pellet. Thus, in some cases, the conditioned medium is cell-free or substantially cell-free. For example, cells may be present in less than 10% of the conditioned medium, for example, less than 5%, by weight or volume.

[0239] The conditioned medium may be subjected to lyophilization for the concentration of bioactive agents that promote preservation and / or tissue repair. A typical lyophilization process involves three separate, interdependent processes: freezing, primary drying (sublimation), and secondary drying (desorption). Various biocompatible preservatives, cryoprotectants, and stabilizers may be used, as needed, to preserve their activity. Non-limiting examples of biocompatible agents include, among others, glycerol, dimethyl sulfoxide, and trehalose. In some embodiments, the lyophilized product also includes one or more excipients, such as buffers, extenders, and osmotic modifiers. The freeze-dried medium is reconstituted by the addition of a suitable solution or pharmaceutical diluent.

[0240] In some embodiments, the conditioned medium may be treated by precipitating the bioactive agents (e.g., growth factors, cytokines, and / or Wnt proteins) in the medium. Precipitation may be carried out using various procedures, such as salting out with ammonium sulfate, or by using a hydrophilic polymer, such as polyethylene glycol.

[0241] In other embodiments, the conditioned medium is subjected to filtration using various selective filters. Processing the conditioned medium by filtration is useful for concentrating factors that promote tissue repair, as well as for removing small molecules and solutes used in the conditioned medium. Filters having selectivity for a specified molecular weight include <5,000 daltons, <10,000 daltons, and <15,000 daltons. Other filters may be used as described herein, and the processed medium may be assayed for tissue repair-promoting activity. Exemplary filter and concentrator systems include, among others, those based on hollow fiber filters, filter discs, and filter probes.

[0242] In other embodiments, the conditioned medium is subjected to chromatography to remove salts, impurities, or to fractionate various components of the medium. Various chromatographic techniques, such as molecular sieving, ion exchange, reversed-phase, and affinity chromatography, may be used. Mild chromatographic media are used to process the conditioned medium without significant loss of biological activity. Non-limiting examples include, among others, dextran, agarose, and polyacrylamide-based separation media (e.g., available under various trade names, e.g., SEPHADEX® and SEPHAROSE®). In some embodiments, impurities are removed from the conditioned medium using the method disclosed in U.S. Patent Application Publication No. 2004 / 0248803.

[0243] The conditioned medium may be used directly without the addition of a pharmaceutically acceptable carrier, or a pharmaceutical composition may be prepared containing the conditioned medium and various pharmaceutically acceptable carriers. The pharmaceutical composition refers to the form of the conditioned medium and at least one pharmaceutically acceptable carrier. The composition may also contain formulation agents, such as suspensions, stabilizers, or dispersants. Injectable formulations may be presented with or without preservatives, in unit dose forms, in ampoules in multi-dose containers. Alternatively, the composition may be presented in powder form for reconstitution with a suitable vehicle, including, for example, sterile, pyrogenic-free water, saline, buffer, or dextrose solution, without limitation.

[0244] In other embodiments, a conditioned medium containing a bioactive agent may be introduced into or encapsulated within the lumen of a liposome for the delivery of the bioactive agent and to extend its lifespan. Liposomes can be classified into various types: multilamellar (MLV), stable plurilamellar (SPLV), small unilamellar (SUV), or large unilamellar (LUV) vesicles. Liposomes can be prepared from a variety of lipid compounds that may be synthetic or naturally occurring, including phosphatidyl ethers and esters, e.g., phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, dimyristoylphosphatidylcholine; steroids, e.g., cholesterol; cerebrosides; sphingomyelin; glycerolipids; and other lipids (see, for example, U.S. Patent No. 5,833,948).

[0245] The conditioned medium may be used alone or in combination with other suitable bioactive agents useful for treating muscle wasting. In some embodiments, the conditioned medium may be used with other compounds or compositions.

[0246] The conditioned medium processed as described herein may be administered to subjects suffering from muscle wasting disorders, such as human subjects, to treat muscle wasting disorders. Muscle wasting disorders may be, for example, any of those disclosed herein. The conditioned medium may be administered systemically, for example, intravenously, or by intramuscular or subcutaneous administration. Intramuscular administration may target specific muscles of interest suffering from wasting, such as muscles experiencing atrophy or dystrophy.

[0247] The acclimatized medium may be administered to subjects daily, weekly, every two weeks, monthly (e.g., every approximately 28 days), every two months (e.g., every approximately 56 days), or every three months (e.g., every approximately 84 days). The dosage and frequency of administration may be adjusted depending on the desired outcome in the subject and the type and severity of muscle wasting disorder.

[0248] Treatment of SMA Methods for treating subjects (e.g., human subjects) having SMA are provided herein. These methods may be used to treat (i.e., prevent, induce remission, suppress, and / or reduce) motor impairments caused by SMA in subjects. In some embodiments, the SMA is type 1 SMA. In other embodiments, the SMA is type 2 SMA. In further embodiments, the SMA is type 3 or type 4 SMA.

[0249] Any suitable subject having or at risk of motor impairment due to SMA may be treated using the method provided herein. Motor impairment may be in the upper or lower limbs, for example, above or below the elbow, above or below the knee, or the entire arm or leg. Subjects may have any of types 1 to 4, for example, type 1, type 2, type 3, or type 4 SMA. In some implementations, subjects with SMA are selected for treatment. This method may be initiated at any point after the onset of motor impairment in the subject, or even before detectable motor impairment in SMA patients at risk of motor impairment.

[0250] SMA can be treated by administering MBV, conditioned medium, or myeloprogenitor cells prepared as disclosed herein to the patient. The administration may be direct to the spinal cord or adjacent muscular system, or it may be systemic, for example, by intravenous administration. The dosage and frequency of administration according to the methods disclosed herein may be adjusted as necessary to affect the desired outcome in the subject. For example, treatment outcomes may be assessed for a particular subject by monitoring the metabolic activity of myoblasts from blood samples taken from the subject, or by monitoring other characteristics of the subject, such as weight gain, improvements in muscle tone and muscle fitness, or other improvements in quality of life. For example, improvements in functional capacity, demonstrated by changes in the Spinal Muscular Atrophy Functional Rating Scale (SMAFRS), may indicate the effectiveness of the methods disclosed herein. In some cases, if the treated subject has SMA, the treatment methods disclosed herein result in improvements in the SMAFRS functional rating.

