Compositions and methods for repairing damage to skeletal muscle

Abstract

A method for treating damaged skeletal muscle tissue generally involves applying a therapeutic composition comprising a purified exosome product (PEP) and a pharma- ceutically acceptable carrier to the damaged skeletal muscle tissue. In one or more embodiments, the method includes administering 1×10 11 PEP exosomes ~ 1 × 10 13 The method includes applying the PEP exosomes to the injured skeletal muscle. In one or more embodiments, the therapeutic composition further comprises a support matrix, such as a collagen scaffold, a tissue sealant, a fibrin glue, or a hydrogel.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 284,989, filed December 1, 2021, which is incorporated by reference in its entirety. Summary of the Invention

[0002] In one aspect, the present disclosure describes a method for treating damaged skeletal muscle tissue. In general, the method comprises applying a therapeutic composition comprising a purified exosome product (PEP) and a pharma- ceutically acceptable carrier to the damaged skeletal muscle tissue.

[0003] In one or more embodiments, the PEPs comprise spherical or spheroidal exosomes having a diameter of 300 nm or less.

[0004] In one or more embodiments, the PEP comprises 1% to 20% CD63 - Exosomes and 80%-99% CD63 + In another embodiment, the PEP comprises at least 50% CD63 - Contains exosomes.

[0005] In one or more embodiments, the method comprises: 11 PEP exosomes ~ 1 × 10 13 This involves applying PEP exosomes to injured skeletal muscle.

[0006] In one or more embodiments, the therapeutic composition further comprises a support matrix, such as a collagen scaffold, a tissue sealant, a fibrin glue, or a hydrogel.

[0007] In one or more embodiments, the therapeutic composition is applied in an amount effective to increase expression of NF-κB in injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

[0008] In one or more embodiments, the therapeutic composition is applied in an amount effective to increase expression of PD-L1 in the injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

[0009] In one or more embodiments, the therapeutic composition is applied in an amount effective to increase polarization into M2 macrophages in injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

[0010] In one or more embodiments, the damaged skeletal muscle tissue comprises the urethral sphincter muscle.

[0011] In one or more embodiments, at least a portion of the PEP exosomes comprise PD-L1, NF-κB, or both PD-L1 and NF-κB.

[0012] In one or more embodiments, at least a portion of the PEP exosomes comprise CD63, CD9, flotillin, or any combination of two or more thereof.

[0013] In one or more embodiments, the therapeutic composition is applied in an amount effective to increase cell proliferation in the injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

[0014] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly illustrates illustrative embodiments. In several places in this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief description of the drawings]

[0015] [Figure 1-1]Characterization, quantification of purified exosome products (PEP) and in vitro human skeletal myoblast (HSMM) culture with increasing concentrations of PEP. (A) NanoSight nanoparticle analysis of size distribution and concentration of PEP diluted 1:1000 in phosphate buffered saline. (B) Transmission electron microscopy of PEP. Scale=200 nm. (C) Western blot probing for CD63, CD9, and flotillin-1 in three PEP preparations. (D) Western blot comparison of NF-κB p65 and PD-L1 levels in three PEP preparations and adipose-derived mesenchymal stem cell conditioned medium (AMSC-CM). (E-G) (E) IncuCyte proliferation, (F) chemotaxis and (G) wound scratch analysis of HSMM grown in serum-free and 10% fetal bovine serum (FBS)-supplemented medium with increasing concentrations of PEP ranging from 1.25 × 1011 exosomes / mL to 5 × 1011 exosomes / mL. [Figure 1-2] Characterization, quantification of purified exosome products (PEP) and in vitro human skeletal myoblast (HSMM) culture with increasing concentrations of PEP. (A) NanoSight nanoparticle analysis of size distribution and concentration of PEP diluted 1:1000 in phosphate buffered saline. (B) Transmission electron microscopy of PEP. Scale=200 nm. (C) Western blot probing for CD63, CD9, and flotillin-1 in three PEP preparations. (D) Western blot comparison of NF-κB p65 and PD-L1 levels in three PEP preparations and adipose-derived mesenchymal stem cell conditioned medium (AMSC-CM). (E-G) (E) IncuCyte proliferation, (F) chemotaxis and (G) wound scratch analysis of HSMM grown in serum-free and 10% fetal bovine serum (FBS)-supplemented medium with increasing concentrations of PEP ranging from 1.25 × 1011 exosomes / mL to 5 × 1011 exosomes / mL. [Figure 1-3]Characterization, quantification of purified exosome products (PEP) and in vitro human skeletal myoblast (HSMM) culture with increasing concentrations of PEP. (A) NanoSight nanoparticle analysis of size distribution and concentration of PEP diluted 1:1000 in phosphate buffered saline. (B) Transmission electron microscopy of PEP. Scale=200 nm. (C) Western blot probing for CD63, CD9, and flotillin-1 in three PEP preparations. (D) Western blot comparison of NF-κB p65 and PD-L1 levels in three PEP preparations and adipose-derived mesenchymal stem cell conditioned medium (AMSC-CM). (E-G) (E) IncuCyte proliferation, (F) chemotaxis and (G) wound scratch analysis of HSMM grown in serum-free and 10% fetal bovine serum (FBS)-supplemented medium with increasing concentrations of PEP ranging from 1.25 × 1011 exosomes / mL to 5 × 1011 exosomes / mL.

[0016] [Figure 2-1] Characterization and quantification of purified exosome products (PEP) and in vitro culture of human skeletal myoblasts (HSMM) with increasing concentrations of PEP. Nuclei were counterstained with DAPI (blue). (A) Representative immunocytostaining showing a 96-hour time course of HSMM cultured in serum-free medium (control) or serum-free medium + 2.5x1011 exosomes / mL PEP (PEP). (B) Immunostaining for MyoD. ImageJ blinded quantification of immunostaining for MyoD+ areas / DAPI+ areas, scale = 100 μm. (C) Representative immunocytostaining showing a 96-hour time course of HSMM cultured in serum-free medium (control) or serum-free medium + 2.5x1011 exosomes / mL PEP (PEP). (D) ImageJ blinded quantification of immunostaining for Pax7+ areas / DAPI+ areas. NS = not significant, *p<0.05, **p<0.005. [Figure 2-2]Characterization and quantification of purified exosome products (PEP) and in vitro culture of human skeletal myoblasts (HSMM) with increasing concentrations of PEP. Nuclei were counterstained with DAPI (blue). (A) Representative immunocytostaining showing a 96-hour time course of HSMM cultured in serum-free medium (control) or serum-free medium + 2.5x1011 exosomes / mL PEP (PEP). (B) Immunostaining for MyoD. ImageJ blinded quantification of immunostaining for MyoD+ areas / DAPI+ areas, scale = 100 μm. (C) Representative immunocytostaining showing a 96-hour time course of HSMM cultured in serum-free medium (control) or serum-free medium + 2.5x1011 exosomes / mL PEP (PEP). (D) ImageJ blinded quantification of immunostaining for Pax7+ areas / DAPI+ areas. NS = not significant, *p<0.05, **p<0.005.

[0017] [Diagram 3] Characterization, quantification of purified exosome products (PEP) and in vitro human skeletal myoblast (HSMM) culture with increasing concentrations of PEP. Nuclei were counterstained with DAPI (blue). (A) Representative immunocytochemical staining of HSMM cultured in serum-free medium (control) or serum-free medium + 2.5x1011 exosomes / mL PEP (PEP) over a 96-hour time course. Immunostaining for MyoD myosin heavy chain (MHC), scale = 20 μm. (B) ImageJ blinded quantification of myosin heavy chain+ areas / number of DAPI objects compared to day 0. NS = not significant, *p<0.05, **p<0.005.

[0018] [Figure 4] Figure 2. Resveratrol dose-dependent inhibition of PEP-mediated HSMM proliferation. Representative live cell images of HSMM cultured in basal medium, PEP at 2.5x1011 exosomes / mL, and PEP at 2.5x1011 exosomes / mL with increasing concentrations of resveratrol. Scale = 400 μm (NS = not significant, *p<0.05, **p<0.0001).

[0019] [Diagram 5]Resveratrol dose-dependent inhibition of PEP-mediated HSMM proliferation. (A) Representative proliferation growth curves of HSMM cultured in basal medium, PEP at 2.5x1011 exosomes / mL, and PEP at 2.5x1011 exosomes / mL with increasing concentrations of resveratrol. Scale=400μm (NS=not significant, *p<0.05, **p<0.0001). (B) Resveratrol dose-dependent inhibition of HSMM proliferation. Data points from live cell analysis corresponding to each growth condition (Figure 4 and Figure 5A) at t=72h.

[0020] [Figure 6] Resveratrol dose-dependent inhibition of PEP-mediated HSMM proliferation. (A) Western blot of NF-κB p65 expression in HSMM cells cultured in medium supplemented with 2.5×1011 exosomes / mL PEP alone or increasing concentrations of resveratrol. (B) Comparison of basal NF-κB p65 protein expression with that obtained from cells cultured with 2.5×1011 exosomes / mL PEP alone or increasing concentrations of resveratrol (all compared to PEP, *p<0.05, **p<0.0001).

[0021] [Figure 7] FIG. 1 is a schematic diagram of the hypothesized mechanism of PEP donation to skeletal muscle growth and the role of NF-κB p65.

[0022] [Figure 8-1] Diagram of PEP repair of muscle volume loss latissimus dorsi defect in rats. (A) Study timeline. (B) Schematic of latissimus dorsi muscle volume loss rat model. (C) Scanning electron microscopy of TISSEEL (Baxter International, Inc., Deerfield, IL, left) and TISSEEL reconstituted with 1 x 1012 exosomes / mL PEP (right). Scale = 500 nm. [Figure 8-2]Diagram of PEP repair of rat latissimus dorsi muscle volume loss defect. (A) Study timeline. (B) Schematic of latissimus dorsi muscle volume loss rat model. (C) Scanning electron microscopy of TISSEEL (Baxter International, Inc., Deerfield, IL, left) and TISSEEL reconstituted with 1 x 1012 exosomes / mL PEP (right). Scale = 500 nm.

[0023] [Figure 9] PEP repair of muscle volume loss latissimus dorsi defect (VML) in rats. TISSEEL release assay to demonstrate sustained exosome release. Triplicates were reported every 24 hours and serum-free medium was changed daily.

