Use of annexins in preventing and treating muscle membrane injury

Abstract

To provide compositions and methods for increasing the activity of an annexin protein to treat a cellular membrane injury in a patient in need thereof.SOLUTION: A method of treating a cellular membrane injury comprises administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 783,619, filed December 21, 2018, and U.S. Provisional Patent Application No. 62 / 903,525, filed September 20, 2019, each of which is incorporated herein by reference in its entirety.

[0002] Government Interest Statement This invention was made with United States government support under grant numbers U54 AR052646 and R01 NS047726 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Materials This application contains as another part of this disclosure a Sequence Listing in computer readable form (Filename: 2018-119R_Seqlisting.txt; Size: 168,826 bytes; Created: December 20, 2019). [Background technology]

[0004] Plasma membrane repair occurs after membrane disruption and is a highly conserved process. The active process required for resealing of the membrane disruption is Ca 2+One model suggests that membrane repair is mediated by lysosomal vesicle fusion and / or lateral membrane diffusion to the site of injury (Demonbreun et al., 2016b; McDade et al., 2014; Reddy et al., 2001; Rodriguez et al., 1997). These models are not mutually exclusive and may vary depending on the type and extent of injury. Skeletal muscle is highly dependent on plasma membrane repair, as mutations in genes encoding repair proteins cause muscle disease ( Bansal et al., 2003 , Bashir et al., 1998 , Cai et al., 2009 , Defour et al., 2017 , Demonbreun and McNally, 2016 , Demonbreun et al., 2015 ).

[0005] Annexins regulate lipid binding, cytoskeletal reorganization, and Ca ions that regulate blebbing, steps required for membrane repair. 2+ annexin-binding proteins (Bizzarro et al., 2012; Boye et al., 2018; Boye et al., 2017; Grewal et al., 2017; Jimenez and Perez, 2017; Lauritzen et al., 2015). Each annexin repeat domain contains a unique annexin-specific type II or type III binding site and a Ca 2+ Coordinate binding of type II and type III binding sites to Ca 2+ Due to their different affinities, each annexin binds to intracellular Ca 2+Annexins have the unique ability to respond to a range of membrane levels and phospholipid binding (Blackwood and Ernst, 1990). They possess the ability to self-oligomerize and hetero-oligomerize (Zaks and Creutz, 1991). Typical annexins, such as A1 and A2, contain a single annexin core composed of four annexin repeat domains. In contrast, annexin A6 contains two annexin cores, resulting in eight annexin repeat domains (Benz et al., 1996). The duplicative structure of annexin A6 allows the amino- and carboxyl-terminal annexin core domains to bind to one or two separate membranes, making annexin A6 a primary target for promoting membrane coalescence and folding, which is necessary during membrane repair (Boye et al., 2018, Boye et al., 2017, Buzhynskyy et al., 2009).

[0006] Annexins have high affinity for phosphatidylserine, phosphatidylinositol, and cholesterol, which are highly enriched in the sarcolemma (Fiehn et al., 1971; Gerke et al., 2005). Multiple annexins have been implicated in membrane repair in skeletal muscle, Xenopus oocytes, human trophoblasts, and HeLa cancer cells, suggesting a conserved mechanism (Babbin et al., 2008; Bement et al., 1999; Carmeille et al., 2015; Davenport et al., 2016; Demonbreun et al., 2016b; Lennon et al., 2003; McNeil et al., 2006; Roostalu and Strahle, 2012). Annexins are sequentially recruited to damaged membranes and form macromolecular repair complexes at membrane lesions ( Boye et al., 2017 , Demonbreun et al., 2016b , Roostalu and Strahle, 2012 ). Summary of the Invention

[0007] Defects in the cell's ability to repair its plasma membrane lead to many diseases, including muscular dystrophies and other pathological conditions that result in cell death. Membrane damage can result from events such as abuse, trauma, burns, chemical exposure, and chronic diseases. Currently, there are no drugs to prevent or treat membrane damage (Demonbreun and McNally, 2016). Annexins are calcium-binding proteins with high affinity for membrane lipids. Annexins are involved in membrane repair in many cell types, including muscle, trophoblasts, HeLa cells, and oocytes, suggesting a broad role for annexins (Babbin et al., 2008; Bement et al., 1999; Carmeille et al., 2015; Davenport et al., 2016; Demonbreun et al., 2016b; McNeil et al., 2006; Roostalu and Strahle, 2012). Annexin A1 ("A1"), annexin A2 ("A2"), annexin A5 ("A5"), and annexin A6 ("A6") are sequentially recruited to the injury site to form a large repair complex (Demonbreun et al., 2016b; Roostalu and Strahle, 2012). A polymorphism in Anxa6 that creates a truncated form of annexin A6 has been identified (Swaggart et al., 2014). Expression of the truncated annexin A6 acts as a dominant-negative and reduced the formation of the annexin repair complex. This correlated with impaired membrane repair and more severe muscle disease (Demonbreun et al., 2016a; Demonbreun et al., 2016b; Quattrocelli et al., 2017b; Swaggart et al., 2014). Conversely, overexpression of annexin A6 was sufficient to improve repair and reduce susceptibility to membrane damage (Quattrocelli et al., 2017a). Furthermore, treatment with recombinant annexin A6 was sufficient to reduce muscle damage in cell-based and whole-animal studies.

[0008] One aspect of the present disclosure relates to a method of treating cell membrane damage, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein. Another aspect of the present disclosure relates to a method of delaying the onset of, enhancing recovery from, or preventing cell membrane damage, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein. Embodiments of each of these aspects are contemplated, wherein the agent is selected from the group consisting of a recombinant protein, a steroid, and a polynucleotide capable of expressing an annexin protein. In some embodiments, the steroid is a corticosteroid or a glucocorticoid. In some embodiments, the recombinant protein is an annexin protein. In some embodiments, the annexin protein is annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof). In some embodiments, the recombinant protein is a wild-type annexin protein, a modified annexin protein, an annexin-like protein, or a fragment of a wild-type annexin protein or an annexin-like protein. In some embodiments, the modified annexin protein is annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof). In some embodiments, the patient is suffering from an acute injury. In some embodiments, the acute injury results from surgery, burns, toxins, chemicals, radiation-induced injury, acute myocardial injury, acute muscle injury, acute lung injury, acute epithelial injury, acute epidermal injury, acute kidney injury, acute liver injury, vascular injury, excessive mechanical force, or trauma. In some embodiments, the patient is suffering from a chronic disorder.In some embodiments, the patient suffers from a chronic disease such as Becker muscular dystrophy (BMD), Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), myotonic dystrophy, facioscapulohumeral dystrophy (FSHD), oculopharyngeal muscular dystrophy, distal muscular dystrophy, cystic fibrosis, pulmonary fibrosis, muscle atrophy, cerebral palsy, epithelial disorders, epidermal disorders, renal disorders, hepatic disorders, sarcopenia, or cardiomyopathy (hypertrophic, dilated, congenital, arrhythmogenic, restrictive, ischemic, or heart failure). In some embodiments, the cardiomyopathy is hypertrophic, dilated, congenital, arrhythmogenic, restrictive, ischemic, or heart failure.

[0009] In some embodiments, the methods disclosed herein further comprise administering an effective amount of a second agent, wherein the second agent is selected from the group consisting of mitsugumin 53 (MG53), a modulator of latent TGF-β binding protein 4 (LTBP4), a modulator of transforming growth factor β (TGF-β) activity, a modulator of androgen response, a modulator of inflammatory response, a promoter of muscle growth, a chemotherapeutic agent, a modulator of fibrosis, and combinations thereof.

[0010] In some embodiments of the methods disclosed herein, the polynucleotide is associated with a nanoparticle. In some embodiments, the polynucleotide is contained in a vector. In some embodiments, the vector is within a chloroplast. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of a herpesvirus vector, an adeno-associated virus (AAV) vector, an adenovirus vector, and a lentivirus vector. In some embodiments, the AAV vector is recombinant AAV5, AAV6, AAV8, AAV9, or AAV74, including embodiments in which the AAV74 vector is AAVrh74.

[0011] In some embodiments of the methods disclosed herein, the composition increases the activity of annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof), annexin A7 (SEQ ID NO: 9 or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 or SEQ ID NO: 16), annexin A13 (SEQ ID NO: 17 or SEQ ID NO: 18), or a combination thereof. In some embodiments, the composition increases the activity of annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), and annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In some embodiments, the composition increases the activity of annexin A2 (SEQ ID NO:2 or SEQ ID NO:3) and annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof). In some embodiments, the composition increases the activity of annexin A1 (SEQ ID NO:1) and annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof). In some embodiments, the composition increases the activity of annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof). In any of the aspects or embodiments of the present disclosure, the composition for use in any of the methods described herein is a pharmaceutical composition disclosed herein.

[0012] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a modified annexin protein and a pharmaceutically acceptable carrier, buffer, and / or diluent. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an annexin protein and a pharmaceutically acceptable carrier, buffer, and / or diluent. In some embodiments, the annexin protein or modified annexin protein is annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof), annexin A7 (SEQ ID NO: 9 or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 or SEQ ID NO: 16), annexin A13 (SEQ ID NO: 17 or SEQ ID NO: 18), or a combination thereof. In some embodiments, the annexin protein or modified annexin protein is annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof). In further embodiments, the pharmaceutical composition comprises annexin A1 (SEQ ID NO:1), annexin A2 (SEQ ID NO:2 or SEQ ID NO:3), and annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof). In some embodiments, the pharmaceutical composition comprises annexin A2 (SEQ ID NO:2 or SEQ ID NO:3) and annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof). In further embodiments, the pharmaceutical composition comprises annexin A1 (SEQ ID NO:1) and annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof). In some embodiments, the pharmaceutical composition further comprises a steroid. In further embodiments, the steroid is a corticosteroid or a glucocorticoid.In some embodiments, the pharmaceutical composition further comprises administering an effective amount of a second agent, wherein the second agent is selected from the group consisting of Mitsugumin 53 (MG53), a modulator of latent TGF-β binding protein 4 (LTBP4), a modulator of transforming growth factor β (TGF-β) activity, a modulator of androgen response, a modulator of inflammatory response, a promoter of muscle growth, a chemotherapeutic agent, a modulator of fibrosis, and combinations thereof. In further embodiments, the purity of the annexin protein in the composition is about 90% or greater as measured by standard release assays, including, but not limited to, SDS-PAGE, SEC-HPLC, and immunoblot analysis. In some embodiments, the composition has an endotoxin level of less than about 0.50000 endotoxin units per milligram of A280 annexin protein (EU / mg). In some embodiments, the modified annexin protein is produced in prokaryotic cells.