[0251] Combination therapy for SMA The method of the present invention may be combined with other treatments for SMA. Gene therapy, such as onasemnogen abeparvovec (Zolgensma®), an IV-administered adeno-associated virus vector-based gene therapy that delivers one copy of the Surviving Motor Neuron 1 (SMN1) gene, has been somewhat successful in preventing the need for persistent respiratory support and death in neonatal patients (types 1-2). Furthermore, SMA neurorestorative agents that slow or prevent motor neuron death caused by SMA, such as nusinersen (Spinraza®), an intrathecal antisense oligonucleotide (ASO) that targets the SMN2 gene, and risdiplam (Evrysdi®), an orally administered Surviving Motor Neuron 2 (SMN2) splicing modifier, are available. These available treatments differ in their mechanism of action against the disease and their means of administration, but may be combined with the method claimed herein.

[0252] Treatment and combination therapy for FSHD Methods for treating subjects (e.g., human subjects) having FSHD are provided herein. These methods may be used to treat (i.e., prevent, induce remission, suppress, and / or reduce) motor impairments caused by FSHD in subjects. Subjects may have type 1 FSHD or type 2 FSHD.

[0253] FSHD is the third most common hereditary myopathy, with an estimated incidence of approximately 1:20,000. The disorder is characterized by progressive weakness and atrophy of the facial and shoulder girdle muscles, followed by its spread to the abdominal and pelvic girdle muscles with highly variable expression. The genetic defect follows an autosomal dominant inheritance pattern, with new mutations accounting for approximately 10% of recognized cases. Type 1 FSHD is the most common form of FSHD, accounting for 95 percent of cases. FSHD is caused by ectopic expression of the germline transcription factor double homeobox (DUX)4 gene in muscle cells. FSHD1 is associated with a deletion of a chromosomal tandem repeat called D4Z4, near the end of chromosome 4 at position 4q35. The D4Z4 region is a polymorphic variable-number tandem repeat (VNTR) array consisting of 3.3 kilobase units, each encoding the DUX4 gene. Unaffected individuals possess a chromosome 4 D4Z4 array with a length of 11 to 150 consecutive units. In individuals with FSHD, this D4Z4 repeat array is reduced to a range of 1 to 10 consecutive units. This reduction of the D4Z4 repeat array causes chromatin hypomethylation and relaxation, which activates the expression of the toxic DUX4 gene. Adjacent to the D4Z4 region, towards the distal end of the chromosome, are polyadenylation sites, which are part of the DUX4 gene and are required for stable gene expression in the most distal D4Z4 units. Chromosome 4 is provided with two alleles, called 4qA and 4qB, based on distal DNA variations relative to the D4Z4 repeat array. Only the 4qA variant combined with the shortened D4Z4 repeat region is associated with FSHD. These subjects are suitable for treatment using the methods disclosed herein.

[0254] Facioscapulohumeral muscular dystrophy type 2 (FSHD2) accounts for approximately 5% of all cases of FSHD and describes patients who do not have a D4Z4 repeat reduction on chromosome 4. Phenotypically, FSHD2 shows virtually no difference from FSHD1, and both types of FSHD arise via a common downstream mechanism: the desuppression of the transcription factor DUX4 in skeletal muscle cells. This results in the expression of DUX4 and target genes, leading to skeletal myotoxicity. FSHD2 is a bigenic disorder, and mutations in the genes SMCHD1, DNMT3B, and more recently LRIF1 can cause FSHD2. These subjects are suitable for treatment using the methods disclosed herein.

[0255] The methods disclosed herein are used in conjunction with other treatments for FSHD. These include, but are not limited to, exercise, dietary modifications, surgical procedures to stabilize the shoulder, and mechanical aids. The methods disclosed herein may also be used in conjunction with other therapeutic agents. See, for example, U.S. Patent Application Publication 2021 / 0038653 disclosing the use of an antibody linked to a DUX4 inhibitor, PCT Publication WO2022 / 115745A1 disclosing the use of DUX4 antisense RNA, and U.S. Patent No. 10,907,157 disclosing antisense and interfering agents that reduce the expression of DUX4 and DUX4c.

[0256] FSHD may be treated by administering MBV, conditioned medium, or myeloprogenitor cells prepared as disclosed herein to the patient. Administration may be systemic, e.g., intravenously, or it may be directly into the affected muscle, e.g., intramuscularly. The dosage and frequency of administration according to the methods disclosed herein may be adjusted as necessary to influence the desired outcome in the subject. For example, treatment outcomes may be assessed for a particular subject by monitoring the metabolic activity of myoblasts from blood samples taken from the subject, or by monitoring other characteristics of the subject, e.g., weight gain, improvements in muscle tone and muscle fitness, or other improvements in quality of life. For example, improvements in functional capacity, demonstrated by changes in the FSHD compositive outcome measure (FSHD-COM) showing improvements in quality of life indicators, or through measurements of muscle strength, leg function (timed walk test, time to climb stairs test, or time to stand up from a chair), may demonstrate the effectiveness of the methods disclosed herein. In some cases, if the treated subject has FSHD, the treatment methods disclosed herein result in improvements in the FSHD-COM functional rating, specifically in the timed walking test, timed stair test, and / or time to stand test.

[0257] Promote skeletal muscle regeneration Methods for promoting the regeneration or repair of skeletal muscle in subjects requiring such regeneration or repair are provided herein. Subjects requiring such regeneration or repair are experiencing muscle loss or injury. For example, a subject may have a muscular degenerative disease as disclosed herein that contributes to muscle injury or loss. These methods provide a step of administering an effective amount of a composition comprising exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or CD63 lo CD81 lo Therefore, MBV does not contain alkaline phosphatase.