[0024] [Figure 10] PEP repair of muscle volume loss latissimus dorsi defect (VML) in rats. (A) Representative photographs of macroscopic healing at the injury site, (B) Masson's trichrome staining of VML sites treated with saline, TISSEEL, and TISSEEL reconstituted with 1x1012 exosomes / mL PEP. The dashed area highlights the area of ​​the biopsy punch injury. Images obtained at 10x magnification.

[0025] [Figure 11] Representative sections immunostained from VML sections treated with saline sham, TISSEEL, or TISSEEL reconstituted with PEP at 1x1012 exosomes / mL. Nuclei were counterstained with DAPI (blue), skeletal muscle protein desmin (red), and EdU cell proliferation (cyan). Scale bar = 500 μm.

[0026] [Figure 12] ImageJ blinded assay (red) of de novo skeletal muscle proliferation by calculating the percentage of EdU positive cells per mm2 of desmin positive area. *p<0.05, **p<0.005.

[0027] [Figure 13]Illustrative diagram of intraurethral pressure after creation of the porcine SUI model and repair of urethral sphincter lesion with PEP. (A) Timeline of study. (B) Schematic of cystoscope-guided transurethral full thickness, focal urethral sphincter defect to create the porcine SUI model. Note the apposition of the urethral sphincter to the anterior surface of the vagina and urethra. (C) Schematic of the gross appearance of the urethral sphincter defect at day 0, before injection of intervention (day 7), and before euthanasia (day 42). (D) Modified Medspira mCompass pressure catheter highlighting stabilization of the catheter at the bladder neck (grey) and the movable pressure sensor (green) used to obtain pressure along the length of the porcine vagina and urethra.

[0028] [Figure 14] Illustrates intraurethral pressure after preparation of a porcine SUI model and repair of urethral sphincter lesions with PEP. (A) Scanning electron microscopy of collagen and collagen reconstituted with 1x1012 exosomes / mL PEP. Scale=1 μm. (B) Collagen release assay showing sustained exosome release (n=3).

[0029] [Figure 15] FIG. 1 shows the intraurethral pressure after creation of a porcine SUI model and repair of the urethral sphincter lesion with PEP. Representative cystoscopic images of the urethral sphincter lesion at 7 days before intervention delivery and at 42 days before euthanasia. Arrows indicate the site of the urethral sphincter defect.

[0030] [Figure 16] Graph of intraurethral pressure after creation of a porcine SUI model and repair of urethral sphincter lesions with PEP. Graph of mean intraurethral pressure pre-injury and post-injury on day 0 (n=10), pre-injection on day 7 (n=10), and pre-euthanasia on day 42. Collagen = injection of collagen; PEP = injection of collagen reconstituted with PEP at 1x1012 exosomes / mL. D42 control = urethral sphincter defect repaired with collagen (n=4). Day 42 PEP = urethral sphincter defect repaired with collagen reconstituted with PEP at 1x1012 exosomes / mL (n=6). *p<5x10-8; **p<5x10-14; #p<1x10-5; ##p<1x10-7;

number

[0031] [Figure 17] Immunohistochemical analysis of PEP-induced repair of urethral sphincter injury. Representative sections of urethral sphincter lesions repaired with collagen or collagen reconstituted with PEP at 1x1012 exosomes / mL, and stained intact urethra. (A) Nuclei were counterstained with DAPI (blue), skeletal muscle with desmin (red), and EdU (white). Scale bar = 100 μm. Magnified views of representative sections of intact urethra (intact), and urethral sphincter lesions repaired with collagen (collagen) or collagen reconstituted with PEP at 1x1012 exosomes / mL (PEP). (n=9, 18, 15, respectively).

[0032] [Figure 18] Immunohistochemical analysis of PEP-induced repair of urethral sphincter injury. Representative sections of urethral sphincter lesions repaired with collagen or collagen reconstituted with PEP at 1x1012 exosomes / mL, and stained intact urethra. Tissues were stained for PD-L1 (green), p65 NF-κB (red), and DAPI (blue). Merge overlay highlights that PEP-treated samples contained colocalized PD-L1 / NF-κB p65 staining in tissue. (n=15, 15, 15), scale=20μm.

[0033] [Figure 19]Immunohistochemical analysis of PEP-induced repair of urethral sphincter injury. Representative sections of urethral sphincter lesions repaired with collagen or collagen reconstituted with PEP at 1x1012 exosomes / mL and intact urethra stained. (A) ImageJ blinded quantification of de novo skeletal muscle proliferation by calculating the number of EdU positive cells per mm2 of desmin positive area. (B) ImageJ blinded quantification of PD-L1 expression by analysis of PD-L1 positive tissue area per total tissue area. (C) ImageJ blinded quantification of NF-κB p65 expression by analysis of NF-κB p65+ tissue area per total tissue area. *p<0.05.

[0034] [Figure 20] FIG. 13: PEP induces M2 macrophage polarization in a porcine SUI model. Representative sections of urethral sphincter lesions repaired with collagen, collagen reconstituted with PEP at 1×1012 exosomes / mL, or intact urethra. Tissues were stained for general macrophage antigen (Mφ; green), CD163 M2-specific macrophages (red), and DAPI (blue). Merge overlay highlights that PEP samples contained colocalized staining of Mφ+ / CD163+ tissue. Representative sections of intact urethra (Intact) and urethral sphincter lesions repaired with collagen (Collagen) or collagen reconstituted with PEP at 1×1012 exosomes / mL (PEP). (n=12, 15, 20, respectively), scale=20 μm.

[0035] [Figure 21]FIG. 13: PEP induces M2 macrophage polarization in a porcine SUI model. Representative sections of urethral sphincter lesions repaired with collagen, collagen reconstituted with PEP at 1x1012 exosomes / mL, or intact urethra. ImageJ blinded assay of Mφ and M2 staining. M2:M1 ratios were determined from comparison of M2:(Mφ-M2) image areas. Haldane correction was used to account for division by 0 in M2:M1 ratios. This correction adds 0.5 to all values ​​in the list to ensure the calculation is error-free. Graphs represent log base 2 ratios of M2:M1 macrophages. The dotted line at y=0 indicates the value where the ratios are equal. The log base 2 scale indicates M2:M1>1 for values ​​of Log2>0. *p<0.05.

[0036] [Figure 22-1] Characterization of cGMP PEP preparations. (A) Atomic force microscopy comparing platelet conditioned medium EV isolation using centrifugation versus the PEP process. Scale bars are embedded in the images. (B) Representative images obtained from single particle interference reflectance imaging sensing (SP-IRIS) analysis for the presence of surface CD41a, CD9, CD63 and CD81 tetraspanins. (C) Graphical representation of SP-IRIS analysis. (D) Quantification of CD9, CD63 and CD81 on CD41a capture plates scored the majority of PEP as CD41a / CD9 positive with less CD63 and background CD81. Data presented as mean ± standard deviation. n=3 separate cGMP manufactured PEP lots. (E) Pie chart representation of exosomal tetraspanin surface marker profile of CD41a-captured PEP exosomes. [Figure 22-2]Characterization of cGMP PEP preparations. (A) Atomic force microscopy comparing platelet conditioned medium EV isolation using centrifugation versus the PEP process. Scale bars are embedded in the images. (B) Representative images obtained from single particle interference reflectance imaging sensing (SP-IRIS) analysis for the presence of surface CD41a, CD9, CD63 and CD81 tetraspanins. (C) Graphical representation of SP-IRIS analysis. (D) Quantification of CD9, CD63 and CD81 on CD41a capture plates scored the majority of PEP as CD41a / CD9 positive with less CD63 and background CD81. Data presented as mean ± standard deviation. n=3 separate cGMP manufactured PEP lots. (E) Pie chart representation of exosomal tetraspanin surface marker profile of CD41a-captured PEP exosomes. [Figure 22-3] Characterization of cGMP PEP preparations. (A) Atomic force microscopy comparing platelet conditioned medium EV isolation using centrifugation versus the PEP process. Scale bars are embedded in the images. (B) Representative images obtained from single particle interference reflectance imaging sensing (SP-IRIS) analysis for the presence of surface CD41a, CD9, CD63 and CD81 tetraspanins. (C) Graphical representation of SP-IRIS analysis. (D) Quantification of CD9, CD63 and CD81 on CD41a capture plates scored the majority of PEP as CD41a / CD9 positive with less CD63 and background CD81. Data presented as mean ± standard deviation. n=3 separate cGMP manufactured PEP lots. (E) Pie chart representation of exosomal tetraspanin surface marker profile of CD41a-captured PEP exosomes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure describes compositions and methods for repairing damaged or injured skeletal muscle tissue. Generally, the compositions include purified exosome products (PEP). When applied to damaged or injured skeletal muscle tissue, the PEP compositions promote healing and / or repair of damaged or injured skeletal muscle tissue.

[0038] Damaged Skeletal Muscle Although described below with respect to exemplary embodiments in which the skeletal muscle is a urethral sphincter or latissimus dorsi muscle, the methods described herein may include repair of damage to any skeletal muscle. Other exemplary skeletal muscles that may be repaired using the methods described herein include, but are not limited to, the deltoid, trapezius, sternocleidomastoid, abdominal, oblique, and adductor muscles.

[0039] Urinary incontinence afflicts up to 40% of adult women in the United States. Stress urinary incontinence (SUI) accounts for approximately one-third of urinary incontinence cases and leads to approximately 200,000 surgical procedures annually. Urinary continence is maintained through the interaction of the urethral support and the urethral sphincter junction, especially during activities that increase abdominal pressure. Currently, surgical correction of SUI focuses on reconstruction of the urethral support. However, mesh-based repairs can be associated with foreign body reactions and poor local tissue healing leading to mesh exposure, necessitating the exploration of techniques that both restore external urethral sphincter function and limit surgical risks.

[0040] This disclosure describes the use of a purified exosome product (PEP), engineered to be enriched for NF-κB-positive and PD-L1-positive exosomes, as a clinical-grade, off-the-shelf biologic to drive skeletal muscle regeneration. NF-κB and PD-L1 are proteins that play a role in suppressing the immune system. PD-L1 in PEP exosomes + / NF-κB + The population was PD-L1 - / NF-κB - They may promote tissue repair more than exosomes. In one or more embodiments, the PEP exosomes described herein are positive for CD63, CD9, and / or flotillin.