[0013] In some embodiments, the present disclosure provides a pharmaceutical composition comprising modified annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof) and a pharmaceutically acceptable carrier, buffer, and / or diluent. In some embodiments, the present disclosure provides a pharmaceutical composition comprising annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof) and a pharmaceutically acceptable carrier, buffer, and / or diluent. In some embodiments, the pharmaceutical composition further comprises annexin A1 (SEQ ID NO:1) and annexin A2 (SEQ ID NO:2 or SEQ ID NO:3). In some embodiments, the pharmaceutical composition further comprises annexin A2 (SEQ ID NO:2 or SEQ ID NO:3). In some embodiments, the pharmaceutical composition further comprises annexin A1 (SEQ ID NO:1). In further embodiments, the steroid is a corticosteroid or a glucocorticoid. In some embodiments, the pharmaceutical composition further comprises administering an effective amount of a second agent, wherein the second agent is selected from the group consisting of Mitsugumin 53 (MG53), a modulator of latent TGF-β binding protein 4 (LTBP4), a modulator of transforming growth factor β (TGF-β) activity, a modulator of androgen response, a modulator of inflammatory response, a promoter of muscle growth, a chemotherapeutic agent, a modulator of fibrosis, and combinations thereof. In some embodiments, the purity of the annexin protein in the composition is about 90% or greater as measured by a standard release assay. In some embodiments, the composition has an endotoxin level of less than about 0.50000 endotoxin units per milligram (EU / mg). In some embodiments, the modified annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof) is produced in prokaryotic cells.

[0014] Other features and advantages of the present disclosure will be better understood by reference to the following detailed description, including figures and examples. [Brief explanation of the drawings]