[0258] Subjects administered with MBV may demonstrate an increase in satellite cells and / or myoblasts in muscle tissue, particularly in muscle tissue where regeneration or repair is desired. Subjects administered with MBV may demonstrate an increase in muscle fibers in muscle tissue where regeneration or repair is desired. MBV may be administered systemically. MBV may be administered topically to the muscle where regeneration or repair is desired. This method may include a step of measuring muscle regeneration, for example, a step of measuring an increase in satellite cells and / or myoblasts in a sample from a subject.

[0259] While observation of such cellular changes in muscle tissue can be tested by tissue biopsy and staining, an increase in muscle mass is another criterion that can be used to determine if muscle regeneration is occurring. An increase in muscle mass can be determined, for example, by calculating body composition via bioelectrical impedance analysis, dual-energy X-ray absorptiometry (DEXA / DXA) scans, or magnetic resonance imaging (MRI) to determine the proportion of body weight that is fat, muscle, water, or bone.

[0260] The level of muscle regeneration or repair achieved in a subject from MBV administration can also be functionally assessed by using muscle strength tests. For example, peak torque (PT), rate of torque generation (RTD), or mean torque (AT) over a single contraction can be used to assess muscle function during isometric contraction of the muscle. Assessments can be performed both before and after treatment to assess muscle function, where an increase in muscle function after treatment compared to before treatment indicates skeletal muscle regeneration and repair. According to the present invention, a subject administered with MBV experiences an increase of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or greater in PT, RTD, or AT after treatment with MBV compared to before treatment. The increase can be measured one week, two weeks, one month, two months, or three months after MBV administration.

[0261] Skeletal muscles undergoing regeneration or repair may include, for example, the quadriceps, biceps, gastrocnemius, flexor thigh muscles, gluteus maximus, triceps, deltoid, latissimus dorsi, or trapezius muscles. The subjects may be humans.

[0262] According to the present invention, subjects administered with MBV experience an increase of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or greater in muscle mass after treatment with MBV. For example, this increase may be measured approximately 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after the start of treatment with MBV. The increase is compared to the subject's muscle mass before the start of treatment with MBV.

[0263] MBV may be administered chronically (for example, indefinitely as long as there is a need for muscle regeneration) or for a limited period until the desired level of muscle regeneration is achieved.

[0264] For example, the subject has approximately 1 x 10 1 ~Approx. 1×10 20 A dose of 1 MBV / kg of body weight is administered. For example, a subject may receive approximately 1 x 10⁶ units of MBV. 6 ~Approx. 1×10 20 The MBV per 1 kg of body weight is, for example, approximately 1 × 10 6 ~Approx. 1×10 12 The dosage is one MBV / kg of body weight / administered. In some cases, the subjects were given approximately 1 × 10⁶ units. 6 ~Approx. 1×10 19 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 18 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 17 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 16 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 15 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 14 Each MBV, approximately 1 x 10 6 ~Approx. 1×10 13 1 MBV, or approximately 1 x 10 6 ~Approx. 1×1012 MBV / dosage is administered. In other cases, the subjects receive approximately 1 × 10⁻⁶ doses. 7 ~Approx. 1×10 11 The dose is 1 MBV / kg of body weight / administered. In another example, the subject receives 1 × 10⁶ 7 ~1 × 10 8 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 8 ~1 × 10 10 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 9 ~1 × 10 10 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 6 ~1 × 10 8 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 7 ~1 × 10 9 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 8 ~1 × 10 11 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 9 ~1 × 10 11 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 10 ~1 × 10 11 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 11 ~1 × 10 12 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 6 ~1 × 10 14 A dose of 1 MBV / kg of body weight is administered. In another embodiment, the subject receives 1 × 10⁶ 12 ~1 × 10 14The dose is 1 MBV / kg of body weight / administered. In one embodiment, the administration of MBV according to any of the above amounts is by systemic administration. For example, in one embodiment, the administration is intravenous. In another embodiment, the administration is by bolus subcutaneous injection. The administration may be, for example, intramuscular, for example, in the muscle where regeneration is desired. The intramuscular injection may be a single bolus injection or may be divided and introduced at multiple sites in the muscle where repair or regeneration is needed.

[0265] MBV may be administered once daily, once weekly, twice weekly (biweekly), every two weeks, once a month, or as needed to achieve the desired level of muscle regeneration or repair.

[0266] The following embodiments are provided to illustrate certain features of a particular aspect of the present disclosure, but the claims should not be limited to the illustrated features. [Examples]

[0267] Matrix-bound nanovesicles (MBVs) are extracellular vesicles present in the extracellular matrix (ECM) of all animal tissues, including muscle. MBVs have been shown to modulate the phenotype of macrophages by promoting a pro-remodeling phenotype and downmodulating inflammation. Many other cell types, such as mesenchymal stem cells, fibroblasts, and neural precursors, take up MBVs and have a biological response to them. The following examples demonstrate that MBVs have a direct effect on primary muscle cells of FSHD and a homeostatic effect on macrophages. Effects on the secretomes of neural progenitor cells and microvascular endothelial cells are also shown herein. Furthermore, MBVs have been demonstrated to be effective in animal models of FSHD and spinal muscular atrophy. These results support the therapeutic utility of MBVs as a treatment for muscle wasting conditions disclosed herein.

[0268] (Example 1) material and method MBV Isolation: MBV was isolated from pre-decellularized porcine bladder (UBM). MBV isolation was performed by liberase (collagenase and dispase enzyme cocktail) digestion of UBM, ultracentrifugation, and size exclusion chromatography (SEC). The isolated MBV was quantified by nanotracking analysis (NTA) and stored at -20°C before use.

[0269] Processing of FSHD2 primary cells: For all experiments, cell cultures were prepared under sterile conditions, and cells were incubated at 37°C and 5% CO2. Myoblasts from healthy patients and patients diagnosed with facioscapulohumeral muscular dystrophy (FSHD) were cultured in 96-well plates in growth medium (Ham's F-10, 20% FBS, 1% PS). At 80% confluence, cells were treated with different doses of MBV (1 × 10⁶). 9 , 1 x 10 10 and 1 × 10 11 Cells were treated with (particles / ml) for 24 hours. Then, the cells were washed in PBS and incubated with 10% ALAMARBLUE® to measure metabolic activity.