[0041] As described herein, application of PEP to skeletal satellite muscle in vitro promoted proliferation and differentiation in an NF-κB-dependent manner. In vivo, suspension of PEP-exosomes in hydrogels achieved sustained exosome release and induced functional restoration of the external urinary sphincter with new skeletal muscle formation, and polarization of local macrophages toward a regenerative M2 phenotype. The data presented herein support the use of PEP as an off-the-shelf, cell-independent, exosome-based approach to restore skeletal muscle function and provide non-surgical management of SUI.

[0042] In one or more embodiments, the composition comprising PEP forms a hydrogel. The composition may form a hydrogel prior to application to the area to be treated, or the composition may form a hydrogel after application to the area to be treated. In one or more particular embodiments, the composition may form a hydrogel upon incubation at an incubation temperature. The incubation temperature may be, for example, at least 30°C, at least 31°C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, or at least 37°C. The incubation temperature may be, for example, at most 45°C, at most 44°C, at most 43°C, at most 42°C, at most 41°C, at most 40°C, at most 39°C, or at most 38°C. The incubation temperature may be between 33°C and 40°C, for example between 35°C and 39°C, or 37°C.

[0043] It may be desirable for the composition to form a hydrogel after being incubated at the desired treatment site for a given time.If the composition forms a hydrogel too quickly, it may be more difficult to deliver or distribute the composition into the treatment site.In one or more embodiments, the composition may form a hydrogel, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 8 minutes, 10 minutes, 12 minutes, or 15 minutes after application.

[0044] This disclosure describes the use of purified exosome products (PEP) for the treatment of skeletal muscle, for example, the skeletal muscle of the urethral sphincter to treat stress urinary incontinence. Interaction between urethral support and urethral sphincter contraction is typically required to achieve optimal coaptation and maintain urinary continence during activities that result in increased abdominal pressure. Events such as vaginal birth can disrupt the above coordinated events and impair urinary continence. Most surgical approaches to correct SUI focus on re-establishing urethral support and pay little attention to restoring urethral sphincter function. For example, mid-urethral sling surgery is a minimally invasive procedure that uses thin strips of polypropylene mesh placed under the urethra to support the urethra, but does not address sphincter function. Concerns over mesh-based treatment options highlight the need for safer and more effective non-surgical and non-mesh-based treatment options. As noted above, while described with respect to an exemplary embodiment in which the skeletal muscle is a urethral sphincter muscle, the compositions and methods described herein may include repair of damage to any skeletal muscle.

[0045] PEP inhibits CD63 expression, which is enriched for NF-κB and PD-L1. + / CD9 + / Flotillin + To produce exosome populations, they were prepared from platelet populations maintained in suspension culture. In vitro treatment of myoblasts with PEP exosomes induced chemotaxis, proliferation, and differentiation in an NF-κB-dependent manner. In vivo, treatment with PEP exosomes induced skeletal muscle repair in a rat model of muscle volume loss. In a porcine SUI model, minimally invasive delivery of PEP resulted in functional recovery of the external sphincter muscle associated with enhanced muscle proliferation, local upregulation of NF-κB and PD-L1, and polarization of local macrophages to an M2 fate. The data presented herein provide mechanistic and interpretational support for the use of acellular biologics that can induce local progenitor cell migration and differentiation following minimally invasive delivery in a porcine model of SUI.

[0046] The production of purified exosome product (PEP) involves separating plasma from blood and isolating a solution of exosomes from the separated plasma by filtration and centrifugation. PEP is fully characterized and methods for its preparation are described in International Patent Application No. PCT / US2018 / 065627 (published as WO 2019 / 118817), U.S. Patent Application Publication No. 2021 / 0169812 (A1), and U.S. Patent No. 10,596,123, which are incorporated herein by reference in their entireties. Typically, the blood is from a human, although other blood sources are certainly contemplated.

[0047] Briefly, PEP is a purified exosome product prepared using a freeze-drying process that produces a product with a different structure than exosomes prepared using conventional methods. For example, PEP typically has a spherical or spheroidal structure and an intact lipid bilayer, rather than a crystalline structure resulting from lipid reaggregation of the exosome lipid bilayer after exosomes are disrupted during conventional exosome preparation methods. Spherical or spheroidal exosome structures generally have a diameter of 300 nm or less. Typically, PEP preparations contain spherical or spheroidal exosome structures with a relatively narrow size distribution. In some preparations, PEP contains spherical or spheroidal exosome structures with a mean diameter of about 110 nm ± 90 nm, with the majority of exosome structures having a mean diameter of 110 nm ± 50 nm, such as 110 nm ± 30 nm.

[0048] Unmodified PEP preparations, i.e., PEP preparations whose characteristics have not been altered by selection or separation of the exosome population in the preparation, exhibited CD63 + Exosomes and CD63 - Naturally contains a mixture with exosomes. CD63 - Exosomes can inhibit unlimited cell proliferation, and thus CD63 + and CD63 -Unmodified PEP preparations that naturally contain exosomes can stimulate cell proliferation for wound repair and / or tissue regeneration or limit uncontrolled cell proliferation.

[0049] In addition, CD63 + Exosome sorting revealed that CD63 was expressed in naturally isolated PEP preparations. + Extract the exosomes and then elucidate the desired amount of CD63 + By returning exosomes, CD63 in the PEP product + Exosomal CD63 - The ratio of PEP to exosomes can be controlled. In one or more embodiments, the PEP preparation contains CD63 - It may only have exosomes.

[0050] In one or more embodiments, the PEP preparation comprises CD63 + Exosomes and CD63 - Exosomes may contain both CD63 and CD63. + Exosomal CD63 - The ratio of CD63 to exosomes can vary, at least in part, depending on the amount of cell expansion desired in a particular application. + / CD63 - Exosome ratio is CD63 + Desired cell proliferation induced by exosomes and CD63 achieved through cell contact inhibition - In certain scenarios, such as tissues with non-adherent cells (e.g., blood-derived components), this ratio can be adjusted to provide the appropriate balance of cell proliferation or cell inhibition for the tissue being treated. For example, in tissues with non-adherent cells, cell-to-cell contact is not a trigger, so CD63 + The ratio of exosomes can be reduced to avoid unlimited cell proliferation. Conversely, if one wishes to expand a clonal population of cells, such as in allogeneic cell-based therapies or immunotherapy, one can use CD63 to ensure that one can obtain a large cell population from a very small source. +The ratio of exosomes can be increased.

[0051] Thus, in one or more embodiments, CD63 in the PEP preparation + Exosomal CD63 - The ratio of CD63 to exosomes can be at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 11:1, at least 12:1, at least 13:1, at least 14:1, at least 15:1, or at least 16:1. + Exosomal CD63 - The ratio of CD63 to exosomes can be at most 15:1, at most 16:1, at most 17:1, at most 18:1, at most 19:1, at most 20:1, at most 25:1, or at most 30:1. + Exosomal CD63 - The ratio of exosomes to PEP may be 1:1 to 30:1, 2:1 to 20:1, 4:1 to 15:1, or 8:1 to 10:1. In one or more particular embodiments, the native PEP, e.g., CD63 + Exosomal CD63 - An unmodified ratio of PEP to exosomes may also be used.

[0052] Exosome characterization Extracellular vesicles engineered from platelet suspension cultures were assessed for size, lipid bilayer, and markers consistent with exosome populations (Figure 1A-C). NanoSight characterization of a distinct platelet exosome preparation, PEP, revealed approximately 6.65 × 10 12A particle concentration of 100 particles / mL and a size average of approximately 154.4 nm were revealed, consistent with an exosome population (Figure 1A). Electron microscopy of PEP confirmed the NanoSight vesicle size distribution, with the majority of purified vesicles being approximately 100-150 nm in diameter (Figure 1B). Western blot analysis demonstrated consistent expression of CD63, CD9, and flotillin-1 (each of which are established exosome markers) in three separate clinical preparations of PEP (Figure 1C). PEP preparations have been described as enriched for NF-κB p65 (2.41-fold; p<0.05) and PD-L1 (4.98-fold; p<0.005) compared to levels seen in exosomes purified from adipose-derived mesenchymal stem cell conditioned medium (AMSC CM; Figure 1D). Atomic force microscopy of EVs purified from platelets using ultracentrifugation demonstrated disruption of the phospholipid bilayer, which was maintained by avoidance of high shear stress purification (Figure 22A). To confirm that the resulting EVs were platelet exosome preparations (PEPs), single particle interference reflectance imaging sensing (SP-IRIS) analysis of the relative amounts of CD63 (6.67±0.58%), CD9 (76.0±0.0%), CD81 (1.0±0.0%), and CD9 / CD63 (16.67±0.58%) within CD41α-positive exosomes was used to assess for the presence of concomitant integrin α2b (CD41) and CD9 expression (Figure 22).

[0053] In one or more embodiments, the PEP exosomes used in the methods and compositions described herein may be positive for one or more protein markers. The presence of protein markers may be measured, for example, by Western blotting, enzyme-linked immunosorbent assay (ELISA), flow cytometry, dot blotting, or any other suitable molecular biology assay. In one or more embodiments, the PEP exosomes may be positive for PD-L1, CD9, flotillin, NF-κB, or a combination thereof. In one or more embodiments described herein, the PEP preparation may be enriched for exosomes containing known anti-inflammatory proteins. The proteins contained in the PEP preparation may be on the lipid membrane or may be contained within the exosome itself. A schematic diagram of a PEP exosome containing some proteins on its surface and one protein within the exosome is shown in FIG. 7.

[0054] The presence of a particular protein in a PEP preparation can be the result of, for example, expression of the protein by the donor from which the PEP is derived. Additionally or alternatively, recombinantly expressed proteins may be added to the PEP preparation.