[0015] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0016] [Figure 1] These results demonstrate that Ca2+ dynamics influenced the recruitment of the annexin A6 repair cap at the injury site. Myofibers expressing the Ca2+ indicator GCaMP5G were generated, and time-lapse single-slice images were assessed at time points after membrane disruption. A) GCaMP5G fluorescence was present at the injury site at 2 seconds, indicating the presence of Ca2+ at the injury site immediately after injury. B) Time-lapse images of myofibers co-electroporated with GCaMP5G and annexin A6-tdTomato. GCaMP5G fluorescence was present at the injury site, localized around the annexin A6-free zone (arrowhead), and on the annexin A6 cap (arrow). GCaMP5G colocalized with the annexin A6 repair cap (merge, arrow). Scale bar 5 μm. C) Myofibers expressing fluorescently labeled annexins A1, A2, or A6 were injured at multiple Ca2+ concentrations. The size of annexin A1 and A6 repair caps was reduced at 0.1 mM Ca2+ compared to 2 mM and 0.5 mM. Annexin A2 repair cap area was significantly reduced at 0.05 mM Ca2+ compared to 2 mM, 0.5 mM, and 0.1 mM Ca2+. D) Cap dynamics plotted as ferret diameter of the cap over a range of Ca2+ concentrations. Annexin A2 had a statistically significant leftward shift at Km1 / 2, followed by annexin A6 and then A1. Scale 5 μm. [Figure 2]Domain modeling of the multiple Ca2+-binding sites of annexins is shown. Annexins typically contain four annexin domains, except for annexin A6, which has been duplicated to contain eight annexin domains. Annexins A1, A2, and A6 coordinate multiple Ca2+ ions and bind to membranes on the convex surface, positioned toward the top of each ribbon diagram. Residues D171 in annexin A1 and D161 in annexin A2 were previously described as residues required for Ca2+ binding coordination (Jost et al., 1992). Both D171 and D161 are located in the second (yellow) annexin domains of annexins A1 and A2, respectively. The orthologous residue D149 of annexin A6, which occupies the first half of annexin A6, was not observed to coordinate Ca2+ in bovine A6 (Avila-Sakar et al., 1998). In this study, we investigated D149 as well as E233 in annexin A6, which are classified as the second and third annexin domains, respectively. [Figure 3] A) Maximum projection of Z-stack imaging of the repair cap (left). Rotated Z-stack projection (right). Cap size is measured from the central z-stack 2D image, represented by the white dotted line. B) Wild-type annexin-tdTomato and wild-type annexin-turboGFP were co-electroporated into muscle fibers. Cap size was assessed in both the red and green channels. The type of fluorescent tag did not affect cap size. Data are presented as mean ± SEM. Differences were tested by two-tailed t-test (n ≥ 10 fibers per condition). [Figure 4]Figure 1 shows the differential Ca2+ sensitivity of annexin A1, A2, and A6 in repair cap formation. Muscle fibers expressing fluorescently labeled annexin A1, A2, or A6 were injured with multiple Ca2+ concentrations. A&B) The size of the annexin A1 and A6 repair caps decreased with decreasing Ca2+ concentration. The area of ​​the annexin A1 and A6 repair caps and the rate of repair cap formation were reduced at 0.5 mM Ca2+ and 0.1 mM compared to 2 mM. C) Annexin A2 repair cap area was significantly reduced at 0.05 mM Ca2+ compared to 2 mM, 0.5 mM, and 0.1 mM Ca2+. The rate of annexin A2 repair cap formation was comparable across the Ca2+ concentrations tested. Data from the 0.05 mM concentration were not plotted because the caps were too small to measure over time. Large arrows indicate large caps. Small arrows indicate small caps. *p<0.05 for cap area or time, #p<0.05 for statistically different slopes. Scale bar 5 μm. Data are presented as mean ± SEM. Differences were tested by two-way ANOVA with Bonferroni's multiple comparison test. *p<0.05 for cap area or time, #p<0.05 for statistically different slopes (n=4-7 muscle fibers per condition). [Figure 5] Laser injury of myofibers on a Nikon A1R+ GaSP confocal and a Nikon A1R MP+ multiphoton confocal shows that comparable annexin A6-GFP repair caps (arrowheads) were induced at the site of membrane injury. Scale 5 μm. n ≥ 3 mice per condition. [Figure 6]These results demonstrate that annexin expression promoted bleb release from myofiber repair sites. Myofibers were electroporated with the Ca2+ indicator GCaMP5G with or without tdTomato-labeled annexin A1, annexin A2, or annexin A6. Ca2+ area and fluorescence were assessed after membrane injury. A) High-magnification z-projection images show external blebs filled with Ca2+ indicator released from the lesion when annexins A1, A2, or A6 were coexpressed, and a corresponding decrease in Ca2+ indicator within the myofiber compared to GCaMP5G alone. B) Expression of annexin A6 or A2 increased the number of GCaMP5G-positive blebs. C) Expression of annexin A6 resulted in the formation of the largest GCaMP5G-positive blebs. *p<0.05. (n≥3 mice per condition). [Figure 7]This shows that expression of annexins reduced Ca2+ in myofibers. Myofibers were electroporated with the Ca2+ indicator GCaMP5G with or without tdTomato-labeled annexin A1, annexin A2, or annexin A6 (annexin channels not shown). Ca2+ area and fluorescence were assessed after membrane injury. A) Time-lapse single-slice images show that coexpression of either annexin A1, A2, or A6 significantly reduced GCaMP5G fluorescence measured within myofibers at the injury site. B) Expression of either annexin A1, A2, or A6 significantly reduced GCaMP5G fluorescence measured within myofibers at the injury site over 240 seconds of imaging, with annexin A6 eliciting the greatest decrease in GCaMP5G fluorescence. C) Both annexins A2 and A6 contributed to the early decrease in GCaMP5G fluorescence, as seen by imaging during the first 20 seconds after injury. D) Early GCaMP5G mean fluorescence was not significantly different between groups. Scale bar 5 μm. *p<0.05. Myofibers from n≥3 mice per condition. Data are presented as mean ± SEM. Differences were tested by two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test (B, C) or one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (D). *p<0.05, (n≥9 myofibers from n≥3 mice per condition). [Figure 8]Myofibers overexpressing annexin A6 show no change in baseline Ca2+ levels. Myofibers electroporated with annexin A6-GFP or vehicle control. Isolated myofibers were loaded with Indo-1AM dye and stimulated to measure Ca2+ cycling and cell shortening. A) Representative Ca2+ transients at 80 Hz. B&C) Resting Ca2+ levels and peak Ca2+ transients did not change between myofibers electroporated with annexin A6-GFP or vehicle control. D) Representative sarcomere length shortening traces. E&F) Resting sarcomere length and peak unloaded sarcomere length shortening did not differ between treatment groups. Myofibers from n ≥ 3 mice per condition. Data are presented as mean ± SEM. Differences were tested by two-way ANOVA (A, D) or two-tailed t-test (B, C, E, F) (n ≥ 30 myofibers from n ≥ 3 mice per condition). [Figure 9]A) The first type II Ca2+ binding site is conserved in annexins A1 and A2, but not in annexin A6. B) Muscle fibers were co-electroporated with wild-type or wild-type mutant annexin constructs, and cap size was assessed after sarcolemma injury. Brackets indicate proteins that were co-expressed in the muscle fiber but not visualized within the channel to determine their effect on co-expressed annexins. Cap kinetics was plotted as the ferret diameter of the cap over a range of Ca2+ concentrations from 0 to 2 mM. Expression of mutant annexins A1D171A and A2D161A, but not A6D149A, was sufficient to significantly reduce the maximum diameter (DMAX) of co-expressed wild-type annexin caps. C) Expression of annexins A1D171A and A2D161A was not sufficient to significantly reduce the cap area of ​​co-expressed annexin A6, although there was a trend in the direction of annexin A2D161A. #, *p<0.05, ns=not significant. Muscle fibers from n≧3 mice per condition. Data are presented as mean±SEM. Differences were tested by two-way ANOVA with Bonferroni's multiple comparison test (B) or two-tailed t-test (C). *p<0.05#, ns=not significant (n≧5 muscle fibers from n≧3 mice per condition). [Figure 10]These results demonstrate that Ca2+-binding mutants of annexin A6 reduced annexin repair cap recruitment and impaired myofiber membrane repair capacity. A) Myofibers were co-electroporated with wild-type tdTomato and either wild-type GFP or mutant GFP annexin constructs. Cap size was assessed after membrane injury; only the red channel is shown to demonstrate the effect on wild-type annexin. B) Co-expression of mutant annexin A6 E233A was sufficient to reduce cap assembly of wild-type annexin A6. Cap kinetics was plotted as the ferret diameter of the cap over a range of Ca2+ concentrations from 0 to 2 mM. C) Co-expression of annexin A6 E233A was sufficient to significantly reduce the cap area of ​​co-expressed annexins A1, A2, and A6. *p<0.05 WT (WT) vs. WT (MUT). D) Myofibers were electroporated with annexin A6-GFP or mutant A6E233A-GFP. The cap area of ​​annexin A6E233A is significantly smaller (small arrow) compared to annexin A6 (large arrowhead), correlating with the increased FM4-64 fluorescence area (large arrowhead). Scale bar 5 μm. *p<0.05. Myofibers from n≥3 mice per condition. Data are presented as mean ± SEM. Differences were tested by two-tailed t-test (A, C, D). *p<0.05 (n=4-18 myofibers from n≥3 mice per condition). [Figure 11]These results demonstrate that annexin A6 enhances myofiber membrane repair. A) Overexpression of annexin A6 in wild-type myofibers reduced the uptake of FM4-64 dye, a membrane damage marker, after laser-induced injury compared to control myofibers. B) Wild-type myofibers injured in the presence of extracellular recombinant annexin A6 (rANXA6) showed significantly less FM4-64 dye uptake than control myofibers. C) rANXA6 with a C-terminal HIS tag localized to the plasma membrane of mdx / hLTBP4 myofibers 6 hours after systemic injection, as visualized by immunofluorescence microscopy. No anti-HIS staining was observed in control muscles from mice injected with phosphate-buffered saline (PBS). D) mdx / hLTBP4 myofibers injured in the presence of extracellular rANXA6 showed significantly less FM4-64 dye uptake (volume) than control myofibers, as shown by Imaris surface rendering of FM4-64 fluorescence. Scale bar 5 μm (A, B, D). Scale bar 1 mm (C). *p<0.05. (n≧3 muscle fibers per condition). [Figure 12] These results demonstrate that intramuscular injection of recombinant annexin A6 (rANXA6) protected against muscle injury in vivo. A) Wild-type mice were pre-injected with rANXA6 or a control solution and then injured with a cardiotoxin to induce muscle damage. B) Gross imaging revealed decreased dye uptake in rANXA6-pretreated muscles compared to the contralateral control muscles. C) Immunofluorescence imaging revealed decreased dye uptake in rANXA6-pretreated muscles. A surface plot of dye uptake shows decreased fluorescence in rANXA6-pretreated muscles. The white dotted line outlines the muscle section. D) rANXA6-pretreated muscles showed a significant reduction in dye fluorescence by approximately 50% across the entire muscle area compared to control muscles. Scale: 1 mm. *p>0.05 (n=3 mice per condition). Data are presented as mean ± SEM. Differences were tested by two-tailed t-test. *p<0.05 (n=3 mice per condition). [Figure 13]Systemic injection of recombinant annexin A6 (rANXA6) protects against muscle injury in vivo. A) Wild-type mice were systemically injected with rANXA6 or control solution before (pre-injection) or after (post-injury) cardiotoxin-induced muscle injury. B-C) ​​Immunofluorescence imaging revealed significantly reduced dye uptake in tibialis anterior muscles pre- or post-treated with rANXA6 compared to vehicle controls. The white dotted line indicates the outline of the muscle section. A surface plot of dye uptake shows reduced fluorescence in muscles pre-treated with rANXA6. Scale: 1 mm. *p>0.05 (n≥3 mice per condition, n≥6 legs). [Figure 14] We demonstrate that systemic injection of recombinant annexin A6 (rANXA6) protected against chronic muscle damage in vivo. A) Sgcg-deficient mice, a model of limb-girdle muscular dystrophy type 2C (LGMD2C), were injected with rANXA6 or a control solution every 3 days for a total of 12 days. B-C) ​​Next, clinically validated serum biomarkers of muscle damage, creatine kinase (CK) and lactate dehydrogenase (LDH), were assessed. Treatment with rANXA6 reduced serum CK and LDH compared to controls, indicating enhanced repair of chronically damaged muscle tissue. [Figure 15] This shows that there is no visible pH change immediately after membrane injury. Myofibers were laser injured in the presence of the pH fluorescent indicator pH Rodo AM. pH did not change before (0 s) and after (10 s) injury, expressed as F / F0 average = 0.97, with the same value of 1. Scale 5 μm. [Figure 16]This shows that annexin expression promoted the release of blebs from the lesion when myofibers were repaired. Myofibers were electroporated with the Ca2+ indicator GCaMP5G (green) with or without tdTomato-labeled annexin A1, annexin A2, or annexin A6. Ca2+ area and fluorescence were assessed after membrane injury. (A) High-magnification z-projection images show external blebs filled with Ca2+ indicator emanating from the lesion when annexins A1, A2, or A6 were coexpressed, and a corresponding decrease in Ca2+ indicator within the myofiber compared to GCaMP5G alone. (B) Membranes marked with FM4-64 demonstrate the formation of GCaMP5G-negative vesicles in the absence of annexin overexpression. (C) Expression of annexin A6 or A2 increased the number of GCaMP5G-positive blebs. (D) Expression of annexin A6 resulted in the formation of the largest GCaMP5G-positive blebs. Data are presented as mean ± SEM. Differences were tested by one-way ANOVA with Tukey's multiple comparison test. *p<0.05 (n≧16 muscle fibers from n≧3 mice per condition). [Figure 17] These results demonstrate that annexin A6 expression reduced Fluo-4 Ca2+ levels at the injury site. Myofibers were preloaded with the fluorescent Ca2+ indicator dye Fluo-4, followed by confocal laser injury to the sarcolemma. Annexin A6-overexpressing myofibers exhibited significantly reduced internal Fluo-4 Ca2+ fluorescence levels at the site of membrane injury compared with control myofibers, similar to results obtained with the protein-based Ca2+ indicator GCaMP5G. Scale: 5 μm. Data are presented as mean ± SEM. Differences were tested by two-way ANOVA with Bonferroni's multiple comparison test. *p<0.05 (n=3 mice per condition, n≥8 myofibers). [Figure 18]These results demonstrate that annexin A6 enhances the membrane repair capacity of healthy and dystrophic myofibers in vitro. (A) Plasmid expression of annexin A6 in wild-type (WT) myofibers reduced the uptake of FM4-64 dye, a marker of membrane damage, after laser-induced injury compared with control myofibers. (B) Wild-type myofibers injured in the presence of extracellular recombinant annexin A6 (rANXA6) showed significantly less uptake of FM4-64 dye compared with control myofibers. (C) Dystrophic (Dys) myofibers injured in the presence of recombinant annexin A6 (rANXA6) showed significantly less uptake of FM4-64 dye than control myofibers. Scale: 5 μm. Data are presented as mean ± SEM. Differences were tested by two-tailed t-test, *p<0.05 (n≥10 myofibers from n≥3 mice per condition). [Figure 19] The Ca2+ dependence of the protective effect of recombinant annexin A6 is shown. A and B) Wild-type myofibers were isolated and loaded with FM1-43 (green), a fluorescent marker of membrane damage. After pretreatment with recombinant annexin A6 (rANXA6), myofibers were damaged with 1 mM Ca2+ solution or 0 mM Ca2++, the calcium chelator EGTA. Fluorescent uptake of FM1-43 was significantly reduced in the presence of 1 mM Ca2+ compared to the presence of EGTA. Scale: 5 μm. Data are presented as mean ± SEM. Differences were tested by two-way ANOVA with Bonferroni's multiple comparison test. *p<0.05 (n≥10 myofibers per condition, n=3 mice per condition). [Figure 20]Systemic delivery of recombinant annexin A6 using retro-orbital injections protects against muscle injury in vivo. (A) Wild-type mice were intravenously injected with recombinant human annexin A6 (rANXA6) or a control solution. This was followed by muscle injury with cardiotoxin (CTX). (B-C) Immunofluorescence imaging revealed approximately 38% less dye uptake (red) in muscles pretreated with rANXA6. The dotted line outlines the tibialis anterior muscle (upper panel). DAPI (blue) marks nuclei. A surface plot of dye uptake shows reduced fluorescence in muscles pretreated with rANXA6. (D) Whole-tissue spectroscopy of injured gastrocnemius / soleus muscles revealed that pretreatment with rANXA6 reduced dye uptake by 58% compared to control muscles. (E) Wild-type mice were intravenously injected with rANXA6 or a control solution. Two hours later, the tibialis anterior muscle was injured with cardiotoxin. Muscles were harvested 7 days after injury. (F&G) Hematoxylin and eosin images were quantified. These images show a reduced rate of muscle fiber damage (dotted line) in rANXA6-treated mice compared with controls. B, Scale 1 mm. F, Scale 500 μm. Data are presented as mean ± SEM. Differences were tested by two-tailed t-test. *p<0.05 (B, C, D: n ≥ 3 mice, n ≥ 6 legs per condition; F, G: n ≥ 6 mice, n ≥ 11 muscles per condition). [Figure 21] The amino acid conservation of annexin A6 is shown. [Figure 22]These results demonstrate that recombinant annexin A6 protected against muscle damage in vivo in a mouse model of muscular dystrophy. (A-B) Sgcg-deficient mice, a model of limb-girdle muscular dystrophy 2C, were intravenously injected five times over 48 hours with recombinant human annexin A6 (rANXA6) or a BSA control solution. Serum CK was measured before injection. Two hours after the fifth injection, mice were subjected to 60 minutes of downhill running. Thirty minutes after exercise, serum CK was measured. The fold change in CK before / after running was significantly reduced with rANXA6 compared with BSA-injected controls, consistent with reduced muscle damage from acute running. (C-D) Sgcg-deficient mice were intravenously injected with rANXA6 or a control every three days for 14 days. On day 14, serum creatine kinase was assessed. Serum CK in rANXA6-treated Sgcg-deficient mice was lower than that in PBS controls. (E) Sgcg-deficient mice were intravenously injected with rANXA6 or recombinant annexin A2 every 3 days for 14 days. The post-treatment / pre-treatment fold change (day 14 / day 0) of serum CK was significantly reduced in Sgcg-deficient mice treated with recombinant annexin A6 compared with annexin A2. (F) Histological analysis of the gastrocnemius / soleus muscles of Sgcg-deficient mice shown in part D injected with PBS or recombinant annexin A6. The left side of the boxed area is low magnification, and the right side is high magnification. F, Scale 500 μm (left), Scale 50 μm (right). Data are presented as mean ± SEM. Differences were tested by two-tailed t-test (B&E). *p<0.05 (n≥3 mice per condition, except for part D). [Figure 23]This figure shows Ca2+-dependent annexin repair cap recruitment at the injury site. Myofibers expressing the Ca2+ indicator GCaMP5G (green) were generated, and time-lapse single-slice images were evaluated at time points after membrane disruption. (A) GCaMP5G fluorescence was present at the injury site at 2 seconds (arrow), indicating the presence of Ca2+ at the injury site immediately after injury (upper panel). These data were verified with Fluo-4AM (green, lower panel), a non-protein Ca2+ indicator. (B) Time-lapse images of myofibers co-electroporated with GCaMP5G and annexin A6-tdTomato (A6, red). GCaMP5G fluorescence was present at the injury site and at the annexin A6 cap (arrow), localized around the annexin A6-free zone (arrowhead). GCaMP5G colocalized with the annexin A6 repair cap (merge, yellow, arrow). Scale bar 5 μm. (C) Myofibers expressing fluorescently labeled annexin A1, A2, or A6 were injured at multiple Ca2+ concentrations. The size of the annexin A1 and A6 repair caps was reduced at 0.1 mM Ca2+ compared to 2 mM and 0.5 mM. The annexin A2 repair cap area was significantly reduced at 0.05 mM Ca2+ compared to 2 mM, 0.5 mM, and 0.1 mM Ca2+. (D) Cap dynamics were plotted as the ferret diameter of the cap across a range of Ca2+ concentrations. Annexin A2 exhibited a statistically significant leftward shift at Km1 / 2, followed by annexin A6 and then A1. Scale: 5 μm. Data are presented as mean ± SEM. Differences were tested by one-way ANOVA with Tukey's multiple comparison test (C). *p<0.05 (n≥5 myofibers per condition). [Figure 24]Genetically encoded annexin A6GFP responds to prednisone exposure. A) Targeting strategy for generating genetically encoded annexin A6GFP in mice. B) Mice were injected intraperitoneally with prednisone (pred) or control (Ctrl). 24 hours after injection, muscle fibers were isolated and subjected to laser injury. The size of the annexin A6GFP repair cap decreased following prednisone administration, indicating protection against injury. Scale: 5 μm. *p<0.05. t-test, n≥3 mice per treatment. [Figure 25] We show that recombinant annexin A6 pretreatment reduces genetically encoded annexin A6 GFP cap size and dye influx, indicating enhanced protection against injury. Muscle fibers were isolated, pretreated with recombinant annexin A6 (rANAXA6), and then subjected to laser-induced membrane damage. Z-stack imaging revealed that the size of the annexin A6 GFP repair cap was reduced in the presence of recombinant annexin A6, indicating protection against injury. This correlated with reduced uptake of FM4-64, a membrane damage marker, in the presence of rANXA6. Scale 5 μm. *p<0.05. t-test. n≥3 mice per treatment. [Figure 26] Genetically encoded annexin A6GFP localizes to sites of membrane injury in cardiomyocytes. Adult cardiomyocytes were isolated from Anxa6em1(GFP) mice and then laser-injured. Annexin A6GFP (green) localizes to the repair cap at the injury site (white arrow, left image). The area enclosed by the dotted line is enlarged on the right. Time-lapse imaging shows that annexin A6GFP localizes to the repair cap at the injury site (white arrow) over a 50-second imaging period in adult cardiomyocytes isolated from Anxa6em1(GFP) mice. Scale: 5 μm or 10 μm. [Figure 27]These results demonstrate that annexin A6 enhanced membrane repair capacity and protected dystrophic, dysferlin-deficient myofibers from injury in vitro. A) Plasmid expression of annexin A6 in dysferlin-deficient (Dysf129) myofibers reduced FM4-64 dye uptake (small arrow), a marker of membrane damage, after laser-induced injury compared to control myofibers (arrowhead). B) Pretreatment with recombinant annexin A6 reduced FM4-64 uptake (small arrow) after laser injury in dysferlin-deficient (Dysf129) myofibers compared to controls (arrowhead). Scale: 5 μm. Data are presented as mean ± SEM. Differences were tested by two-tailed t-test. *p<0.05 (n≥9 myofibers per condition). [Figure 28] We demonstrate that recombinant annexin A6 localizes to the dystrophic sarcolemma of limb-girdle muscular dystrophy 2C muscle. Recombinant annexin A6 (rANXA6) with a carboxy-terminal HIS tag was injected into healthy wild-type mice or into an Sgcg-deficient mouse model of limb-girdle muscular dystrophy 2C, which undergoes chronic membrane disruption. Mice were on a 129Sv / EmsJ background. Anti-HIS fluorescence intensity was increased at the plasma membrane of Sgcg-deficient muscle, visualized by anti-laminin staining, compared with wild-type controls. Hoechst-labeled nuclei. [Figure 29] Figure 1 shows SDS-PAGE (left panel) and Western blot analysis (center and right panels) of recombinant annexin A6 protein expressed in mammalian cells. Because recombinant annexin A6 was produced with a C-terminal HIS epitope tag for detection and purification purposes, the center panel is blotted with anti-HIS. The right panel is blotted against A6 itself. [Figure 30]Figure 1 shows the results of SDS-PAGE (Coomassie staining) (left panel) and Western blot analysis (center and right panels) of recombinant annexin A6 protein expressed in prokaryotic cells. Lanes in the left and center panels are labeled T (total), S (soluble), and E (eluate). The contents of the lanes in the right panel reflect those in the left and center panels. The center panel is blotted for annexin A6. Because recombinant annexin A6 was produced with a C-terminal HIS tag for purification purposes, the right panel is blotted for anti-HIS. [Figure 31] Panels A, B, and C show that wild-type myofibers injured in the presence of extracellular recombinant annexin A6 (rANXA6) produced in E. coli or mammalian HEK cells had significantly less FM4-64 dye uptake compared to myofibers treated with the BSA control, indicating protection against membrane damage and enhanced repair with rANXA6 treatment. (B & C) The beneficial effect of rANXA6 on membrane repair was not significantly different between rANXA6 produced in E. coli and rANXA6 protein produced in mammalian HEK cells, obtained commercially through R&D Systems or produced by a contract research organization (CRO, Evotec SE), at 13 μg / ml and 130 μg / ml. Scale = 5 μm. Data are presented as mean ± SEM. Differences were tested by two-tailed t-test. *p<0.05 (n≥8 myofibers from n≥3 mice per condition). [Figure 32] This shows that annexin A6 lacking the amino acid sequence VAAEIL (SEQ ID NO: 47) localizes to sites of muscle membrane injury. A) Schematic diagram showing the structure of annexin A6. The amino acid sequence VAAEIL is located in annexin repeat domain 7, exon 21. B) Muscle fibers were electroporated with GFP-labeled annexin A6 lacking VAAEIL (SEQ ID NO: 47) and tdTomato-labeled annexin A6. High-magnification images show that annexin A6 lacking the VAAEIL (SEQ ID NO: 47) sequence partially colocalizes with annexin A6 in the repair cap at sites of muscle membrane injury. DETAILED DESCRIPTION OF THE INVENTION