[0270] Macrophage secretome generation: Macrophages were isolated from C57BL / 6 mouse bone marrow according to a standard laboratory protocol and differentiated over 7 days. The differentiated macrophages were then divided into 1 × 10⁶ cells in complete medium (DMEM high glucose, 10% FBS, and 1% PS) using LPS-treated and IL-4-treated macrophages as M1 and M2 controls, respectively. 11 Cells were treated with 1 MBV / ml for 24 hours. After 24 hours, the medium was aspirated, the cells were washed with PBS, and blank DMEM (0% FBS) was added. The cells were incubated for 6 hours, and the medium containing the macrophage secretome was collected, filtered, and stored at -20°C.

[0271] Human microvascular endothelial cell (HMEC-1) culture and angiogenesis assay: HMEC-1 cells were grown in MCDB131 medium containing 10% FBS and 1% PS, and seeded in 24-well plates until 90% confluence was reached. A straight scratch was then performed on the cell monolayer using a 20 μl pipette tip, the cells were washed with PBS, and treated with a 1:1 mixture of MCDB131 medium containing 20% ​​FBS and macrophage-derived secretomes (MBV, M1 and M2). Photographs of the scratched area were taken at 0, 2, 4, and 12 hours. These photographs were analyzed using ImageJ software to calculate the scratched area, which decreased with cell movement, indicating cell migration.

[0272] For metabolic activity analysis, cells were seeded in 48-well plates. Upon reaching 80% confluence, HMEC-1 cells were cultured for 24 and 72 hours with a 1:1 mixture of macrophage secretome and complete medium. The medium was then replaced with 10% ALAMARBLUE® and incubated for 3 hours. Post-incubation fluorescence (excited at 530 nm, emitted at 590 nm) was recorded and correlated with metabolic activity.

[0273] Treatment and differentiation of mouse neuroblastoma cells: N1E-155 neuroblastoma cells were cultured in DMEM containing 10% FBS and 1% PS. Cells were seeded in 48-well plates until 80% confluence was reached. Cells were then cultured for 24 and 72 hours with a 1:1 mixture of macrophage secretome and complete medium. The medium was replaced with 10% ALAMARBLUE® and incubated for 3 hours. Post-incubation fluorescence (excitation at 530 nm, emission at 590 nm) was recorded and correlated with metabolic activity.

[0274] For differentiation analysis, cells were seeded at 20% confluence over 24 and 72 hours with a 1:1 mixture of macrophage secretome and complete medium. Representative images of the cells were taken at each time point, and the images were analyzed using CellProfiler software. Single-cell characteristics, such as roundness, eccentricity, and maximum ferret diameter, were measured and correlated with the differentiation stage of neuroblastoma cells.

[0275] Statistical Analysis: Data are presented as mean ± standard deviation. Statistics (figures) and statistical analysis were performed using Prism GraphPad software. One-way ANOVA and Tukey post-hoc tests were performed to evaluate statistical differences in metabolic activity assays, while two-way ANOVA and uncorrected Fisher post-hoc assays were used to evaluate statistical differences in scratch assays and differentiation assays.

[0276] (Example 2) Direct effects of MBV on FSHD cells Metabolic activity in healthy myoblasts and myoblasts derived from FSHD2 patients showed a linear increase in metabolic activity with increasing dose of MBV (Figure 1). These results demonstrate that MBV increased mitochondrial respiration and / or cell proliferation in both healthy and affected myoblasts. Measurements were taken 24 hours after treatment with different doses of MBV.

[0277] (Example 3) Effect of MBV-treated macrophage secretomes on HMEC-1 cells Macrophage-derived secretomes from all groups slightly increased the metabolic activity of HMEC-1 cells after 24 hours (Figure 2, left panel), while only MBV-derived secretomes increased the metabolic activity of endothelial cells after 72 hours (Figure 2, right panel).

[0278] Scratch assays showed no difference between the treatments at 2 and 4 hours. However, at 12 hours, cells treated with MBV-secretomes showed higher migration than the rest of the group (Figure 3). These results indicate that secretomes derived from MBV-treated macrophages induce an activating effect on endothelial cells, which is related to angiogenesis.

[0279] (Example 4) Efficacy of MBV-treated macrophage secretomes on NIE-155 cells Macrophage-derived secretomes from all groups (M1, M2, MBV) showed no difference in the metabolic activity of N1E-155 cells after 24 hours. However, secretomes from M2 and MBV increased the metabolic activity of neuroblastoma cells after 72 hours. This effect was significantly more pronounced when cells were treated with MBV-secretomes (Figure 4).

[0280] Neuroblastoma cells, when treated with M0, M1, and M2 macrophage secretomes, showed morphological changes (roundness, eccentricity, and ferret diameter) after 72 hours (Figures 5A-5B). However, when treated with MBV-secretomes, roundness and ferret diameter did not change after 72 hours. Since the morphological changes in N1E-155 cells are highly correlated with their level of terminal differentiation, these results suggest that MBV-secretomes from macrophages preserved the stem cell nature of the neural precursor in vitro.

[0281] Overall, these results support the direct use of MBV and the use of MBV-treated macrophages or secretomes derived from MBV-treated macrophages for the treatment of muscular dystrophy. These results demonstrate that MBV directly interacts with myoblasts, increasing their metabolic activity. In the case of FSHD2 myoblasts, since FSHD is highly associated with metabolic dysfunction and oxidative stress, the increase in metabolic activity implies an improvement in the metabolic levels of affected cells. These results also demonstrate that macrophages, when treated with MBV, can produce secretomes that promote endothelial cell activity and the stem cell properties of neural progenitor cells. Since macrophages are key regulators of innate immune responses and regeneration, the in vivo effects of MBV on macrophages may promote muscle regeneration in this dystrophy.

[0282] (Example 5) Uptake analysis measured by flow cytometry This analysis was conducted to determine whether MBV accumulates in the bone marrow, interacts with bone marrow precursors, and potentially reprograms or alters the phenotype of innate myeloid cells.