[0055] Human skeletal myoblasts (HSMM) co-cultured with PEP exhibit dose-dependent proliferation, chemotaxis, cell migration, and skeletal muscle differentiation. Myoblasts grown with PEP achieved higher confluency rates compared to serum-free and 10% fetal bovine serum (FBS)-supplemented media (Figure 1E). 11 At higher PEP concentrations than exosomes / mL, myoblasts proliferated to over 90% confluency, whereas only about 50% confluency was achieved in standard growth medium containing 10% FBS. Similarly, dual-chamber migration assays demonstrated that 2.5 × 10 11 We showed that PEP concentrations above exosomes / mL significantly decreased the total phase area in the upper chamber, suggesting that PEP affects myoblast chemotaxis (Figure 1F). 11This was confirmed in scratch assays, which showed that wound confluency was enhanced in the 0.1% (exosomes / mL) condition compared to FBS (Figure 1G).

[0056] 2.5×10 11 After treatment with exosomes / mL PEP, myogenic lineage maturation from satellite cells to multinucleated myotubes occurred (Figures 2 and 3). Specifically, myogenic lineage from myoblasts (identified by expression of Pax7 and MyoD) to myotubes (identified by expression of myosin heavy chain (MHC, myosin heavy chain +)) was identified after 96 hours by culturing HSMM with PEP (Figures 2 and 3). By day 4, PEP was able to induce significant MHC induction (14859±3049, pixel intensity / area, p<0.01) to proceed myogenic lineage maturation, in contrast to basal medium (1949±1106, pixel intensity / area) (Figures 3A,B).

[0057] Resveratrol inhibits PEP-mediated human skeletal myoblast (HSMM) proliferation To assess whether PEP donation of NF-κB p65 is involved in mediating myogenic proliferation and differentiation, HSMM proliferation, cell number, and morphology analyses were performed with PEP alone or in combination with increasing concentrations of resveratrol (a known inhibitor of NF-κB p65). Myoblast proliferation was dose-dependently inhibited by resveratrol. 250 μM resveratrol inhibited proliferation by approximately 50%, and 500 μM resveratrol inhibited proliferation by nearly 100% (PEP vs. 250 μM = p<0.0001, PEP vs. 500 μM = p<0.0001; Figure 4, Figure 5A). Myoblast proliferation was not significantly inhibited when treated with PEP alone and low doses of resveratrol (e.g., 50 μM), but the proliferation rate was reduced at higher resveratrol concentrations (Figure 5B). Western blot analysis of cells grown in each condition showed increased levels of NF-κB p65 (a subunit of NF-κB) in PEP-treated HSMM, accompanied by a dose-dependent decrease in NF-κB expression after resveratrol treatment (Figure 6A, B). These findings support that PEP increases NF-κB expression in HSMM NF-κB p65 levels and the importance of this mitogen in driving skeletal myoblast growth (Figure 7).

[0058] Repair of muscle volume loss (VML) latissimus dorsi defects in rats with purified exosome products enhances skeletal muscle regeneration. To evaluate the effect of PEP on skeletal muscle in vivo, we used a rat muscle volume loss model in which large muscle injuries were created to exceed the capacity of endogenous satellite stem cells for self-repair. Latissimus dorsi defects (8 mm) were created by punch biopsy and were then implanted with saline (Sham; n = 9), clinical grade fibrin glue (TISSEEL, n = 9), or 1 × 10 12 After reconstitution, PEP exosomes were uniformly attached to fibrin fibers as shown by scanning electron microscopy (Fig. 8C). Furthermore, 1 × 10 12TISSEEL combined with exosomes / mL resulted in sustained release of exosomes (Figure 9). Animals were treated with 2'-deoxy-5-ethynyluridine (EdU) as a thymidine analogue to monitor cell division and proliferation.

[0059] All animals survived until sacrifice at 8 weeks after treatment. Sham-treated animals had persistent muscle defects with fatty infiltration, whereas animals repaired with TISSEEL or TISSEEL plus PEP had macroscopically healed defects (Fig. 10A). Histological characterization revealed that TISSEEL alone predominantly induced fatty infiltration accompanied by an inflammatory response, in contrast to the absence of tissue in the sham treatment. Conversely, defects treated with PEP had extensive skeletal muscle regrowth, resulting in a reduction in the area of ​​the defect compared to both sham- and TISSEEL-treated rats (Fig. 10A,B). Immunohistochemical evaluation revealed that desmin + EdU in tissue areas + We demonstrated colocalization of cells, suggesting de novo skeletal muscle regrowth at the injury site (Figure 11). 2 There was significantly more EdU staining in desmin-positive cells per 1000 cells compared to sham controls (p<0.001), but not compared to TISSEEL treatment alone (p=0.24) (Figure 12). These data suggest that PEP affects muscle regeneration at the injury site.

[0060] Recovery of intraurethral pressure after urethral sphincter repair with PEP in a novel porcine SUI model A porcine SUI model was used to evaluate whether skeletal muscle regenerated with PEP integrates with the surrounding uninjured muscle to restore pre-injury function of the urethral sphincter. This model was chosen due to the anatomical similarity of the urethral sphincter between humans and pigs. A total of 10 female Yorkshire cross pigs weighing 70-80 kg were used (4 controls, 6 experimental). Under sterile conditions and general anesthesia, full-thickness lesions approximately 2 cm in length extending through the mucosa and urethral sphincter were created cystoscope-wise at the 6 o'clock position starting 1 cm from the urethral opening and extending cephalad (Figure 13B). Animals were allowed to recover for approximately 7 days and then returned to the operating room where they were treated with a total of 5 mL of collagen or 1 × 10 12 Collagen containing PEP exosomes / mL was injected in aliquots of approximately 0.5 cc over 10 injection points along the length of the previously created urethral sphincter defect (FIG. 13C). In contrast to TISSEEL, which polymerizes instantly, 5 mg / mL collagen hydrogel takes several minutes to gel at 37°C, making it more suitable as a vehicle for injection via the cystoscope. PEP showed a binding affinity for collagen similar to that for TISSEEL on SEM (FIG. 14A). Similarly, 1×10 12 Collagen combined with exosomes / mL resulted in sustained release of exosomes (Figure 14B). Intraurethral pressure profiles were used to quantify sphincter function using a modified MEDSPIRA mCompass manometric pressure catheter. Pressure was recorded pre- and post-injury on day 0, pre-treatment on day 7, and prior to euthanasia on day 42 post-treatment (Figure 13D). Intraurethral pressure was recorded for 10 seconds at 1 cm intervals starting from the vagina to the bladder neck (Figure 13D). Three pressure readings from each 10 second recorded segment were averaged to obtain the mean intraurethral pressure at that location. EdU at a dose of 5 mg / kg was administered orally to all animals twice weekly throughout the study as a thymidine analogue to monitor cell division and proliferation.

[0061] No deaths or infections were observed during the study. Two animals developed overt urinary incontinence between lesion formation and euthanasia, suggesting that this approach provides a model with the desired impact on urethral sphincter function. Animals treated with collagen alone had persistent muscle defects, whereas animals repaired with collagen and PEP had macroscopically healed defects (Figure 15). Mean intraurethral pressure changed only slightly on day 0, but dropped significantly by day 7, quantitatively validating the model (Figure 16). At day 42, animals treated with collagen alone had no significant improvement in sphincter function as measured by intraurethral pressure. In contrast, animals treated with PEP had significant recovery of intraurethral pressure relative to pre-injection on day 7 and to collagen alone on day 42 (Figure 16).

[0062] Histological characterization of urethral sphincter lesion repair Representative sections from both the injured and intact segments of the urethra were stained for desmin (a cytoskeletal intermediate filament found in the sarcomeres of adult myoblasts) to identify skeletal muscle, EdU (a thymidine analog used to monitor cell division and proliferation) to identify newly synthesized cells, and DAPI to identify newly synthesized nuclei. While EdU / desmin colocalization was limited in collagen-treated urethral lesions and intact urethral tissue (Figure 17), abundant colocalization of desmin and EdU positive cells was seen in the PEP-injected injury sites (Figure 17). Magnification of these areas revealed multinucleated cells staining positive for the above markers only in the PEP-treated injury sites, suggesting de novo skeletal muscle cell regeneration with differentiation into multinucleated myotubes (Figure 17, magnified inset). Quantification by blinded observers revealed no significant increase in desmin in the PEP-treated injury sites. + 1mm in the area 2 We demonstrated that there were significantly more EdU positive cells per urethra compared to collagen controls and compared to intact urethras (FIG. 19A).

[0063] NF-κB and PD-L1 expression at injury sites and immune responses induced by PEP PD-L1 and NF-κB expression and colocalization were observed in significantly higher percentages in sections of injured urethra treated with PEP compared to sections of injured urethra treated with collagen alone (Figure 18). NF-κB and PD-L1 expression promotes M2 macrophage polarization, which promotes skeletal muscle regeneration. Intact urethra lacked a triggering event and had limited macrophage staining for either M1 or M2 macrophages (Figure 20, Figure 21). Macrophage response to collagen treatment appeared to be limited to less prominent undifferentiated macrophage staining with a paucity of M2 macrophages (Figure 20, Figure 21). In contrast, abundant M2-dominant macrophages were found only in PEP-treated (Figure 20). Quantification supported M2 polarization in the PEP-treated cohort, where polarization was not observed with collagen alone (Figure 21).

[0064] Thus, this disclosure describes the use of platelet-derived exosomes (PEP) to stimulate myoblast growth in vitro and in two separate in vivo models. PEP produced dose-dependent effects on myoblast proliferation, cell migration, and chemotaxis. Resveratrol inhibition of PEP-mediated myoblast proliferation suggested a critical role for NF-κB p65, a molecule detected in PEP. In vivo, treatment with PEP produced significantly higher skeletal muscle repopulation in a VML latissimus dorsi model and improved urinary sphincter function in a PEP-treated porcine model of SUI. Colocalization of EdU and desmin, sustained expression of NF-κB and PDL-1, and M2 macrophage polarization indicate in vivo activation of these established muscle regeneration molecular cues in PEP-treated tissues. Collectively, these findings support the use of PEP, an exosome-based, off-the-shelf cell-free platform, to restore skeletal muscle function.

[0065] The PEP exosome technology described herein offers a potential solution to the complications of muscle biopsy, high manufacturing costs, dose-to-dose variability, and obstacles associated with cell transport and cell manipulation that exist with the use of myocyte precursor (MCP) therapy. PEP exosomes are scalable, stable at room temperature, and stable as lyophilized products. Furthermore, PEP eliminates the need for exogenous myoblast transplantation to drive the desired muscle regeneration events by activating local myocyte precursors.