[0017] Membrane repair is essential for cell survival and requires a fast and efficient process to limit cell damage.Muscle membranes are prone to breakdown due to the elongated shape of muscle cells and the continuous stress caused by muscle contraction.Mutations in genes encoding membrane-related proteins produce fragile membranes, reduce membrane repair ability, and cause progressive muscular dystrophy.The treatments and methods disclosed herein for enhancing membrane repair and reducing susceptibility to membrane damage are believed to benefit muscles in both acute and chronic injury situations.

[0018] As used herein, an agent that "increases the activity of annexin protein" refers to an agent that increases the properties of annexin protein as a calcium-binding membrane-associated repair protein that enhances the restoration of membrane integrity. The enhancement to restore membrane integrity may be due to promoting the formation of macromolecular repair complexes at membrane lesions, including, but not limited to, proteins such as annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), EHD2, dysferlin, and MG53.

[0019] As used herein, the term "treat" or "treatment" refers to an intervention intended to prevent further development or change in the symptoms of a disease or infectious disease. Thus, "treatment" refers to both therapeutic treatment and preventive or preventative measures. Of course, when "treatment" is used with the separate term "prevention," it is understood that "treatment" refers to the narrower meaning of changing the symptoms of a disease or condition. "Prevent" refers to preventive measures taken on subjects who do not have a condition or disease. A therapeutic agent may directly reduce the symptoms of a disease or make the disease more susceptible to treatment by another therapeutic agent or, for example, the host's own cell membrane repair system. Treatment of patients suffering from clinical, biochemical, or subjective symptoms of a disease may include alleviating one or more of such symptoms or reducing predisposition to the disease. Improvement after treatment may be manifested as a reduction or elimination of one or more of such symptoms.

[0020] Annexin proteins The annexin protein family is a Ca 2+ Annexins are characterized by their ability to bind phospholipids and actin in a Ca-dependent manner. Annexins preferentially bind phosphatidylserine, phosphatidylinositol, and cholesterol (Gerke et al., 2005). In humans, dominant or recessive mutations in annexin genes have not been associated with muscle diseases. However, genetic variants of annexin A5 have been associated with miscarriage (de Laat et al., 2006). The annexin family is known to contain over 160 distinct proteins present in over 65 unique species (Gerke and Moss, 2002). Humans have 12 distinct annexin genes, characterized by distinct tissue expression and localization. Annexins are involved in various cellular processes, including membrane permeability, mobility, vesicle fusion, and membrane bending. These properties are mediated by Ca 2+Annexins do not contain EF-hand domains, but calcium ions bind to individual annexin repeat domains. 2+ Affinity allows each annexin protein to respond to changes in intracellular calcium levels under unique spatiotemporal conditions (Blackwood and Ernst, 1990).

[0021] Structurally, annexin family proteins contain a conserved carboxy-terminal core domain composed of multiple annexin repeats and a variable amino-terminal head. The amino-terminus varies in length and amino acid sequence among annexin family members. Furthermore, post-translational modifications alter protein function and protein localization (Goulet et al., 1992; Kaetzel et al., 2001). Annexin proteins mediate Ca transport. 2+ It may self-oligomerize in the presence of Ca and interact with membrane surfaces and actin (Zaks and Creutz, 1991; Hayes et al., 2006; Jaiswal et al., 2014). 2+ It is believed that annexins bind to actin or one lipid membrane in a membrane-dependent manner, while the annexin core region binds to an additional lipid membrane.

[0022] Although annexins do not contain a predicted hydrophobic signal sequence that targets them for classical secretion through the endoplasmic reticulum, they can be found both inside and outside cells (Christmas et al., 1991, Deora et al., 2004, Wallner et al., 1986). The process by which annexins are externalized remains unclear. While the method of externalization may vary depending on the cell type, annexins are hypothesized to be released by exocytosis or cell lysis. Functionally, their localization both inside and outside the cell adds to the complexity of the roles that annexins play in tissues and cell types. Annexin A5 is commonly used as a marker of apoptosis due to its high affinity for phosphatidylserine (PS). During cell death and injury, PS reverses membrane orientation from the inner to outer membrane, providing access for annexin binding from the extracellular space. Annexins have been shown to have anti-inflammatory, profibrinolytic, and antithrombotic effects. Annexin A1-deficient mouse models exhibit exacerbated inflammatory responses when challenged and are resistant to the anti-inflammatory effects of glucocorticoids (Hannon et al., 2003). Annexin A2-deficient mice exhibit fibrin accumulation in the microvasculature and defective arterial thrombus clearance (Ling et al., 2004). Although little is known about the precise function of extracellular annexins, there is a strong correlation between high annexin expression levels and clinical severity of disease, suggesting that annexin protein expression levels may serve as diagnostic markers for many diseases (Cagliani et al., 2005).

[0023] Drugs In some embodiments, the present disclosure provides a method of treating cell membrane damage, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein. In a further embodiment, a method of delaying the onset of, preventing, or enhancing recovery from cell membrane damage is provided, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein. By "increasing the activity of an annexin protein" is meant that administration of the agent results in an overall increase in the activity of one or more annexin proteins as disclosed herein (i.e., an increase in any endogenous activity in addition to the activity achieved by administration of the agent).

[0024] As used herein, the term "agent" refers to recombinant proteins (eg, recombinant annexin proteins), steroids, annexin peptides, and polynucleotides capable of expressing annexin proteins.