[0283] The interaction between MBV and different cell types in the bone marrow was evaluated using a panel of markers. To do this, bone marrow from BalbC mice was flushed before staining, strained through a 40 μm strainer, and 1 × 10⁶ 24 hours and 3 hours before flow cytometry. 11 Individuals were exposed in vitro to CFSE-tagged MBV / ml.

[0284] Next, the cells were tagged with the following panel of markers for different immune cell subtypes: [Table 1]

[0285] Flow cytometry showed that cells from the bone marrow took up MBV in vitro. After 3 hours, the vast majority of these cells were myeloid cells, while more cells from other cell lineages were able to take up MBV after 24 hours. See Figure 6. A significant percentage of these myeloid cells were bone marrow precursors (CD34 and CD64 positive), while other major myeloid cell types that took up MBV were neutrophils (Ly6G positive) and macrophages (F4 / 80 positive).

[0286] (Example 6) ATAC-seq analysis Assays of transposase-accessible chromatin sequencing were performed in vitro on bone marrow flushes, in vitro on differentiated macrophages, or after systemic and local injections to determine whether MBV treatment affects chromatin accessibility throughout the genome and leads to stable phenotypic changes. If such changes occur in progenitor cells, this may indicate stable changes in subsequent cell lineages of the innate immune response.

[0287] ATAC-seq analysis showed that bone marrow precursors and terminally differentiated macrophages were differently affected in vitro by MBV (Figure 7), leading independently to upregulation of 28 genes in MBV-treated precursors and upregulation of 5917 genes in MBV-treated macrophages, although 168 of these upregulated genes were shared (Venn diagram). These results suggest that MBV induces stable phenotypic changes in precursors that may be descendant myeloid lineages.

[0288] The distribution of statistically significant regulated genes at the epigenetic level in different in vitro treatments (macrophages or bone marrow) is shown in Figure 9. In the Venn diagram, the larger circles correspond to the dots on the left side of the graph, and the smaller circles correspond to the dots on the right side of the graph.

[0289] (Example 7) accumulation of MBV in the bone marrow DiD-tagged MBV was injected into Balb / c mice at 3 and 24 hours. At different time points, the bone marrow was flushed, and the presence of a fluorescent signal in the bone marrow was measured ex vivo using an IVIS instrument. Ex vivo analysis showed that MBV accumulated in the bone marrow after systemic injection, producing a significant signal. Furthermore, this accumulation was associated with phenotypic changes (Figure 10A-B).

[0290] Data from Examples 5, 6, and 7 demonstrate that MBV accumulates in the bone marrow and interacts with different myeloid cell types containing its precursors. This interaction is associated with changes in chromatin accessibility at the epigenetic level, which may be related to phenotypic changes. These results suggest that MBV may be used for stable modulation of the immune response in myeloids.

[0291] (Example 8) Results in animal models of FSHD and SMA The effects of MBV were tested in two animal models of FSHD. See Figure 11. These animal models are disclosed in DeSimone et al., Disease Models & Mechanisms (2020) 13, dmm046904. doi:10.1242 / dmm.046904, 2022, which is incorporated herein by reference. The effects of MBV were also tested in an animal model of SMA. See Feng et al., Human Mol. Gen. 25(5): 964-975, 206, which is incorporated herein by reference.

[0292] For these studies, an intermediate SMA model was generated by treating Delta 7 mice with SMN-C3, an SMN-upregulatory compound. Mice were started at postnatal day 1 (PND) and treated with SMN-C3L (3 mg / kg IP, daily). At PND21, MBV in saline, hydrogel, or PBS was intramuscularly injected into both gastrocnemius, tibialis anterior, quadriceps, triceps, and masseter muscles of the mice. Muscle function was measured at PND50, after which the mice were euthanized and tissues were collected.

[0293] For FSHD, the first study was conducted using the iDUX4pA-HSA mouse model. This model induced iDUX4pA transgene expression via a muscle fiber-restricted reverse tetracycline-regulating transactivator (rtTA) using doxycycline (dox) (Bosnakovski et al., J. Clin. Invest., 2020 13(5): 2465). DUX4 expression was induced in 4-week-old female iDUX4pA-HAS mice by feeding them dox diet on day 0 and maintained throughout the duration of the study to simulate a “moderate” DUX4 environment. Animals were treated with MBV or saline according to the protocol shown in Figure 11 (top).

[0294] In a second animal model, a FLExDUX4(+TMX) mouse model enabling TMX-inducible cre expression was used to allow spatiotemporal control of DUX4-fl expression during development or in adulthood (Jones & Jones, 2018, PLoS One, 13(2):e0192657). Animals were treated with MBV or saline according to the protocol shown in Figure 11 (bottom).

[0295] Animals were graded on a severity scoring scale, where a score of 1 was assigned to normal; 2 reflected increased but minimal inflammatory cells with some satellite cell activation; 3 showed myonecrosis, abundant inflammatory cells, and loss of cytoplasmic integrity, with satellite cell activation including myoblasts; and 4 was the most severe, with scattered myonecrosis and demonstration of abundant inflammatory cells including myoblasts, fibrosis, and loss of cytoplasmic integrity.

[0296] As shown in Figure 12, FSHD animals treated with MBV (TMX-induced) had an average severity score of approximately 2.2, while FSHD animals treated with saline alone had an average severity score of approximately 3.7. This demonstrates that MBV has a positive effect on reducing FSHD severity scores compared to untreated animals.

[0297] As shown in the exemplary histological images found in Figure 13, of skeletal muscle samples obtained from PBS (saline) TMX-treated mice (left) and MBV-treated mice (right), PBS-treated animals exhibit shrunken skeletal muscle fibers, localized necrosis, and an increased number of inflammatory cells between fibers. In contrast, exemplary muscle fiber samples from MBV-treated animals show fibers of normal size and evidence of activated satellite cells around the skeletal muscle fibers. Therefore, MBV has a positive effect in inducing phenotypic changes in FSHD mice that indicate healthy muscle cells.