[0066] Satellite cells are mononucleated skeletal muscle stem cells that reside in a quiescent state in close opposition to mature myofibers until activated by muscle injury. Upon activation, cells expand to both replace the stem cell pool and repair damaged tissue. The repair-prospective cells differentiate into myotubes that line damaged fibers and fuse with surrounding undamaged muscle to heal the injury. Macrophages are pleiotropic immune cells involved in muscle repair and regeneration, among other functions. Specifically, an orchestrated shift from an early pro-inflammatory response to a wound-healing and repair response is required for muscle regeneration after injury. The early pro-inflammatory response is mediated by M1 macrophages and is required for myofiber and wound debridement. Wound healing and repair involve direct interactions between M2 macrophages and satellite cells at the site of myofiber injury. Under local inflammatory conditions and during injury repair, myoblasts express several B7-related (CD80 and CD86) costimulatory and inhibitory molecules, including PD-L1, a potent immune regulator detected in large amounts during PEP. Here, exogenous PD-L1 donated by PEP drove a pro-regenerative transition from proinflammatory M1 to M2 macrophages at the site of PEP-treated urethral sphincter.

[0067] Therefore, the present disclosure describes compositions and methods for repairing injured or damaged skeletal muscle in a subject.Generally, the composition comprises PEP and a pharma- ceutically acceptable carrier.In a surgical setting, PEP may be combined with a carrier suitable for repairing skeletal muscle, such as, for example, surgical adhesive, tissue adhesive, and / or support matrix (e.g., collagen scaffold).

[0068] Thus, the method includes administering an effective amount of the composition to repair damaged skeletal muscle. In this embodiment, an "effective amount" is an amount effective to induce expression of NK-κB, induce expression of PD-L1, and / or promote polarization into M2 macrophages.

[0069] As used herein, a "subject" can be a human or any non-human animal. Exemplary non-human animal subjects include, but are not limited to, livestock animals or companion animals. Exemplary non-human animal subjects include, but are not limited to, members of the family Hominidae (including, for example, chimpanzees, gorillas, or orangutans), subfamily Bovinae (including, for example, cattle), subfamily Caprinae (including, for example, goats), genus Ovis (including, for example, sheep), porcinae (including, for example, pigs), family Equidae (including, for example, horses), members of the family Cervidae (including, for example, deer, elk, moose, caribou, reindeer, etc.), members of the family Bisonidae (including, for example, bison), family Felidae (including, for example, domestic cats, tigers, lions, etc.), family Canidae (including, for example, domestic dogs, wolves, etc.), birds (including, for example, turkeys, chickens, ducks, geese, etc.), rodents (including, for example, mice, rats, etc.), members of the family Leporidae (including, for example, rabbits or hares), members of the family Mustelidae (including, for example, ferrets), or members of the order Chiroptera (including, for example, bats).

[0070] PEP can be formulated with a pharma- ceutically acceptable carrier to form a pharmaceutical composition. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, hydrogel, carrier solution, suspension, colloid, and the like. The use of such media and / or agents for pharma- ceutical active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the composition. As used herein, "pharma- ceutical acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance can be administered to an individual together with PEP without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is included. As mentioned above, in a surgical setting, exemplary suitable carriers include surgical adhesives, tissue adhesives, or support matrices (e.g., collagen scaffolds).

[0071] In one or more embodiments, the PEP composition may include additional components, either in admixture with the PEP vesicles or loaded into at least a portion of the PEP vesicles. In one or more embodiments, the additional components may promote healing. In one or more embodiments, the additional components may include one or more biocompatible scaffold components, such as, for example, collagen, fibrin, fibronectin, laminin, proteoglycan, hyaluronic acid, alginate, or other biocompatible scaffold components. In one or more embodiments, the additional components may include one or more pharmacologic active components. In one or more embodiments, the pharmacologic active components may be selected, for example, to decrease healing time, provide pain relief, or reduce the likelihood or severity of complications (e.g., infection). Pharmaceutically active components include, for example, steroids (e.g., estrogen), painkillers, or antibiotics. One or more additional components may be loaded into at least a portion of the PEP vesicles. The presence of a particular component in a PEP preparation can be, for example, the result of expression of the component (e.g., a protein) by the donor cells from which the PEP is derived. Additionally or alternatively, one or more components can be loaded into at least a portion of the PEP vesicles, either directly (loading of the component itself) or indirectly (e.g., recombinantly expressed proteins).

[0072] Pharmaceutical compositions containing PEP can be formulated in various forms suitable for the preferred route of administration. Thus, pharmaceutical compositions can be administered via known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., application to exposed nerve tissue during surgery, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.). Pharmaceutical compositions can be administered to mucosal surfaces, such as, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). Pharmaceutical compositions can also be administered via sustained or delayed release.

[0073] Thus, the pharmaceutical composition may be provided in any suitable form, including but not limited to the form of a solution, suspension, emulsion, spray, aerosol, or any mixture.The pharmaceutical composition may be provided in a formulation that includes any pharma- ceutically acceptable excipient, carrier, or vehicle.For example, the formulation may be provided in a conventional topical dosage form, such as, for example, cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, etc.The formulation may further include one or more additives, including, for example, adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, thickeners, etc.

[0074] The formulation may be provided in a convenient unit dosage form and can be prepared by a method known in the art of pharmacy.The method of preparing the composition containing a pharmaceutically acceptable carrier comprises the step of associating PEP with the carrier, which constitutes one or more accessory ingredients.In general, the formulation can be prepared by uniformly and / or intimately associating PEP with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0075] The amount of PEP administered may vary depending on various factors, including but not limited to the content and / or source of PEP administered, the weight, health condition, and / or age of the subject, and / or the route of administration.Therefore, the absolute weight of PEP contained in a given unit dosage form may vary widely and depends on factors such as the species, age, weight, and health condition of the subject, and / or the method of administration.Therefore, it is not practical to generally state the amount that constitutes the amount of PEP that is effective for all possible uses.However, those skilled in the art can easily determine the appropriate amount by fully considering such factors.

[0076] In one or more embodiments, the dose of PEP can be measured in terms of PEP exosomes delivered per dose. Thus, in one or more embodiments, the method can include administering, for example, about 1×10 6 PEP exosomes ~ approx. 1 x 10 15This may include administering sufficient PEP to provide a dose of PEP exosomes to the subject, although in one or more embodiments the method may be practiced by administering PEP at a dose outside this range.

[0077] Thus, in one or more embodiments, the method comprises: 6 PEP exosomes, at least 1 × 10 7 PEP exosomes, at least 1 × 10 8 PEP exosomes, at least 1 × 10 9 PEP exosomes, at least 1 × 10 10 PEP exosomes, at least 1 × 10 11 PEP exosomes, at least 2 × 10 11 PEP exosomes, at least 3 × 10 11 PEP exosomes, at least 4 × 10 11 PEP exosomes, at least 5 × 10 11 PEP exosomes, at least 6 × 10 11 PEP exosomes, at least 7 × 10 11 PEP exosomes, at least 8 × 10 11 PEP exosomes, at least 9 × 10 11 PEP exosomes, at least 1 × 10 12 PEP exosomes, 2 × 10 12 PEP exosomes, at least 3 × 10 12 PEP exosomes, at least 4 × 10 12 PEP exosomes, at least 5 × 10 12 PEP exosomes, at least 1 × 10 13 PEP exosomes, or at least 1 × 10 14 This may include administering sufficient PEP to provide a minimal dose of PEP exosomes.

[0078] In one or more embodiments, the method comprises: 15 PEP exosomes, 1 × 10 14 PEP exosomes, 1 × 10 13PEP exosomes, 1 × 10 12 PEP exosomes, 1 × 10 11 or 1×10 10 This may include administering sufficient PEP to provide a maximum dose of PEP exosomes of:

[0079] In one or more embodiments, the method may include administering sufficient PEP to provide a dose characterized by a range having endpoints defined by any minimum dose identified above and any maximum dose greater than the minimum dose. For example, in one or more embodiments, the method may include administering a dose of 1×10 11 ~1×10 13 Dose of PEP exosomes, e.g., 1 x 10 11 ~5×10 12 Dose of PEP exosomes, 1 × 10 12 ~1×10 13 Dose of PEP exosomes, or 5 × 10 12 ~1×10 13 In one or more embodiments, the method may include administering sufficient PEP to provide a dose equal to any minimum dose or any maximum dose recited above. Thus, for example, the method may include administering a 1×10 10 PEP exosomes, 1 × 10 11 PEP exosomes, 5 × 10 11 PEP exosomes, 1 × 10 12 PEP exosomes, 5 × 10 12 PEP exosomes, 1 × 10 13 PEP exosomes, 1 × 10 14 This may include administering a dose of PEP exosomes.

[0080] Alternatively, the dose of PEP can be measured in terms of the concentration of PEP upon reconstitution from a lyophilized state. Thus, in one or more embodiments, the method can include administering PEP to provide the subject with PEP in a dose of, for example, about a 0.01% solution to a 100% solution, although in one or more embodiments, the method can be practiced by administering PEP in a dose outside this range. As used herein, a 100% solution of PEP refers to a concentration of PEP (about 2×10 11 For comparison, the dose of 0.01% PEP is approximately equivalent to a standard dose of exosomes prepared using conventional methods for obtaining exosomes, such as isolating exosomes from cells in vitro using standard cell conditioned medium.

[0081] Thus, in one or more embodiments, the methods may include administering sufficient PEP to provide a minimum dose of at least 0.01%, at least 0.05%, at least 0.1%, at least 0.25%, at least 0.5%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, or at least 70%.

[0082] In one or more embodiments, the methods may include administering sufficient PEP to provide a maximum dose of 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 9.0% or less, 8.0% or less, 7.0% or less, 6.0% or less, 5.0% or less, 4.0% or less, 3.0% or less, 2.0% or less, 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

[0083] In one or more embodiments, the method may include administering sufficient PEP to provide a dose characterized by a range having endpoints defined by any minimum dose identified above and any maximum dose greater than the minimum dose. For example, in one or more embodiments, the method may include administering sufficient PEP to provide a dose between 1% and 50% of the dose, e.g., between 5% and 20% of the dose. In one or more embodiments, the method may include administering sufficient PEP to provide a dose equal to any minimum dose or any maximum dose listed above. Thus, for example, the method may include administering a dose of 0.05%, 0.25%, 1.0%, 2.0%, 5.0%, 20%, 25%, 50%, 80%, or 100%.