[0025] Proteins / Recombinant Proteins Methods of the present disclosure include methods in which a recombinant protein (e.g., one or more annexin proteins) is administered to a patient in need thereof in a therapeutically effective amount. Thus, in any of the aspects or embodiments of the present disclosure, the agent that increases the activity of an annexin protein is a recombinant protein (e.g., an annexin protein). As used herein, "protein" refers to a polymer composed of amino acid residues. As used herein, "annexin protein" includes, but is not limited to, a wild-type annexin protein, a modified annexin protein, an annexin-like protein, or a fragment, analog, variant, fusion, or mimetic (each described herein). An "annexin peptide" is a shorter version (e.g., about 50 amino acids or less) of a wild-type annexin protein, an annexin-like protein, or a fragment, analog, variant, fusion, or mimetic sufficient to increase the overall activity of the annexin protein with which the annexin peptide is associated.

[0026] Polypeptides of the present disclosure can be either natural or non-natural. Natural proteins include, but are not limited to, biologically active proteins that exist in nature or can be produced in a form found in nature, for example, by chemical synthesis or recombinant expression techniques. Natural proteins also include post-translationally modified proteins, such as, but not limited to, glycosylated proteins. Non-natural proteins contemplated by the present disclosure include, but are not limited to, synthetic proteins, as well as fragments, analogs, and variants of natural or non-natural proteins as defined herein. Non-natural proteins also include proteins or proteinaceous substances that have D-amino acids, modified, derivatized, or non-natural amino acids in D- or L-configuration and / or peptidomimetic units as part of their structure. The term "protein" typically refers to large polypeptides. The term "peptide" generally refers to short polypeptides (e.g., less than about 50 amino acids).

[0027] Non-naturally occurring proteins are prepared, for example, using an automated protein synthesizer, or alternatively, using recombinant expression techniques using modified oligonucleotides encoding the desired protein.

[0028] As used herein, a "fragment" of a protein is meant to refer to any portion of the protein that is smaller than the full-length protein expression product.

[0029] As used herein, "analog" refers to any of two or more proteins that are substantially similar in structure to either the entire molecule or a fragment thereof and have the same biological activity, but may have varying degrees of activity. Analogs differ in the composition of their amino acid sequence based on one or more mutations, including the substitution, deletion, insertion, and / or addition of one or more amino acids for other amino acids. Substitutions can be conservative or non-conservative based on the physicochemical or functional relatedness of the amino acid being replaced and the amino acid replacing it.

[0030] As used herein, "variant" refers to a protein or analog thereof that has been modified to contain additional chemical moieties not normally part of the molecule. Such moieties can, for example, but not limited to, modulate the solubility, absorption, and / or biological half-life of the molecule. Moieties that can mediate such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to molecules are well known in the art. In various embodiments, the polypeptide is modified by biotinylation, glycosylation, PEGylation, and / or polysialylation.

[0031] Fusion proteins, including fusion proteins in which one fusion component is a fragment or mimetic, are also contemplated. As used herein, "mimetic" refers to a peptide or protein that has biological activity comparable to that of the protein it is a mimetic of.

[0032] In any of the aspects or embodiments of the present disclosure, the recombinant protein is an annexin protein (e.g., a recombinant wild-type annexin protein, a modified annexin protein, an annexin-like protein, or a fragment of a wild-type annexin protein or an annexin-like protein that exhibits one or more biological activities of an annexin protein). By "annexin-like protein" is meant a protein having sufficient amino acid sequence identity to a reference wild-type annexin protein to exhibit the activity of an annexin protein, for example, activity as a calcium-binding membrane-associated repair protein that enhances restoration of membrane integrity by promoting the formation of macromolecular repair complexes at membrane lesions, including proteins such as, but not limited to, annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), EHD2, dysferlin, and MG53. In some embodiments, the annexin-like protein is a protein having about or at least about 75% amino acid sequence identity to a reference wild-type human annexin protein (annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof), annexin A7 (SEQ ID NO: 9 or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 or SEQ ID NO: 16), or annexin A13 (SEQ ID NO: 17 or SEQ ID NO: 18)). In further embodiments, the annexin-like protein is a protein having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, or about 99% amino acid sequence identity to a wild-type human annexin protein.

[0033] In some embodiments, the agent of the present disclosure is an annexin protein comprising a post-translational modification that, in various embodiments, increases production of the annexin or annexin-like protein, increases the solubility of the annexin or annexin-like protein, decreases aggregation of the annexin or annexin-like protein, increases the half-life of the annexin or annexin-like protein, increases the stability of the annexin or annexin-like protein, enhances target membrane binding of the annexin or annexin-like protein, or is a codon-optimized version of the annexin or annexin-like protein.

[0034] Polynucleotides In some embodiments, the agent of the present disclosure is a polynucleotide that can express annexin protein as described herein.The term "nucleotide" or its plural forms used herein can be interchangeable with the variants discussed herein or otherwise known in the art.In certain instances, the art uses the term "nucleobase", which encompasses naturally occurring nucleotides and non-naturally occurring nucleotides, including modified nucleotides.Therefore, nucleotide or nucleobase refers to naturally occurring nucleobases A, G, C, T and U. Non-naturally occurring nucleobases include, for example and without limitation, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosine, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and the "non-naturally occurring" nucleobases described in U.S. Patent No. 5,432,272 to Benner et al. and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp 4429-4443. The term "nucleobase" includes not only the known purine and pyrimidine heterocycles, but also their heterocyclic analogs and tautomers.Further, naturally occurring and non-naturally occurring nucleobases include those described in U.S. Pat. No. 3,687,808 (Merigan, et al.), Chapter 15 by Sanghvi, Antisense Research and Application, Ed. S.T. Crooke and B. Lebleu, CRC Press, 1993, Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623), and Concise Encyclopedia of Polymer Science and Engineering, J.I. Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design 1991, 6, 585-607, each of which is incorporated herein by reference in its entirety. In various embodiments, oligonucleotides also contain one or more "nucleoside bases" or "base units," which are a category of non-naturally occurring nucleotides, including compounds such as heterocyclic compounds, that are not nucleoside bases in the most classical sense, but can function like nucleic acid bases, including certain "universal bases" that function as nucleoside bases.Universal bases include 3-nitropyrrole, optionally substituted indole (e.g., 5-nitroindole), and optionally substituted hypoxanthine.Other desirable universal bases include pyrrole, diazole, or triazole derivatives, including universal bases known in the art.

[0035] Modified nucleotides are described in EP 1072679 and WO 97 / 12896, the disclosures of which are incorporated herein by reference. Modified nucleotides include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (succinimide), 5-isopropyl ... uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), and pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Some of these bases are useful for increasing binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C, and in certain embodiments, are combined with 2'-O-methoxyethyl sugar modifications (see U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, and 5,552). See, for example, Nos. 5,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,830,653, 5,763,588, 6,005,096, 5,750,692, and 5,681,941, the disclosures of which are incorporated herein by reference.

[0036] The method of preparing oligonucleotides of predetermined sequence is well known.For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st ed. (Oxford University Press, New York, 1991).Solid-phase synthesis method is suitable for both polyribonucleotides and polydeoxyribonucleotides (the well-known method of DNA synthesis is also useful for RNA synthesis).Polynucleotides and polyribonucleotides can also be prepared enzymatically, for example, through polymerase chain reaction (PCR).Non-naturally occurring nucleobases can also be incorporated into oligonucleotides. See, for example, U.S. Patent No. 7,223,833, Katz, J. Am. Chem. Soc., 74:2238 (1951), Yamane, et al., J. Am. Chem. Soc., 83:2599 (1961), Kosturko, et al., Biochemistry, 13:3949 (1974), Thomas, J. Am. Chem. Soc., 76:6032 (1954), Zhang, et al., J. Am. Chem. Soc., 127:74-75 (2005), and Zimmermann, et al., J. Am. Chem. Soc., 124:13684-13685 (2002).

[0037] steroid In some embodiments, the agent that increases the activity of annexin protein is a steroid. In further embodiments, the steroid is a corticosteroid, a glucocorticoid, or a mineralocorticoid. In yet another embodiment, the corticosteroid is betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. In some embodiments, the corticosteroid is salmeterol, fluticasone, or budesonide.

[0038] In some embodiments, the steroid is an anabolic steroid. In further embodiments, the anabolic steroid includes, but is not limited to, testosterone or related steroid compounds with muscle growth-inducing properties, such as cyclostanazole or metadrostenol, prohomone or derivatives thereof, estrogen modulators, and selective androgen receptor modulators (SARMS).

[0039] vector In the method of the present disclosure, an appropriate expression vector can be used to deliver exogenous nucleic acids to recipient muscle cells. To achieve effective gene therapy, the expression vector must be designed for efficient cellular uptake and gene product expression. In some embodiments, the vector is located within the chloroplast. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of a herpesvirus vector, an adeno-associated virus (AAV) vector, an adenovirus vector, and a lentivirus vector.

[0040] The use of adenovirus or adeno-associated virus (AAV)-based vectors for gene delivery has been described [Berkner, Current Topics in Microbiol. and Imunol. 158 39-66 (1992); Stratford-Perricaudet et al., Hum. Gene Ther. 1:241-256 (1990); Rosenfeld et al., Cell 8:143-144 (1992); Stratford-Perricaudet et al., J. Clin. Invest. 90:626-630 (1992)]. In various embodiments, the adeno-associated virus vector is AAV5, AAV6, AAV8, AAV9, or AAV74. In some embodiments, the adeno-associated virus vector is AAV9. In further embodiments, the adeno-associated virus vector is AAVrh74.

[0041] The particular methods for gene therapy useful in the context of the present disclosure will depend largely on the expression system used, however, most methods involve the insertion of a coding sequence into an appropriate location within an expression vector, and then delivery of the expression vector to the target muscle tissue for expression.

[0042] Additional delivery systems useful in practicing the methods of the present disclosure are described in U.S. Patent Publication Nos. 2012 / 0046345 and 2012 / 0039806, each of which is incorporated herein by reference in its entirety.

[0043] Disability / damage In various aspects, the present disclosure provides compositions for treating, delaying the onset of, enhancing recovery from, or preventing cell membrane damage, comprising administering to a patient in need thereof a drug and optionally an additional drug.