[0298] The effect of MBV on SMA was studied using an intermediate SMA model generated by treating Delta 7 mice with SMN-C3, an SMN-upregulatory compound. Mice were started at PND1 and treated with SMN-C3L (3 mg / kg IP, daily). At PND21, MBV in saline, MBV in hydrogel, or MBV in PBS was intramuscularly injected into both gastrocnemius, tibialis anterior, quadriceps, triceps, and masseter muscles of the mice (Figure 15). Muscle function was measured at PND50, after which the mice were euthanized and tissues were collected. The mean FSHD score of PBS-treated control mice was 3.3 (n=7). The mean FSHD score of MBV-treated mice was 2.5 (N=12). The mean FSHD score of mice treated with MBV contained in extracellular matrix hydrogel was 2.2 (n=11).

[0299] As shown in Figure 14, control SMA animals exhibited marked fibrous atrophy (longitudinal section) with abundant inflammatory cells between the fibers and areas of muscle necrosis. In contrast, MBV-treated animals showed muscle atrophy but with reduced inflammatory cells compared to PBS-treated animals. Less damage to individual fibers was also present. Therefore, MBV may be used to mitigate the effects of SMA on muscle fibers.

[0300] (Example 9) MBV does not express the common exosome markers CD63 or CD81. The presence or absence of common exosome markers on mouse exosomes, mouse bone marrow matrix vesicles (bone MVs), and mouse matrix-bound nanovesicles (MBVs) was compared using the EXO-CHECK® exosome antibody array (System Biosciences). These results are shown in Figure 15A, clearly demonstrating that MBVs are virtually devoid of standard, well-accepted exosome markers, such as CD63 and CD81. Furthermore, other signaling molecules identified in Figure 15A were absent in MBVs, while they were present to either moderate or high levels in bone microvesicles and exosomes. Concentration plots of expression levels are shown in Figure 15B.

[0301] This data shows that exosomes and bone MVs share similar expression profiles with moderate to high expression of these markers, but MBVs differ significantly in the expression of these EV markers. For example, MBVs also virtually lack one or more of EpCAM, ANXA5, TSG101, FLOT1, ICAM1, and ALIX, i.e., have them at “low” or “undetectable” levels, as indicated by the presence of dark rings or solid dark spots at the same locations on the exosome wells, and by the relative expression levels of these markers, as shown in the graph in the lower panel. Bone MVs also have higher levels of expression of all of these markers compared to MBVs, as indicated by the dark spots on the wells and by the relative expression levels of these markers, as shown in the graph in the lower panel.

[0302] In one embodiment, MBV has one or more of CD63, CD81, EpCAM, ANXA5, TSG101, FLOT1, ICAM1, and ALIX in low or undetectable levels compared to a positive control of the EXO-CHECK® exosome antibody array. In one embodiment, MBV has one or more of CD63, CD81, EpCAM, ANXA5, TSG101, FLOT1, ICAM1, and ALIX in low or undetectable levels compared to exosomes, e.g., plasma exosomes. In one embodiment, MBV has one or more of CD63, CD81, EpCAM, ANXA5, TSG101, FLOT1, ICAM1, and ALIX in low or undetectable levels compared to bone MV. In one embodiment, MBV is characterized by low or undetectable levels of ANXA5, TSG101, and ICAM1 compared to exosomes or bone MV. In one embodiment, MBVs are characterized by low or undetectable levels of CD81, CD63, ANXA5, TSG101, and ICAM1 compared to bone MVs or plasma exosomes.

[0303] (Example 10) MBV has low or no expression of bone MV markers. The expression of bone microvesicle markers, annexin V and tissue-nonspecific alkaline phosphatase (TNAP), was evaluated by Western blot analysis. The results are shown in Figure 16. Lysates prepared from 1711A cells were used as a positive control. The results of this experiment indicate that matrix-bound nanovesicles (MBVs) lack expression of both bone microvesicle markers: TNAP and annexin V. Plasma exosomes express annexin V but not TNAP. These results clearly distinguish MBVs from both exosomes and bone microvesicles.

[0304] (Example 11) MBV has differential immunomodulatory effects compared to exosomes or bone MV. Bone marrow-derived macrophages (BMDMs) recovered from mice were either left untreated (M0) or treated with the following test materials over 24 hours: IFNγ+LPS (M1) to induce the M1 phenotype, IL-4 (M2) to induce the M2-like phenotype, plasma-derived exosomes, bone microvesicles (MVs) derived from 17A cells, or MBV isolated from muscle. After treatment, the change in gene expression was assessed by qPCR. The results shown in Figure 17 demonstrate that the downregulation of the pro-inflammatory markers IL-6 and TNF-α by MBV is clearly distinguishable from the downregulation of the same two inflammatory mediators by exosomes and bone microvesicles. MBV had a potent anti-inflammatory effect; in contrast, exosomes and bone microvesicles did not.

[0305] (Example 12) D2.mdx mouse research material and method: Preparation of bladder matrix (UBM): The UBM was prepared as previously described (Mase VJ, et al. Orthopedics. 2010; 33(7):511). Pig bladders from market-weight animals were obtained from Tissue Source, LLC. Briefly, the serosal layer, muscularis exostata, submucosa, and muscularis mucosa were mechanically removed. The lumens of the mucosa were detached from the basement membrane by washing with deionized water. The remaining tissue, consisting of the basement membrane and the underlying lamina propria, was decellularized by stirring at 300 rpm for 2 hours in 0.1% peracetic acid with 4% ethanol. The tissue was then thoroughly rinsed with PBS and sterile water. The UBM was then lyophilized and ground into granules using a Wiley Mill with a #60 mesh screen.

[0306] Isolation of matrix-bound nanovesicles: MBV was isolated from laboratory-produced porcine bladder matrix (UBM) by enzymatic digestion with Liberase TL (highly purified collagenase I and collagenase II) in buffer (50 mM Tris pH 7.5, 5 mM CaCl2, 150 mM NaCl) on an orbital rocker at room temperature for 24 hours. The digested ECM was then centrifuged at 10,000 × g (30 min) to remove ECM debris. The clarified supernatant containing the free MBV was then centrifuged at 100,000 × g (Beckman Coulter Optima L-90K ultracentrifuge) at 4°C for 2 hours to pelletize the MBV. The MBV was then resuspended in 1 × PBS and stored at 4°C until further use.