[0084] A single dose may be administered all at once, continuously over a period of time, or in multiple separate doses. When multiple doses are used, each dose may be the same or different. For example, a prescribed daily dose may be administered as a single dose, or as two doses that may be equal or unequal, continuously over a 24-hour period. When multiple doses are used to deliver a single dose, the interval between doses may be the same or different. In one or more specific embodiments, PEP may be administered by only one dose, for example, during a surgical procedure.

[0085] In one or more specific embodiments in which multiple doses of the PEP composition are administered to a subject, the PEP composition may be administered as needed to repair or treat the damaged skeletal muscle to the desired extent. Alternatively, the PEP composition may be administered 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times. The interval between doses may be a minimum of at least 1 day, such as at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, or at least 21 days. The interval between doses may be a maximum of 6 months or less, such as 3 months or less, 2 months or less, 1 month or less, 21 days or less, or 14 days or less.

[0086] In one or more embodiments, the method may include multiple administrations of PEP to a subject at intervals (in the case of two administrations) or multiple intervals (in the case of three or more administrations) characterized by a range having endpoints defined by any minimum interval and any maximum interval greater than the minimum interval identified above. For example, in one or more embodiments, the method may include multiple administrations of PEP at intervals of 1 day to 6 months, such as 3 days to 10 days. In one or more particular embodiments, the method may include multiple administrations of PEP at intervals equal to any minimum interval or any maximum interval listed above. Thus, for example, the method may include multiple administrations of PEP at intervals of 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 1 month, 2 months, 3 months, or 6 months.

[0087] In one or more embodiments, the methods may include administering a cocktail of PEPs prepared from a variety of cell types, where each cell type has a unique muscle regenerative profile, e.g., protein composition and / or gene expression, etc. In this manner, the PEP composition can provide a broader spectrum of muscle regenerative activity than if the PEP composition were prepared from a single cell type.

[0088] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements; the terms "comprise", "comprising" and variations thereof are to be construed as open-ended, i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, "a", "an", "the", and "at least one" are used interchangeably to mean one or more; and the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0089] In the foregoing description, certain embodiments may be described alone for clarity. Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "one or more particular embodiments," "one or more embodiments," or "some embodiments" mean that a particular feature, configuration, composition, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearance of such phrases in various places throughout this specification does not necessarily refer to the same embodiment of the present disclosure. Moreover, certain features, configurations, compositions, and characteristics may be combined in any suitable manner in one or more embodiments. Moreover, certain features, configurations, compositions, and characteristics may be combined in any suitable manner in one or more embodiments. Thus, a feature described in the context of one embodiment may be combined with a feature described in the context of a different embodiment, unless the features are necessarily mutually exclusive.

[0090] In any method disclosed herein that includes separate steps, the steps may be performed in any practicable order, and, if desired, any combination of two or more steps may be performed simultaneously.

[0091] As used herein, the terms "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0092] Exemplary embodiments Embodiment 1 is a method of treating damaged skeletal muscle tissue, the method comprising applying a therapeutic composition to the damaged skeletal muscle tissue, the therapeutic composition comprising: Purified exosome product (PEP), A pharma- ceutically acceptable carrier; The method includes:

[0093] Embodiment 2 is the method of embodiment 1, wherein the PEP comprises spherical or spheroidal exosomes having a diameter of 300 nm or less.

[0094] Embodiment 3 is the method of embodiment 1, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm±90 nm.

[0095] Embodiment 4 is the method of embodiment 3, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm±50 nm.

[0096] Embodiment 5 is the method of embodiment 4, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm±30 nm.

[0097] Embodiment 6 is a method for producing a PEP comprising the steps of: 1%–20% CD63 - Exosomes and 80%-99% CD63 + Exosomes and The method according to any one of the first to fifth embodiments, comprising:

[0098] Embodiment 7 is the method according to any one of embodiments 1 to 5, wherein the PEP comprises at least 50% CD63-exosomes.

[0099] Embodiment 8 is a method according to any one of embodiments 1 to 7, wherein the PEP comprises 1×10 11 PEP exosomes to 1×10 13 PEP exosomes.

[0100] Embodiment 9 is the method of embodiment 8, wherein the PEP comprises 1×10 12 PEP exosomes to 1×10 13 PEP exosomes.

[0101] Embodiment 10 is the method of any of embodiments 1-9, wherein the therapeutic composition further comprises a support matrix.

[0102] Embodiment 11 is the method of embodiment 10, wherein the support matrix comprises a collagen scaffold.

[0103] Embodiment 12 is the method of any of embodiments 1-11, wherein the therapeutic composition further comprises a tissue sealant or fibrin glue.

[0104] Embodiment 13 is a method according to any one of embodiments 1 to 12, wherein the therapeutic composition forms a hydrogel after being applied to the injured skeletal muscle.

[0105] Embodiment 14 is the method of embodiment 13, wherein the therapeutic composition forms a hydrogel when incubated at a temperature between 35° C. and 40° C.

[0106] Embodiment 15 is the method of embodiment 13 or embodiment 14, wherein the therapeutic composition forms a hydrogel at least 1 minute after application to the skeletal muscle.

[0107] Embodiment 16 is a method according to any one of embodiments 1 to 15, wherein the therapeutic composition is applied in an amount effective to increase expression of NF-κB in the injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

[0108] Embodiment 17 is the method of any of embodiments 1-16, wherein the therapeutic composition is applied in an amount effective to increase PD-L1 expression in the injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

[0109] Embodiment 18 is a method according to any one of embodiments 1 to 17, wherein the therapeutic composition is applied in an amount effective to increase polarization towards M2 macrophages in the injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

[0110] Embodiment 19 is the method of any one of embodiments 1 to 18, wherein the damaged skeletal muscle tissue comprises a urethral sphincter.

[0111] Embodiment 20 is the method of any one of embodiments 1 to 19, wherein at least a portion of the PEP exosomes comprises PD-L1, NF-κB, or both PD-L1 and NF-κB.

[0112] Embodiment 21 is the method of any one of embodiments 1 to 20, wherein at least a portion of the PEP exosomes comprises CD63, CD9, flotillin, or any combination of two or more thereof.

[0113] Embodiment 22 is a method according to any of embodiments 1 to 21, wherein the therapeutic composition is applied in an amount effective to increase cell proliferation in the damaged skeletal muscle tissue compared to damaged skeletal muscle tissue treated without the therapeutic composition. EXAMPLES

[0114] The present invention is illustrated in the following examples, it being understood that the specific examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein.

[0115] Study design The aim of this study was to evaluate the muscle regeneration potential of a purified exosome product (PEP) cell-free platform. A urethral sphincter model was used to evaluate the potential use of PEP as a non-mesh-based alternative to the surgical treatment of stress urinary incontinence (SUI). The proliferative, migratory, and chemotactic responses of human skeletal muscle myoblasts to PEP were evaluated. Proliferation assays following pretreatment of myoblasts with increasing concentrations of resveratrol resulted in inhibition of myoblast growth, suggesting that the exosome-dependent NF-κB signaling pathway may be required for satellite cell activation and differentiation.

[0116] Second, a muscle volume loss (VML) rat model was established to determine the in vivo efficacy of PEP for muscle regeneration and repair. Animals were treated with saline, TISSEEL (Baxter International, Inc., Deerfield, IL), or 1 × 10 12 They were randomized to TISSEEL reconstituted with PEP exosomes / mL, and the repair response was characterized by Masson's trichrome and immunostaining for EdU and desmin.

[0117] Third, PEP was evaluated for the treatment of urethral sphincter defects using a porcine stress urinary incontinence (SUI) model. The sphincter defects created were large enough to impair function and cause a significant reduction in intraurethral pressure. Animals received either collagen or 1 × 10 12 They underwent surgical repair of urethral sphincter lesions with injection of collagen containing PEP exosomes / mL. Group sizes were selected based on IACUC protocols and previous experience with large animal studies. Investigators were blinded during data collection and analysis but not blinded to treatment groups during injection of the active product.

[0118] Characterization and quantification of purified exosome products Purified exosome product (PEP; Rion LLC, Rochester, MN) is purified from conditioned medium of apheresis-purified platelets as previously described (Kisby et al., 2021, Female Pelvic Med Reconstr Surg. 27(10):609-615; Qi et al., 2020, J Orthop Res. 38(8):1845-1855; Shi et al., 2021, J Orthoped Res. 39(8):1825-1837; WO 2019 / 118817; U.S. Patent Application Publication No. 10,596,123). Briefly, conditioned medium derived from purified platelets was subjected to sequential filtration to remove cytological material and obtain extracellular vesicles in the range of 50 nm to 200 nm. Further processing was performed by stepwise filtration and centrifugation to ensure removal of non-EV material. The extracellular vesicles were lyophilized to approximately 5 × 10 cells per vial. 12 Dry powders containing 1 × 10 vesicles were obtained. Different exosome concentrations were produced by reconstituting the lyophilized exosomes in saline or in the suspension media described. Nanoparticle tracking analysis was performed with a nanoparticle tracking analyzer (NanoSight NS3000, NanoSight, Ltd., Salisbury, United Kingdom) to confirm the presence of exosomes, characterize the particle size distribution, and quantitate the exosome concentration. Transmission electron microscopy (TEM) was performed at the Mayo Clinic Microscopy and Cell Analysis Core Facility using a JEM-1400 series 120 kV microscope (JEOL) at an accelerating voltage of 80 kV and a magnification of 60 kx. PEP, TISSEEL or collagen in solution and 1 × 10 12 TEM was performed using TISSEEL or collagen reconstituted with PEP exosomes / mL.