[0044] Such patients may suffer from, for example, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), myotonic dystrophy, facioscapulohumeral dystrophy (FSHD), oculopharyngeal muscular dystrophy, distal muscular dystrophy, cystic fibrosis, pulmonary fibrosis, muscle atrophy, cerebral palsy, epithelial disorders, epidermal disorders, kidney disorders, liver disorders, sarcopenia, cardiomyopathy, myopathy, cystic fibrosis, pulmonary fibrosis, cardiomyopathies (hypertrophic, dilated, (including congenital, arrhythmogenic, restrictive, ischemic, or heart failure), acute lung injury, acute muscle injury, acute myocardial injury, radiation-induced injury, colon cancer, idiopathic pulmonary fibrosis, idiopathic interstitial pneumonia, autoimmune lung disease, benign prostatic hyperplasia, cerebral infarction, musculoskeletal fibrosis, postoperative adhesions, liver cirrhosis, renal fibrotic disease, fibrovascular disease, neurofibromatosis, Alzheimer's disease, diabetic retinopathy, skin lesions, HIV-associated lymph node fibrosis, chronic obstructive pulmonary disease (COPD), inflammatory pulmonary fibrosis, rheumatoid arthritis; rheumatoid spondylitis; osteoarthritis; gout, and other arthritic conditions. Sepsis; septic shock; endotoxic shock; gram-negative sepsis; toxic shock syndrome; myofascial pain syndrome (MPS); bacterial dysentery; asthma; adult respiratory distress syndrome; inflammatory bowel disease; Crohn's disease; psoriasis; eczema; ulcerative colitis; glomerulonephritis; scleroderma; chronic thyroiditis; Graves' disease; Ormond's disease; autoimmune gastritis; myasthenia gravis; autoimmune hemolytic anemia; autoimmune neutropenia; thrombocytopenia; pancreatic fibrosis; chronic active hepatitis including hepatic fibrosis; renal fibrosis, irritable bowel syndrome; fever (pyresis); restenosis; cerebral malaria; cerebral stroke and ischemic injury; neurological trauma; Huntington's disease; Parkinson's disease; allergies, including allergic rhinitis and allergic conjunctivitis; cachexia; Reiter's syndrome; acute synovitis; muscle degeneration, bursitis; tendonitis; tenosynovitis; osteopetrosis; thrombosis; silicosis; pulmonary sarcoidosis; bone resorption diseases such as osteoporosis or multiple myeloma-related bone disorder; cancer, including but not limited to metastatic breast cancer, colorectal cancer, malignant melanoma, gastric cancer, and non-small cell lung cancer; graft-versus-host reaction; and autoimmune diseases such as multiple sclerosis, lupus, and fibromyalgia;Patients with viral diseases such as shingles, herpes simplex I or II, influenza virus, severe acute respiratory syndrome (SARS), and cytomegalovirus;

[0045] As used herein, "cardiomyopathy" refers to any disease or dysfunction of the myocardium (heart muscle) in which the heart becomes abnormally enlarged, thickened, and / or stiffened. This weakens the myocardium's ability to pump blood normally, often leading to congestive heart failure. The disease or disorder may be, for example, inflammatory, metabolic, toxic, infiltrative, fibrotic, hematological, genetic, or of unknown origin. Such cardiomyopathies may be caused by oxygen deprivation. Other diseases include those resulting from myocardial injury, which involves damage to the muscle of the heart wall or myocardium as a result of disease or trauma. Myocardial injury may result from many causes, including, but not limited to, cardiomyopathy, myocardial infarction, or congenital heart disease. Heart disorders may be of pediatric origin. Cardiomyopathy includes, but is not limited to, cardiomyopathies (dilated, hypertrophic, restrictive, arrhythmogenic, hereditary, idiopathic, and unclassified cardiomyopathies), idiopathic dilated cardiomyopathy, X-linked dilated cardiomyopathy (XLDC), acute and chronic heart failure, right heart failure, left heart failure, biventricular heart failure, congenital heart defects, myocardial fibrosis, mitral stenosis, mitral regurgitation, aortic stenosis, aortic regurgitation, tricuspid stenosis, tricuspid regurgitation, pulmonary stenosis, pulmonary regurgitation, combined valve defects, myocarditis, acute myocarditis, chronic myocarditis, viral myocarditis, diastolic heart failure, systolic heart failure, diabetic heart failure, and storage diseases.

[0046] Additional (second) medication In various embodiments of the present disclosure, it is contemplated that the second agent is administered together with an agent that increases the activity of annexin protein.Non-limiting examples of the second agent include Mitsugumin 53 (MG53), microdystrophin, latent TGF-β binding protein 4 (LTBP4) modulator, transforming growth factor β (TGF-β) activity modulator, androgen response modulator, inflammatory response modulator, muscle growth promoter, chemotherapeutic agent, fibrosis modulator, and combinations thereof.Furthermore, in various embodiments, the method disclosed herein can include one or more of these agents, or one or more of these agents in a composition with any other active agent.

[0047] LTBP4 modulators LTBP4 is an extracellular matrix protein located on human chromosome 19q13.1-q13.2 that binds to and sequesters TGFβ. LTBP4 regulates muscular dystrophy in mice through polymorphisms in the Ltbp4 gene. See U.S. Patent No. 9,873,739 (incorporated herein by reference in its entirety). There are two common variants of the Ltbp4 gene in mice. Most mouse strains, including mdx mice, carry the Ltbp4 insertion allele (Ltbp4 I / I ) Ltbp4 I / I A 36 base pair (12 amino acid) insertion in the proline-rich region of LTBP4, encoded by LTBP4, leads to milder disease. A 36 bp / 12 aa deletion in the proline-rich region is associated with more severe disease (Ltbp4 D / D ) We found that the Ltbp4 genotype strongly correlates with two distinct aspects of muscular dystrophy pathology: membrane leakage and fibrosis, and that these features define DMD pathology.

[0048] Modulators of LTBP4 are described in US Pat. No. 9,873,739, which is incorporated herein by reference in its entirety.

[0049] Modulators of TGF-β activity The transforming growth factor-β (TGF-β) superfamily is a family of secreted proteins composed of over 30 members, including activins, nodal proteins, bone morphogenetic proteins (BMPs), and growth and differentiation factors (GDFs). Superfamily members are generally ubiquitously expressed and regulate many cellular processes, including growth, development, and regeneration. Mutations in TGF-β superfamily members cause many diseases, including autoimmune diseases, heart disease, fibrosis, and cancer.

[0050] The TGF-β ligand family includes TGF-β1, TGF-β2, and TGF-β3. TGF-β is secreted into the extracellular matrix in an inactive form bound to latency-associated peptide (LAP). Latent TGF-β protein (LTBP), which binds to the TGF-β / LAP complex, provides another level of regulation. Extracellular proteases cleave the LTBP / LAP / TGF-β complex, releasing TGF-β. As a result, TGF-β is free to bind to its receptors, TGF-βRI or TGF-βBRII. TGF-β / receptor binding activates downstream canonical and noncanonical SMAD pathways, including the activation of SMAD factors, leading to gene transcription. TGF-β signaling has emerged as a prominent mediator of the fibrotic response and disease progression in muscle diseases, and its expression is upregulated in both murine and human dystrophies. Blockade of TGF-β signaling in mice by expression of a dominant-negative receptor (TGFBRII) improved dystrophic pathology, enhanced regeneration, and reduced muscle damage in δ-sarcoglycan-deficient mice, a mouse model of muscular dystrophy (Accornero, McNally et al. Hum Mol Genet 2014). Furthermore, antibody-mediated blockade of TGF-β signaling with the pan anti-TGF-β antibody 1d11 monoclonal antibody improved respiratory outcome measures in a mouse model of Duchenne muscular dystrophy (Nelson, Wentworth et al. Am J Pathol 2011). Therefore, therapeutic approaches to TGF-β signaling are contemplated herein to improve repair and slow disease progression.

[0051] Therapeutic agents thought to be effective against TGF-β signaling include galunisertib (LY2157299 monohydrate), TEW-7917, monoclonal antibodies against TGF-β ligands (TGF-β1, 2, 3 alone or pan1, 2, 3), fresolimub (GC-1008), TGF-β peptide P144, LY2382770, small molecules, SB-525334, and GW788388.

[0052] Modulators of androgen response Selective androgen receptor modulators (SARMs) are a class of androgen receptor ligands that activate androgen signaling and exist in nonsteroidal and steroidal forms. Research has shown that SARMs can increase both muscle and bone mass. Testosterone is one of the best-known SARMs and promotes skeletal muscle growth in healthy and diseased tissues. Testosterone and dihydrotestosterone (DHT) promote muscle cell differentiation and upregulate follistatin, while downregulating TGF-β signaling, resulting in muscle growth (Singh et al., 2003; Singh et al., 2009; Gupta et al., 2008). SARM-mediated TGF-β inhibition may prevent muscle damage and improve repair. SARMS include testosterone, estrogen, dihydrotestosterone, estradiol, dihydronandrolone, nandrolone, nandrolone decanoate, ostarine, Ligandrol, LGD-3303, andarine, cardarine, 7-alphamethyl, 19-nortestosterone aryl-propionamides, bicyclic hydantoins, quinoline analogs, benizimidazole, imidazolopyrazole, indole, and pyrazoline derivatives, azasteroid derivatives, and aniline, diarylaniline, and benzoxazepinone derivatives.

[0053] Modulators of the inflammatory response Modulators of the inflammatory response include the following agents. In one embodiment of the present disclosure, the modulator of the inflammatory response is a beta2-adrenergic receptor agonist (e.g., albuterol). The term beta2-adrenergic receptor agonist is used herein to define a class of drugs that act on beta2-adrenergic receptors, thereby causing smooth muscle relaxation, resulting in dilation of bronchial passages, vasodilation of muscles and liver, relaxation of uterine muscles, and release of insulin. In one embodiment, the beta2-adrenergic receptor agonist for use in accordance with the present disclosure is albuterol, an immunosuppressant widely used in inhaled form for asthma patients. Albuterol is thought to slow disease progression by suppressing the infiltration of macrophages and other immune cells that contribute to inflammatory tissue loss. Albuterol also appears to have some anabolic effects and promotes muscle tissue growth. Albuterol may also inhibit protein degradation (possibly via calpain inhibition).

[0054] In DMD, loss of dystrophin disrupts muscle cell membranes and destabilizes neuronal nitric oxide synthase (nNOS), a protein that normally produces nitric oxide (NO). It is thought that at least part of the muscle degeneration observed in DMD patients may be due to reduced production of muscle membrane-associated neuronal nitric oxide synthase. This reduction may lead to dysregulated vasoconstrictor responses and eventual muscle damage.

[0055] In one embodiment, a suitable inflammatory response modulator for use in the disclosed compositions is a nuclear factor kappa-B (NF-κB) inhibitor. NF-κB is a key transcription factor regulating cellular immune, inflammatory, and proliferative responses. NF-κB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to inhibit muscle progenitor cell regeneration. Activation of this factor in DMD contributes to the pathology of the disease. Therefore, NF-κB plays an important role in the progression of muscular dystrophy, and the IKK / NF-κB signaling pathway is a potential therapeutic target for the treatment of TGFβ-related diseases. NF-κB inhibitors (e.g., IRFI042, a vitamin E analog) enhance muscle function, reduce serum creatine kinase (CK) levels and muscle necrosis, and enhance muscle regeneration. Edasalonexent is a small molecule NF-κB inhibitor. Oral administration of edasalonexent at 100 mg / kg slowed the progression of muscle disease in boys with Duchenne muscular dystrophy. Furthermore, specific inhibition of NF-κB-mediated signaling by IKK has similar benefits.