[0307] D2.mdx mouse model: This study used male mdx mice (D2.mdx; Jax#013141) of DBA / 2J. See Sci Rep 10, 14070 (2020). Mice were randomly assigned to the following groups: 1) saline treatment, and 2) MBV treatment. 4.2 × 10^ 9 100 μl containing 1 MBV was administered intraperitoneally on days 1, 3, and 5, and then once a week thereafter. Body weight was recorded weekly.

[0308] Isometric Torque Measurement: Functional analysis was performed by measuring isometric torque generation of the gastrocnemius muscle at weeks 1 and 7. See Tissue Eng Part A. 2018 Jan;24(1-2):34-46. Animals were anesthetized with their hind limbs fixed by platform support in a flexed leg position. The muscle was stimulated at eight different frequencies (25–200 Hz) with a 2-minute rest period between each frequency. Single contractions and tetanuses were analyzed using the Dynamic Muscle Analysis program, and the data were normalized to the animal's body weight.

[0309] Histological evaluation: Animals were sacrificed at 8 weeks. Tissues were collected, fixed in neutral buffered formalin, and embedded in paraffin. Hematoxylin, eosin (HE), and Masson's trichrome staining were performed to evaluate degenerated and / or regenerating areas, which were identified by the presence of inflammatory cells and muscle fiber necrosis.

[0310] Results: The study protocol is shown in Figure 19. Body weight and muscle mass were assessed for the two treatment groups. See Figure 20. Results from muscle function tests are shown in Figure 21. Histological evaluations are shown in Figure 22.

[0311] These results strongly suggest that MBV can mitigate the progression of DMD. The D2.mdx model is a severe version of the disease, and the use of MBV provides unexpectedly superior efficacy. As shown in Figure 20, the weight of the gastrocnemius muscle in MBV-treated animals was statistically significantly higher than in control animals, and for other muscles, there was a tendency toward greater mass for those muscles in MBV-treated animals compared to controls, suggesting that MBV contributes to muscle regeneration and repair in the DMD model. As shown in Figure 21, MBV treatment resulted in muscles with a statistically significant increase in torque generation compared to controls. Torque is a quantitative measure of muscle strength. These results are attributable to greater muscle mass in the MBV-treated group rather than higher muscle strength for each muscle fiber. The increased torque over control animals suggests that MBV contributed to muscle repair and regeneration.

[0312] Furthermore, histology showed reduced muscle necrosis, which, while not theoretically constrained, may be a result of reduced pro-inflammatory and / or myocyte anabolic effects in MBV-treated animals compared to controls. Moreover, surprisingly, satellite cell activation, including the presence of myoblasts, was observed in MBV-treated tissue, indicating a cellular environment that promotes muscle regeneration and repair. These were not observed in control tissue. This suggests that MBV may be able to promote the regeneration and repair of skeletal muscle tissue.

[0313] It is clear that the exact details of the methods or compositions described may be varied or modified without departing from the spirit of the embodiments described herein. The inventors claim all such modifications and variations that fall within the scope and spirit of the following claims.

Claims

1. A method for treating a subject with muscle wasting: a) Exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or express CD63 ｌｏ CD81 ｌｏ The MBV is an exogenous MBV derived from the extracellular matrix that does not contain alkaline phosphatase; b) Myeloid progenitor cells or myeloid-derived cells treated with the exogenous MBV; and / or c) A conditioned medium or fraction thereof obtained from macrophages cultured in the presence of the exogenous MBV. A method comprising the step of administering a composition containing an effective amount of to a subject, thereby treating the muscle wasting condition in the subject.

2. The subject is administered the composition containing an effective amount of the exogenous MBV derived from the extracellular matrix, and the MBV either does not express CD63 and CD81, or CD63 ｌｏ CD81 ｌｏ The method according to claim 1, wherein the MBV does not contain alkaline phosphatase.

3. The method according to claim 1, wherein the subject is administered the composition comprising an effective amount of the bone marrow progenitor cells or bone marrow-derived cells treated with the exogenous MBV.

4. The method according to claim 1, wherein the subject is administered the composition containing an effective amount of the conditioned medium or fraction thereof obtained from macrophages cultured in the presence of the exogenous MBV.

5. A method for promoting skeletal muscle regeneration and repair in a subject requiring it, comprising the step of administering to the subject a composition comprising an effective amount of exogenous matrix-bound nanovesicles (MBVs) derived from the extracellular matrix, wherein the MBVs do not express CD63 and CD81, or CD63 ｌｏ CD81 ｌｏ A method wherein the MBV does not contain alkaline phosphatase, thereby promoting the regeneration and repair of skeletal muscle in the subject.

6. The method according to claim 1, 2, or 5, wherein the exogenous MBV is contained in an extracellular matrix (ECM) hydrogel or pregel prepared from the extracellular matrix (ECM), and the MBV contained in the hydrogel or pregel is administered to the subject.

7. The method according to claim 6, wherein the extracellular matrix hydrogel or pregel is an enzymatic ECM hydrogel or pregel containing an inactivated protease.

8. The method according to claim 6 or 7, wherein the enzymatic hydrogel or pregel has a pH of about 7.0 to about 7.

8.

9. The method according to any one of claims 6 to 8, wherein the enzymatic pregel forms a gel at a temperature higher than about 25°C.

10. The method according to claim 6, wherein the ECM hydrogel is an acoustically treated hydrogel having a storage modulus (G') of about 50 Pa to about 200 Pa, a loss modulus (G'') of about 5 Pa to about 20 Pa, and a ratio of G' to G'' of about 4:1 to about 15:1 at 37°C.

11. The method according to any one of claims 6 to 10, wherein the ECM hydrogel or pregel contains solubilized ECM at a concentration of 1 mg / mL to 500 mg / mL.

12. The method according to any one of claims 6 to 11, wherein the ECM in the hydrogel or pregel is not dialyzed.

13. The method according to any one of claims 6 to 12, wherein the ECM hydrogel or pregel is prepared from the extracellular matrix of the bladder, small intestine, heart, dermis, liver, kidney, uterus, brain, blood vessels, lungs, bone, muscle, pancreas, placenta, stomach, spleen, colon, adipose tissue, or esophagus.