[0119] Western blotting and quantification Western blotting for the expression of exosome markers was performed using two methods: conventional and Li-Cor. In the conventional method, equal amounts of protein were loaded in each lane and transferred to a PVDF membrane. The membrane was blocked in 5% nonfat dry milk diluted in TBST and incubated with primary antibodies against CD63, CD9, and flotillin (Table 1). The membrane was incubated with HRP-conjugated secondary antibodies and developed by enhanced chemiluminescence (AMERSHAM, GE Healthcare, Chicago, IL). Loading was normalized to a GAPDH loading control. In the Li-Cor digital fluorescence method (Li-Cor Biosciences, Inc., Lincoln, NE), equal amounts of protein were loaded in each lane and transferred to a nitrocellulose membrane. The membrane was blocked in Li-Cor TBS blocking buffer and incubated with primary antibodies against NF-κB p65 and PD-L1. The membranes were fluorescently imaged using an imaging system (Odyssey CLx, Li-Cor Biosciences, Inc., Lincoln, NE) with secondary antibodies at 680 nm and 800 nm. Digital fluorescence intensity was determined by IMAGE STUDIO software (Li-Cor Biosciences, Inc., Lincoln, NE), and loading was normalized by total protein staining (Li-Cor Biosciences, Inc., Lincoln, NE) for protein digital fluorescence across lanes and analyzed by EMPIRIA STUDIO software (Li-Cor Biosciences, Inc., Lincoln, NE).

[0120] [Table 1] Atomic Force Microscopy (AFM) and Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS) Analysis Contact mode AFM with silicon nitride NP-S tips (spring constant, 0.58 Newtons / meter) was performed with a Nanoscope III controller (Digital Instruments). Exosomes (PEPs) purified by conventional ultracentrifugation or by any of the methodologies described above were fixed in situ, rinsed with ultrapure water (18 MΩ-cm), and dried. Images were acquired using a linear scan frequency (5–15 Hz) to generate AFM images of 512 × 512 pixels. Three-dimensional topographic images were generated and quantified using Nanoscope software.

[0121] Two lots of lyophilized PEP were reconstituted in triplicate with 1 mL of incubation solution (NanoView Biosciences) and further diluted 1,000-fold with the same solution before incubating 50 μL on the chip for 16 h according to the manufacturer's protocol for the ExoView Human Tetraspanin Plasma Kit (NanoView Biosciences; EV-TETRA-P). ExoView Tetraspanin Plasma chips were spotted in triplicate with capture antibodies against CD41a and mouse IgG1, kappa isotype controls. The chips were then washed in an automated chip washer and incubated for 1 h with conjugated antibodies (CF488a anti-CD9, NanoView Biosciences) for fluorescent labeling of the captured PEP. After labeling, the chips were washed, dried in an automated chip washer, and placed in the reader for analysis. All data were collected using an ExoView R100 reader with ExoView Scanner 3.0 software and analyzed using ExoView Analyzer 3.0. All data reported were obtained and analyzed in a blinded fashion in accordance with CGMP quality control standards.

[0122] Characterization of the effects of PEP on human skeletal myoblasts The proliferation, migration, and chemotaxis of human skeletal muscle myoblasts (HSMM, Lonza Group AG, Basel, Switzerland, cat. #CC-2580) were examined in basal medium (SkBM-2 Skeletal Muscle Cell Growth Basal Media, Lonza Group AG, Basel, Switzerland, cat. #CC-3246), basal medium supplemented with 10% FBS (SkGM-2 SingleQuots Supplements and Growth Factors, Lonza Group AG, Basel, Switzerland, cat. #CC-3244), or 1.25 × 10 11 PEP exosomes / mL ~ 5 x 10 11 Cells were cultured in basal medium containing increasing concentrations of PEP, ranging from 1000 PEP exosomes / mL, and assessed using a live cell analyzer (INCUCYTE, Essen BioScience, Inc., Ann Arbor, MI). Data were generated from live cell analysis by acquiring phase images (10X lens; 4 images / well in a 96-well plate with 12 wells / condition).

[0123] The chemotactic properties of PEP were evaluated using a chemotaxis module for a live cell analyzer (INCUCYTE, Essen BioScience, Inc., Ann Arbor, MI). HSMM in culture were seeded in the upper chamber and either in basal medium, medium supplemented with 10% FBS, or at 1.25 × 10 11 PEP exosomes / mL ~ 5 x 10 11 Chemotaxis was assessed by placing media containing increasing concentrations of PEP, ranging from 1000 PEP exosomes / mL, into the lower chamber. Images were collected from both the upper and lower chambers. The total phase area in the upper wells was normalized to the initial upper value. A decrease in the cell area in the upper chamber indicates enhanced cell migration through the pores and improved chemotaxis. Graphs were plotted using the chemotaxis module of a live cell analyzer (INCUCYTE, Essen BioScience, Inc., Ann Arbor, MI) by acquiring wide-angle phase images (10X lens; 1 image / well in a 96-well plate with 12 wells / condition).

[0124] HSMM migration was assessed using a scratch wound assay. Graphs show the effect of basal medium, 10% FBS-supplemented medium, and 1.25 × 10 11 PEP exosomes / mL ~ 5 x 10 11 Plotted at different time points using media containing increasing concentrations of PEP ranging from PEP exosomes / mL. Data are generated from a live cell analyzer (INCUCYTE, Essen BioScience, Inc., Ann Arbor, MI) by acquiring phase images (10X lens; 1 image / well in a 96-well plate with 12 wells / condition).

[0125] Resveratrol inhibition of human skeletal myoblast proliferation The mechanism of PEP-induced HSMM proliferation was investigated using 2.5 × 10 11 The NF-κB inhibitor resveratrol (InvivoGen, San Diego, CA) was examined in cultures incubated with PEP exosomes / mL at concentrations of 1 × 10 and 1 × 10 because they had the optimal effect on the proliferation of cultured HSMM. 12 The PEP exosomes / mL concentration was chosen to match the empirically determined sustained release PEP concentration from TISSEEL (Baxter International, Inc., Deerfield, IL) reconstituted with PEP exosomes / mL. HSMMs were seeded overnight at 10,000 cells / well in 96-well plates. 11 Resveratrol was incubated for 1 h at concentrations ranging from 50 μM to 500 μM before adding PEP exosomes / mL to each well. Data were generated from a live cell proliferation assay (INCUCYTE, Essen BioScience, Inc., Ann Arbor, MI) by acquiring phase images (10X lens; 5 images / well in a 96-well plate with 3 wells / condition). Western blotting for expression of NF-κB p65 at 72 h after inhibition was quantified and normalized using total protein staining (IMAGE STUDIO software, LI-COR Biosciences, Inc., Lincoln, NE).

[0126] Exosome release assay For the TISSEEL release assay, the TISSEEL kit (Baxter International, Inc., Deerfield, IL) was prepared by adding 1 mL of fibrinolysis inhibitor to Sealer Protein Concentrate (Baxter International, Inc., Deerfield, IL) and 1 mL of CaCl2 solution to Thrombin 500. Both solutions were incubated at 37°C for 20 minutes. One vial of PEP was resuspended with 400 μL of heparin and 1.25 mL of deionized water. The entire PEP solution was added to the fibrinolysis inhibitor / sealer protein concentrate solution. This solution was combined with the CaCl2 / Thrombin 500 solution in a 2:1 ratio in each well of a 12-well plate to a total volume of 990 μL per well.

[0127] For collagen release assays, one vial of PEP was reconstituted in 2.5 mL of sterile water and filtered through a 0.22 μm filter. 500 μL of PEP was combined with 500 μL of 6 mg / mL collagen (Collagen Solutions plc, Glasgow, United Kingdom). NaOH was added to a final concentration of 0.02 M. The entire 1 mL solution was added to a single well of a 12-well plate and incubated at 37°C until solid. The final concentration of PEP for both assays in each well was 1 × 10 12 The exosomes were collected at 1000 x 1000 cells / mL. One mL of serum-free Dulbecco's Modified Eagle Medium 1X (DMEM) was added to each well. All media was collected daily and replaced with another 1 mL of serum-free DMEM throughout the experimental period.

[0128] Restoration of muscle volume loss in rat latissimus dorsi After a 48-72-h acclimation period, 27 male Lewis rats (Envigo, Indianapolis, IN) aged 2-4 months were housed and handled in accordance with Mayo Clinic Institutional Animal Care and Use Committee (IACUC) regulations (Mayo Clinic, Rochester, MN). Animals were clipped and anesthetized with isoflurane inhalation prior to surgery and maintained throughout the procedure with intravenous fluids and isoflurane. After prepping the skin with betadine, a 3-cm longitudinal incision was made above the scapula, and the skin was secured with Metzenbaum scissors to expose the latissimus dorsi (LD) muscle. An 8-mm punch biopsy was used to create the LD muscle volume loss; care was taken not to exceed a depth of approximately 2 mm to avoid intrusion into the thoracic cavity. The defect was filled with saline, tissue sealant (TISSEEL; Baxter International, Deerfield, IL), or 1 × 10 12 TISSEELs reconstituted with PEP were filled with exosomes / mL. The skin was closed with interrupted 2.0 VICRYL sutures (Ethicon, Inc., Raritan, NJ). 2'-deoxy-5-ethynyluridine (EdU) was administered intraperitoneally once a week at a dose of 50 mg / kg as a means of tracking cell proliferation. Animals were sacrificed at 6 weeks.

[0129] A directional urethral sphincter defect porcine model to evaluate the efficacy of PEP-induced sphincter regeneration After an acclimation period of 48-72 hours, ten female domestic Yorkshire cross pigs weighing 70-80 kg underwent two survival and one terminal surgery. All animals received the procedures outlined, but the injection interventions differed as follows:

[0130] Anesthesia for survival surgery 1 was induced by TELAZOL (Zoetis, Parsippany, NJ; 5 mg / kg), xylazine (1–2 mg / kg), and maintained with an isoflurane vaporizer (1.5%–3%). After general anesthesia was achieved, the pig was prepped and draped in a sterile manner. Ceftiofur (5 mg / kg) was administered intramuscularly for antibiotic prophylaxis. Cystoscopy was performed to identify the urethra and a whistle tip stent was placed. Using cystoscopic guidance, a catheter (Medspira LLC, Minneapolis, MN) was placed in the ureter. The stent was removed and the bladder balloon was inflated to 30 cubic centimeters (cc), and pressures were obtained in 1 cm increments starting at the vagina and extending to the bladder neck. Pressures were collected for 10 seconds at each point, and the three pressures along a given 10-second recording were averaged. The pressure catheter balloon was deflated, the catheter removed, and the urethra re-identified using a stent. A full-thickness transmural defect 2-3 cm long, including the urethral wall and underlying sphincter, was created approximately 1 cm cephalad to the urethral orifice using a Collins knife (cutting setting at 70 watts). Mild hemostasis was obtained with a Collins knife (coagulation setting at 30 watts). A Foley catheter was left in place for 48-72 hours to prevent urinary retention. The position was confirmed with a cystoscope, and the catheter was secured with 2.0 PROLENE sutures (Ethicon, Inc., Raritan, NJ). Animals were allowed to heal for approximately 7 days and returned to the operating room for interventional procedures. Buprenorphine (0.03 mg / kg) was given every 6-8 hours for analgesia.