[0056] In a further embodiment, the inflammatory response modulator is a tumor necrosis factor alpha antagonist.TNF-α is one of the key cytokines that induce and maintain inflammatory response.In one particular embodiment of the present disclosure, the inflammatory response modulator is infliximab, a TNF-α antagonist.

[0057] TNF-α antagonists for use in accordance with the present disclosure include infliximab (Remicade™), as well as chimeric monoclonal antibodies containing murine VK and VH domains and a human constant Fc domain. This drug blocks the action of TNF-α by binding to TNF-α and preventing it from signaling to TNF-α receptors on the cell surface. Another TNF-α antagonist for use in accordance with the present disclosure is adalimumab (Humira™). Adalimumab is a fully human monoclonal antibody. Another TNF-α antagonist for use in accordance with the present disclosure is etanercept (Enbrel™). Etanercept is a dimeric fusion protein containing a soluble human TNF receptor linked to the Fc portion of IgG1. It is a large molecule that binds to and blocks the action of TNF-α. Etanercept mimics the inhibitory effects of naturally occurring soluble TNF receptors, but as a fusion protein, it has a significantly extended half-life in the bloodstream, resulting in a more profound and long-lasting inhibitory effect.

[0058] Another TNF-α antagonist for use in accordance with the present disclosure is pentoxifylline (Trental™), chemically named 1-(5-oxohexyl)-3,7-dimethylxanthine. The usual dosage in sustained-release tablet form is one tablet (400 mg) three times daily with meals.

[0059] Dosage: Remicade is administered by intravenous infusion, usually at two-month intervals. The recommended dose is 3 mg / kg administered as an intravenous infusion, with additional doses at two and six weeks after the first infusion, and then every eight weeks. For patients with an incomplete response, adjusting the dose up to 10 mg / kg or treating every four weeks can be considered. Humira is sold in both a preloaded 0.8 ml (40 mg) syringe and a preloaded pen device, both of which are typically injected subcutaneously by patients at home. Etanercept can be administered in doses of 25 mg (twice weekly) or 50 mg (once weekly).

[0060] In another embodiment of the present disclosure, the inflammatory response modulator is cyclosporine. Cyclosporine A, the primary form of this drug, is a cyclic, nonribosomal peptide composed of 11 amino acids produced by the fungus Tolypocladium inflatum. Cyclosporine is believed to bind to cyclophilin (immunophilin), a cytosolic protein of immunocompetent lymphocytes (especially T lymphocytes). This cyclosporine-cyclophilin complex inhibits calcineurin, which under normal circumstances activates interleukin-2 transcription. It also inhibits lymphokine production and interleukin release, thereby reducing effector T cell function. It does not affect cytostatic activity. It also affects mitochondria, preventing the opening of the mitochondrial PT pore and inhibiting the release of cytochrome c (a potent apoptosis stimulator). Cyclosporine can be administered at a dose of 1 to 10 mg / kg / day.

[0061] Muscle growth promoter In some embodiments of the present disclosure, a therapeutically effective amount of a muscle growth promoter is administered to the patient. Muscle growth promoters contemplated by the present disclosure include, but are not limited to, insulin-like growth factor-1 (IGF-1), Akt / protein kinase B, clenbuterol, creatine, decorin (see U.S. Patent Publication No. 20120058955), steroids (e.g., but not limited to, corticosteroids or glucocorticoid steroids), testosterone, and myostatin antagonists.

[0062] Myostatin antagonists Another class of muscle growth promoters suitable for use in the combinations of the present disclosure are myostatin antagonists. Myostatin, also known as growth / differentiation factor 8 (GDF-8), is a member of the transforming growth factor-β (TGFβ) superfamily, which is involved in regulating skeletal muscle mass. Most members of the TGF-β-GDF family are widely expressed and pleiotropic. However, myostatin is primarily expressed in skeletal muscle tissue, where it negatively regulates skeletal muscle growth. Myostatin is synthesized as an inactive preproprotein and activated by proteolysis. The precursor protein is cleaved to generate a COOH-terminal protein of approximately 109 amino acids. This is the mature, active form in the form of a homodimer of approximately 25 kDa. The mature dimer appears to circulate in the blood as an inactive latent complex bound to the propeptide. As used herein, the term "myostatin antagonist" refers to an agent that inhibits or blocks at least one activity of myostatin or blocks or reduces the expression of myostatin or its receptor (e.g., by interfering with the binding of myostatin to its receptor and / or blocking the signal transduction resulting from the binding of myostatin to its receptor). Thus, such agents include agents that bind to myostatin itself or its receptor.

[0063] Myostatin antagonists for use in accordance with the present disclosure include antibodies to GDF-8; antibodies to the GDF-8 receptor; soluble GDF-8 receptors and fragments thereof (including, for example, ActRIIB fusion polypeptides, such as those described in U.S. Patent Publication No. 2004 / 0223966, which is incorporated herein by reference in its entirety, and soluble ActRIIB receptors in which ActRIIB is linked to the Fc portion of an immunoglobulin); GDF-8 propeptide and modified forms thereof (e.g., as described in WO 2002 / 068650 or U.S. Patent No. 7,202,210, forms in which the GDF-8 propeptide is linked to the Fc portion of an immunoglobulin and / or in which GDF-8 is mutated at aspartic acid (asp) residues). forms, e.g., asp-99 of the murine GDF-8 propeptide and asp-100 of the human GDF-8 propeptide; small molecule inhibitors of GDF-8; follistatin (e.g., as described in U.S. Pat. No. 6,004,937, incorporated herein by reference), or follistatin domain-containing proteins (e.g., GASP-1, or other proteins such as those described in U.S. Pat. Nos. 7,192,717 and 7,572,763, each incorporated herein by reference); and modulators of metalloprotease activity that affect GDF-8 activation, such as those described in U.S. Patent Publication No. 2004 / 0138118, incorporated herein by reference.

[0064] Additional myostatin antagonists include myostatin antibodies that bind to and inhibit or neutralize myostatin (including the monomeric or dimeric form of myostatin proprotein and / or mature protein). Myostatin antibodies can be mammalian or non-mammalian antibodies, such as shark IgNAR antibodies, or humanized antibodies, or include functional fragments derived from antibodies. Such antibodies are described, for example, in WO2005 / 094446 and WO2006 / 116269, the contents of which are incorporated herein by reference. Myostatin antibodies also include antibodies that bind to myostatin proprotein and prevent its cleavage into the mature, active form. Additional antibody antagonists include the antibodies described in U.S. Patent No. 6,096,506 and U.S. Patent No. 6,468,535, each of which is incorporated herein by reference. In some embodiments, the GDF-8 inhibitor is a monoclonal antibody or fragment thereof that blocks the binding of GDF-8 to its receptor. Further embodiments include the murine monoclonal antibody JA-16 (as described in U.S. Pat. No. 7,320,789 (ATCC Deposit No. PTA-4236)), humanized derivatives thereof, and fully human monoclonal anti-GDF-8 antibodies (e.g., Myo29, Myo28, and Myo22, ATCC Deposit Nos. PTA-4741, PTA-4740, and PTA-4739, respectively, or derivatives thereof) (as described in U.S. Pat. No. 7,261,893, incorporated herein by reference).

[0065] In yet another embodiment, the myostatin antagonist comprises a soluble receptor that binds to myostatin and inhibits at least one of its activities. The term "soluble receptor" herein includes truncated forms or fragments of the myostatin receptor that specifically bind to myostatin, thereby blocking or inhibiting myostatin signal transduction. For example, truncated forms of the myostatin receptor include naturally occurring soluble domains and variations produced by proteolysis of the N-terminus or C-terminus. The soluble domain comprises all or part of the extracellular domain of the receptor, alone or bound to additional peptides or other moieties. Because myostatin binds to activin receptors (including activin IEB type receptor (ActRHB) and activin HA type receptor (ActRHA)), activin receptors can form the basis of soluble receptor antagonists. Soluble receptor fusion proteins containing soluble receptor Fc can also be used (see US Patent Publication Nos. 2004 / 0223966 and WO2006 / 012627, both of which are incorporated herein by reference in their entireties).

[0066] Other myostatin receptor-based myostatin antagonists are ALK-5 and / or ALK-7 inhibitors (see, e.g., WO2006 / 025988 and WO2005 / 084699, each of which is incorporated herein by reference). As a TGF-β cytokine, myostatin signals through a family of single transmembrane serine / threonine kinase receptors. These receptors can be divided into two classes: type I or activin-like kinase (ALK) receptors and type II receptors. ALK receptors are distinct from type II receptors in that they (a) lack a serine / threonine-rich intracellular tail, (b) possess a serine / threonine kinase domain that is highly homologous among type I receptors, and (c) share a common sequence motif, termed the GS domain, consisting of a region rich in glycine and serine residues. The GS domain is located amino-terminal to the intracellular kinase domain and is thought to be important for activation by type II receptors. Several studies have shown that TGF-β signaling requires both ALK (type I) and type II receptors. Specifically, type II receptors phosphorylate the GS domain of the type I receptor of TGFβ ALK5 in the presence of TGFβ. ALK5 then phosphorylates the cytoplasmic proteins smad2 and smad3 at two carboxy-terminal serines. Generally, in many species, type II receptors are thought to regulate cell proliferation, while type I receptors regulate matrix production. Various ALK5 receptor inhibitors have been described (see, e.g., U.S. Patent No. 6,465,493, U.S. Patent No. 6,906,089, U.S. Patent Publication Nos. 2003 / 0166633, 2004 / 0063745, and 2004 / 0039198, the disclosures of which are incorporated herein by reference). Thus, myostatin antagonists for use in accordance with the present disclosure may comprise the myostatin binding domains of the ALK5 and / or ALK7 receptors.

[0067] Other myostatin antagonists include soluble ligand antagonists that compete with myostatin for binding to myostatin receptors. The term "soluble ligand antagonist" herein refers to a soluble peptide, polypeptide, or peptidomimetic that can non-productively bind to myostatin receptors (e.g., activin-type HB receptor (ActRHA)), thereby competitively blocking myostatin receptor signal transduction. Soluble ligand antagonists include myostatin variants, also known as "myostatin analogs," that have homology with myostatin but do not have the activity of myostatin. Such analogs include truncations (such as N- or C-terminal truncations, substitutions, deletions, and other changes in amino acid sequence, such as variants with non-amino acid substitutions).

[0068] Additional myostatin antagonists contemplated by the present disclosure include inhibitory nucleic acids as described herein.These antagonists include antisense or sense polynucleotides, which comprise single-stranded polynucleotide sequences (either RNA or DNA) that can bind to target mRNA (sense) or DNA (antisense) sequences.Therefore, RNA interference (RNAi), which is produced by introducing specific small interfering RNA (siRNA), can also be used to inhibit or eliminate the activity of myostatin.