14. The composition according to any one of claims 6 to 12, wherein the ECM hydrogel or pregel is prepared from bladder matrix (UBM), small intestinal submucosal tissue (SIS), bladder submucosal tissue (UBS), or dermis.

15. The MBV is at least about 1 × 10 ５ ~1 x 10 ２０ The method according to claims 6 to 14, wherein the particles are present in the hydrogel or pregel at a concentration of particles / mL.

16. The method according to any one of claims 1 to 15, wherein the MBV is derived from the extracellular matrix of the bladder, small intestine, heart, dermis, liver, kidney, uterus, brain, blood vessels, lungs, bone, muscle, pancreas, placenta, stomach, spleen, colon, adipose tissue, or esophagus.

17. The method according to any one of claims 1 to 16, wherein the MBV is not derived from the bone ECM or the cardiac ECM.

18. The method according to any one of claims 1 to 15, wherein the MBV is derived from the extracellular matrix of the bladder, small intestine, dermis, liver, kidney, uterus, brain, blood vessels, lungs, muscle, pancreas, placenta, stomach, spleen, colon, adipose tissue, or esophagus.

19. The method according to any one of claims 1 to 15, wherein the MBV is derived from the bladder matrix (UBM), the submucosal tissue of the small intestine (SIS), or the submucosal tissue of the bladder (UBS).

20. The method according to any one of claims 1 to 19, wherein the MBV is derived from the extracellular matrix of a mammalian vertebrate selected from humans, monkeys, pigs, cattle, or sheep.

21. The method according to any one of claims 1, 2, or 5 to 20, wherein the MBV is administered to the subject by systemic administration.

22. The method according to claim 21, wherein the systemic administration is intravenous administration.

23. The method according to claim 1, 2, or 5 to 20, wherein the MBV is administered to the subject by local administration to a tissue or area of ​​interest.

24. The method according to claim 23, wherein the target tissue or region is muscle, nerve or spinal cord.

25. The method according to any one of claims 1 or 3, wherein the bone marrow-derived cells are macrophages, monocytes, or granule cells.

26. The method according to any one of claims 1, 3, or 24, wherein the bone marrow progenitor cells or bone marrow-derived cells are autologous to the subject.

27. The method according to any one of claims 1, 3, 24, or 25, wherein the bone marrow progenitor cells or bone marrow-derived cells are administered to the subject by systemic administration.

28. The method according to any one of claims 1, 3, or 24 to 26, wherein the bone marrow progenitor cells or bone marrow-derived cells are treated with exogenous MBV in a cell culture and isolated from the cell culture after treatment with MBV for administration to the subject.

29. The method according to any one of claims 1, 3, or 24 to 27, wherein the bone marrow progenitor cells or bone marrow-derived cells are administered to the patient by intravenous administration.

30. The method according to any one of claims 1 or 4, wherein the macrophage is autologous to the subject.

31. The method according to claim 29, wherein the conditioned medium is purified by dialysis, size fractionation and / or centrifugation before the conditioned medium is administered to the subject.

32. The method according to claim 5, wherein the subject has a muscle wasting disease.

33. The method according to any one of claims 1 to 4 or 6 to 32, wherein the muscle wasting state is spinal muscular atrophy (SMA).

34. The method according to claim 33, wherein the spinal muscular atrophy is infantile progressive spinal muscular atrophy (Type I SMA), intermediate spinal muscular atrophy (Type II SMA), juvenile spinal muscular atrophy (Type III SMA), or adult spinal muscular atrophy (Type IV SMA).

35. The method according to claims 1 to 4 or 6 to 32, wherein the muscle wasting state is muscular dystrophy.

36. The method according to claim 35, wherein the muscular dystrophy (MD) is Becker type MD, congenital MD, Duchenne type MD, distal type MD, Emery-Dreyfus type MD, facioscapulohumeral type MD, limb-girdle type MD, myotonic MD, oculopharyngeal type MD, Bethlem myopathy, or Ulrich type congenital muscular dystrophy.

37. The method according to any one of claims 1 to 4 or 6 to 36, wherein the muscle wasting disease is facioscapulohumeral muscular dystrophy (FSHD) or smamatical muscle atrophy (SMA).

38. The method according to any one of claims 1 to 4 or 6 to 32, wherein the muscle wasting disease is sarcopenia or cachexia.

39. The method according to any one of claims 1 to 38, wherein the composition increases myotubular formation in the subject.

40. The method according to any one of claims 1 to 39, wherein the composition maintains neuronal progenitor cells in an undifferentiated state in the subject.

41. The method according to any one of claims 1 to 38, wherein the composition increases the migratory ability of endothelial cells in the subject.

42. The method according to any one of claims 1 to 39, wherein the composition increases the growth of muscle tissue in the subject.

43. The method according to any one of claims 1 to 40, wherein the subject is a human.

44. The method according to claim 5 or 32, wherein the subject experiences an increase in satellite cell activation and / or myoblast activity in skeletal muscle requiring regeneration or repair after administration of the composition.

45. The method according to claim 5, 32, or 44, wherein the subject experiences an increase in muscle strength in skeletal muscle requiring repair or regeneration after administration of the composition compared to before administration.

46. The method according to claim 45, wherein the increase in muscle strength is measured by a change in peak torque, torque generation rate, or average torque.

47. The method according to claim 5, 32, or 44 to 46, wherein the subject experiences an increase in muscle mass after administration of the composition compared to before administration.

48. For use in the method according to any one of claims 1 to 47, a) An exogenous matrix-binding nanovesicle (MBV) derived from the extracellular matrix, wherein the MBV does not express CD63 and CD81 or CD63 ｌｏ CD81 ｌｏ and the MBV does not contain alkaline phosphatase, an exogenous MBV derived from the extracellular matrix; b) Myeloid progenitor cells or myeloid-derived cells treated with the exogenous MBV; and / or c) A conditioned medium or fraction thereof obtained from macrophages cultured in the presence of the exogenous MBV. A composition containing an effective amount of [the substance].

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