[0131] Anesthesia for survival surgery 2 was performed as described for surgery 1 above. Cystoscopy was used to identify the urethra and a catheter was used to measure pressure as previously described. After pressure readings were collected, cystoscopy was used to inject collagen and 1 x 10 urethra into the length of the previously created defect. 12 Injections were performed with 1 × 10 exosomes / mL of PEP (n = 4) or collagen alone (n = 2). 12Exosomes / mL of PEP were created by reconstituting PEP (Rion LLC, Rochester, MN) with 1 mL of sterile water (Hospira, Inc., Lake Forest, IL) and 4 mL of clinical grade type I bovine collagen (5 mg / mL; Collagen Solutions, Glasgow, UK). Collagen was made by adding 1 mL of sterile water to 4 mL of clinical grade type I bovine collagen (5 mg / mL; Collagen Solutions, Glasgow, UK). A total of 5 mL was injected in approximately 0.5 mL aliquots along the length of the lesion using a 21-gauge needle (INJETAK, Laborie Medical Technologies, Corp., Portsmouth, NH). A prototype PEP delivery device was used to deliver 1 × 10 12 Delivery of exosomes / mL of PEP+collagen (n=2) and collagen alone (n=2) was also performed. The device has been developed to allow for placement of two needles at the 4 and 8 o'clock positions at specific points along the length of the urethra, avoiding the need for cystoscopy or general anesthesia. The device, specially modified for porcine dissection, was placed into the urethra, the needle deployed, 1 cc injected, and the needle retracted. This process was repeated five times, starting approximately 1 cm from the urethral opening and extending cephalad. The position of the device within the urethra was confirmed with a cystoscope.

[0132] To aid in tracking cell populations, 2'-deoxy-5-ethynyluridine (EdU) was given as an oral supplement (5 mg / kg) twice weekly. After 6-7 weeks of healing, terminal surgery was performed. After anesthesia was administered, the urethra was identified, intraurethral pressures were collected, and animals were euthanized using intravenous pentobarbital (250 mL of 390 mg / mL). The perineum was removed en bloc, and the urethra and bladder were placed in formalin. Organs were weighed and representative samples were collected. Blood was drawn and standard blood counts were performed (e.g., red blood cell count, white blood cell count, platelet count, albumin concentration), and chemistries were obtained.

[0133] Histological treatment and staining quantification Paraffin-embedded formalin-fixed samples were processed into 10 μm sections on a microtome. Tissues were stained with hematoxylin and eosin (H&E) and Masson's trichrome stains and evaluated using a slide scanner (Axio Scan.Z1; Carl Zeiss AG, Oberkochen, Germany). Further sections were deparaffinized with successive xylene washes, rehydrated in decreasing amounts of ethanol baths, and finally washed with water. Antigen retrieval was performed by immersing the sections in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) and boiling for 10 min. Sections were then permeabilized with blocking buffer (PBS + 5% normal donkey serum, 5% BSA, 0.2% Triton-X) for 1 h at room temperature. Primary antibodies against desmin (Abcam plc, Cambridge, United Kingdom), PD-L1 (Cell Signaling Technology, Inc., Danvers, MA), NF-κB p65 (Santa Cruz Biotechnology, Inc., Dallas, TX), M1 (LifeSpan BioSciences, Inc., Seattle, WA), and M2 macrophages (Abcam plc, Cambridge, United Kingdom) were diluted in blocking buffer and incubated with the samples overnight at 4°C (Table 1). ALEXAFLUOR (Thermo Fisher Scientific, Inc., Waltham, MA) secondary antibody was then diluted 1:500 in blocking buffer and incubated with the samples for 1 h at room temperature. EdU was labeled using an imaging kit (CLICK-IT PLUS EdU AF647; Thermo Fisher Scientific, Inc., Waltham, MA) according to the manufacturer's instructions.After washing, DAPI mounting medium (PROLONG GOLD antifade mountant; Thermo Fisher Scientific, Inc., Waltham, MA) was added to the sections, coverslips were applied, and images were taken on an inverted fluorescence microscope with a variable fluorescence objective (AXIO Observer, Carl Zeiss AG, Oberkochen, Germany). Overall images were taken with an iPhone (Apple, Inc., Cupertino, CA) camera.

[0134] To accurately image immunohistochemistry, localization of the injury border zone was confirmed by Masson's trichrome staining. Immunohistochemistry quantification was assessed in a blinded manner using ImageJ (Version 1.52a; Schneider et al., 2012, Nature Methods 9(7):671-675) macro scripts. Tissue stained positive for desmin, PD-L1, and NF-κB p65 was quantified per percent tissue area in each image. Characterization of M2:M1 macrophage staining was performed by quantifying the area percentage of Mφ and M2 per tissue area in each image. Quantification of M1 was defined as Mφ positive tissue-M2 positive macrophage stained tissue. Additionally, EdU positive counts, determined by the number of objects per desmin positive stained tissue area, were quantified for the determination of proliferative muscle.

[0135] External urethral sphincter pressure collection A portable anorectal manometry system (mCompass, Medspira, LLC, Minneapolis, Minnesota) equipped with a 5-channel modified pressure catheter was used to assess intraurethral pressure using the squeeze mode of Biofeedback 1.01 software (Figure 13D). Pressure recordings were performed in triplicate and assessed at four time points: pre-injury (day 0), post-injury (day 0), pre-injection (day 7), and pre-sacrifice (day 42). Pressure measurements were obtained at 1 cm intervals along the length of the urogenital tract, starting from the vagina and extending to the bladder neck. At each point, pressure was collected for 10 seconds, and the three pressures along a given 10-second recording were averaged. Entry into the urethra was determined by the change in resistance with advancement of the pressure sensor and the associated increase in intraurethral pressure. The urethral entrance and pressure measurement location were confirmed with a cystoscope.

[0136] statistical analysis Data are reported as mean ± standard error of the mean and interquartile range, and statistical significance is assessed by two-tailed Student's T-test or single-variable ANOVA with post-hoc Tukey's honestly significant difference test comparing all experimental groups. Significance was set at α = 0.05.

[0137] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence deposits in GenBank and RefSeq, and amino acid sequence deposits in SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. In the event of any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be construed therefrom. The invention is not limited to the exact details shown and described, and variations obvious to one skilled in the art will be included in the invention as defined by the claims.

[0138] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood in all instances to be modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by ordinary rounding techniques.

[0139] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

1. 1. A therapeutic composition for treating damaged skeletal muscle tissue, comprising: Purified exosome product (PEP); a pharmaceutically acceptable carrier; The therapeutic composition comprising:

2. 2. The therapeutic composition of claim 1, wherein the PEP comprises spherical or spheroidal exosomes having a diameter of 300 nm or less.

3. 2. The therapeutic composition of claim 1, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm±90 nm.

4. 4. The therapeutic composition of claim 3, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm±50 nm.

5. 5. The therapeutic composition of claim 4, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm±30 nm.

6. The PEP is 1% to 20% CD63 － Exosomes and 80% to 99% CD63 ＋ Exosomes and The therapeutic composition of any one of claims 1 to 5, comprising:

7. The PEP is at least 50% CD63 － The therapeutic composition according to any one of claims 1 to 5, comprising exosomes.

8. The PEP is 1×10 １１ PEP exosomes ~ 1 x 10 １３ The therapeutic composition according to any one of claims 1 to 5, comprising PEP exosomes.

9. The PEP is 1×10 １２ PEP exosomes ~ 1 x 10 １３ The therapeutic composition of claim 8, comprising PEP exosomes.

10. The therapeutic composition of any one of claims 1 to 5, further comprising a support matrix.

11. The therapeutic composition of claim 10 , wherein the support matrix comprises a collagen scaffold.

12. The therapeutic composition of any one of claims 1 to 5, wherein the therapeutic composition further comprises a tissue sealant or a fibrin glue.

13. The therapeutic composition of any one of claims 1 to 5, wherein the therapeutic composition forms a hydrogel after application to the injured skeletal muscle.

14. 14. The therapeutic composition of claim 13, wherein the therapeutic composition forms a hydrogel when incubated at a temperature between 35°C and 40°C.

15. 14. The therapeutic composition of claim 13, wherein the therapeutic composition forms a hydrogel at least 1 minute after application to skeletal muscle.

16. 6. The therapeutic composition of any one of claims 1 to 5, wherein the therapeutic composition is applied in an amount effective to increase the expression of NF-κB in the injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

17. 6. The therapeutic composition of any one of claims 1 to 5, wherein the therapeutic composition is applied in an amount effective to increase PD-L1 expression in the injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

18. 6. The therapeutic composition of any one of claims 1 to 5, wherein the therapeutic composition is applied in an amount effective to increase M2 macrophage polarization in the injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

19. The therapeutic composition of any one of claims 1 to 5, wherein the damaged skeletal muscle tissue comprises a urethral sphincter.

20. The therapeutic composition of any one of claims 1 to 5, wherein at least a portion of the PEP exosomes comprises PD-L1, NF-κB, or both PD-L1 and NF-κB.

21. The therapeutic composition of any one of claims 1 to 5, wherein at least a portion of the PEP exosomes comprises CD63, CD9, flotillin, or any combination of two or more thereof.

22. 6. The therapeutic composition of any one of claims 1 to 5, wherein the therapeutic composition is applied in an amount effective to increase cell proliferation in the injured skeletal muscle tissue compared to injured skeletal muscle tissue treated without the therapeutic composition.

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