[0069] In certain embodiments, myostatin antagonists include, but are not limited to, follistatin, myostatin prodomain, growth and differentiation factor 11 (GDF-11) prodomain, prodomain fusion proteins, antagonistic antibodies or antibody fragments that bind to myostatin, antagonistic antibodies or antibody fragments that bind to activin type IEB receptor, soluble activin type IHB receptor, soluble activin type IEB receptor fusion proteins, soluble myostatin analogs (soluble ligands), polynucleotides, small molecules, peptidomimetics, and myostatin binding agents.Other antagonists include peptide immunogens described in U.S. Patent No. 6,369,201 and WO2001 / 05820 (each of which is incorporated herein by reference), as well as myostatin multimers and immune complexes that can induce an immune response and thereby block myostatin activity. Other antagonists include protein inhibitors of myostatin described in WO 2002 / 085306 (incorporated herein by reference), including truncated activin type II receptors, myostatin prodomains, and follistatin. Other myostatin inhibitors include those released into culture from cells overexpressing myostatin (see WO 2000 / 43781), dominant-negative myostatin proteins, including proteins encoded by the Piedmont allele (see WO 2001 / 53350), and mature myostatin peptides with C-terminal truncations at either amino acid position 335 or 375 or between amino acid positions 335 and 375. Small peptides described in U.S. Patent Publication No. 2004 / 0181033 (incorporated herein by reference), including the amino acid sequence WMCPP, are also suitable for use in the compositions of the present disclosure.

[0070] chemotherapy drugs Chemotherapeutic agents contemplated for use include, but are not limited to, alkylating agents, including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl CCNU); ethyleneimines / methylmelamines, such as triethylenemelamine (TEM), triethylene, thiophosphoramine, alkylsulfonates, such as busulfan; triazines, such as dacarbazine (DTIC); folic acid analogues, such as methotrexate and trimetrexate, pyrimidine analogues, such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogues, such as Antimetabolites, including 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products, including mitotic inhibitors, e.g., paclitaxel, vinca alkaloids (including vinblastine (VLB), vincristine, and vinorelbine), taxotere, estramustine, and estramustine phosphate; epipodophyllotoxins, such as etoposide and teniposide; antibiotics, such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C, and actinomycin; enzymes, such as L-asparaginase; biological response modifiers, such as interferon-alpha, IL-2, G-CSF, and GM-CSF;Various drugs, including platinum coordination complexes (such as cisplatin and carboplatin), anthracenediones (such as mitoxantrone), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (including N-methylhydrazine (MIH) and procarbazine), adrenocortical suppressants (such as mitotane (o,p´-DDD) and aminoglutethimide); hormones and antagonists, including corticosteroid antagonists (such as prednisone and equivalents, dexamethasone, and aminoglutethimide); progestins, e.g. For example, hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens, such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens, such as tamoxifen; androgens (including testosterone propionate and fluoxymesterone equivalents); antiandrogens, such as flutamide, gonadotropin-releasing hormone analogs, and leuprolide; and nonsteroidal antiandrogens, such as flutamide.

[0071] Fibrosis modulators As used herein, "fibrosis modulator" is synonymous with antifibrotic agent. The term "antifibrotic agent" refers to a compound that has antifibrotic activity (i.e., prevents or reduces fibrosis) in a mammal. This takes into account the abnormal formation of fibrous connective tissue, which is usually composed of collagen. These compounds may have different mechanisms of action; some reduce the formation of collagen or another protein, while others enhance the catabolism or removal of collagen in affected areas of the body. All such compounds active in reducing the presence of fibrous tissue are included herein, regardless of the specific mechanism by which each such agent functions. Antifibrotic agents useful in the methods and compositions of the present disclosure include those described in U.S. Patent No. 5,720,950 (incorporated herein by reference). Additional anti-fibrotic agents contemplated by the present disclosure include, but are not limited to, type II interferon receptor agonists (e.g., interferon-gamma), pirfenidone and pirfenidone analogs, anti-angiogenic agents such as VEGF antagonists, VEGF receptor antagonists, bFGF antagonists, bFGF receptor antagonists, TGFβ antagonists, TGFβ receptor antagonists, anti-inflammatory agents, IL-1 antagonists such as IL-1Ra, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, and aldosterone antagonists.

[0072] Gene correction approaches The present disclosure contemplates that gene correction approaches may be used in conjunction with the methods and compositions described herein. As used herein, "gene correction" approaches include, but are not limited to, techniques related to gene editing (i.e., CRISPR technology), exon skipping, and other techniques known in the art for modifying mRNA. Thus, in some embodiments, a method is provided for using the agents of the present disclosure to increase the activity of annexin proteins in individuals suffering from Becker muscular dystrophy (BMD), Duchenne muscular dystrophy (DMD), all Limb-girdle muscular dystrophy (LGMD) type 1 subtypes, all LGMD type 2 subtypes, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), myotonic dystrophy, facioscapulohumeral dystrophy (FSHD), oculopharyngeal muscular dystrophy, and distal muscular dystrophy, wherein the patient is administered, simultaneously administered, or previously administered a composition that corrects the gene involved in any one of the aforementioned disorders. In further embodiments, methods are provided for using agents of the present disclosure to increase annexin protein activity in individuals suffering from Becker muscular dystrophy (BMD), Duchenne muscular dystrophy (DMD), all Limb-girdle muscular dystrophy (LGMD) type 1 subtypes, all LGMD type 2 subtypes, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), myotonic dystrophy, facioscapulohumeral dystrophy (FSHD), oculopharyngeal muscular dystrophy, and distal muscular dystrophy, wherein the patient is administered, has been administered concomitantly with, or has previously been administered a viral or non-viral based composition that results in the correction of a gene involved in any one of the aforementioned disorders.

[0073] Genetic correction approaches are known in the art (see, for example, US Patent Application Publication No. 2016 / 0130608 and US Patent No. 9,499,817, each of which is incorporated herein by reference in its entirety).Details of such methods can be found in Echigoya et al., J Pers Med 8,2018; Li et al., Trends Pharmacol Sci 39:982-994,2018; Min et al., Annu Rev Med,2018 and Zhang et al., Physiol Rev 98:1205-1240,2018.

[0074] composition Any of the agents and / or additional agents described herein (or nucleic acids encoding any of the agents and / or additional agents described herein) are also provided in compositions. In this regard, the agents and / or additional agents are formulated with a physiologically acceptable (i.e., pharmacologically acceptable) carrier, buffer, or diluent, as further described herein. Optionally, the protein polymer is in the form of a physiologically acceptable salt, which is included in the present disclosure. "Physiologically acceptable salt" refers to any pharmaceutically acceptable salt. Some examples of suitable salts include acetate, trifluoroacetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, and oxalate. Thus, in some aspects, the present disclosure provides pharmaceutical compositions comprising one or more annexin proteins and a pharmaceutically acceptable carrier, buffer, and / or diluent. In any of the aspects or embodiments of the present disclosure, one or more (or all) annexin proteins in the composition are modified annexin proteins. In any of the aspects or embodiments of the present disclosure, one or more (or all) annexin proteins in the composition are naturally occurring mammalian annexin proteins. In some embodiments, the modified annexin proteins are expressed in prokaryotic cells (such as, but not limited to, E. coli cells). Generally, modified proteins are proteins that are altered compared to versions of the protein that normally occur in nature. In some embodiments, modified proteins are those in which at least one amino acid of the modified protein has an altered post-translational modification compared to the naturally occurring mammalian protein. For example, a naturally occurring mammalian protein may contain a phosphorylated amino acid, while the same amino acid in the modified protein has a different or no post-translational modification.In some embodiments, the annexin protein is annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof), annexin A7 (SEQ ID NO: 9 or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 or SEQ ID NO: 16), annexin A13 (SEQ ID NO: 17 or SEQ ID NO: 18), or a combination thereof. In some embodiments, the annexin protein is annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In some embodiments, and as described herein, a pharmaceutical composition comprises a combination of annexin proteins, wherein one or more of the annexin proteins is a modified annexin protein. In some embodiments, a pharmaceutical composition comprises a combination of annexin proteins, each of which is a naturally occurring mammalian annexin protein. Pharmaceutical compositions of the present disclosure comprising one or more annexin proteins are formulated so that the one or more annexin proteins are present in the composition with a high level of purity. "Purity" means that the protein (e.g., annexin protein) used in the pharmaceutical composition is primarily composed of the full-length expressed protein (e.g., annexin protein) and contains little or no truncated or degraded protein products. In various embodiments, the one or more annexin proteins present in the pharmaceutical composition are at least 90%, at least 95%, or at least 99% pure, as measured by standard release assays, including, but not limited to, one or more of SDS-PAGE, SEC-HPLC, and immunoblot analysis. Pharmaceutical compositions of the present disclosure are also relatively free of endotoxin.In various embodiments, pharmaceutical compositions of the present disclosure have endotoxin levels of about 10 endotoxin units (EU / mg) or less, about 5 endotoxin units (EU / mg) or less, about 1 endotoxin unit (EU / mg) or less, about 0.50000 endotoxin units (EU / mg) or less, about 0.40000 endotoxin units (EU / mg) or less, or about 0.30000 endotoxin units (EU / mg) or less per milligram of A280 annexin protein, as measured by standard methods.

[0075] As disclosed herein, the present disclosure provides compositions comprising one or more agents and / or additional agents that increase the activity of an annexin protein. In various embodiments, the annexin protein is annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 and / or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7 and / or SEQ ID NO: 8), annexin A7 (SEQ ID NO: 9 and / or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 and / or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 and / or SEQ ID NO: 16), annexin A13 (SEQ ID NO: 17 and / or SEQ ID NO: 18), or a combination thereof. In some embodiments, the composition increases the activity of annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 and / or SEQ ID NO: 3), and annexin A6 (SEQ ID NO: 7 and / or SEQ ID NO: 8). In further embodiments, the composition increases the activity of annexin A2 (SEQ ID NO: 2 and / or SEQ ID NO: 3), and annexin A6 (SEQ ID NO: 7 and / or SEQ ID NO: 8). In yet another embodiment, the composition increases the activity of annexin A1 (SEQ ID NO: 1), and annexin A6 (SEQ ID NO: 7 and / or SEQ ID NO: 8).

[0076] The present disclosure also contemplates compositions that, in various embodiments, increase the activity of the following annexin proteins, in any combination: annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 and / or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7 and / or SEQ ID NO: 8), annexin A7 (SEQ ID NO: 9 and / or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 and / or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 and / or SEQ ID NO: 16), and annexin A13 (SEQ ID NO: 17 and / or SEQ ID NO: 18). It is understood that where multiple sequence identifiers are used herein to identify annexin proteins (e.g., annexin A2 is identified herein by SEQ ID NO: 2 and / or SEQ ID NO: 3), the different sequence identifiers serve to identify isoforms of a particular annexin protein, and that isoforms may be used interchangeably or in combination in the methods and compositions of the present disclosure.

[0077] Refseq accession number NP_000691.1 Annexin A1 [Homo sapiens] (SEQ ID NO: 1): MAMVSEFLKQAWFIENEEQEYVQTVKSSKGGPGSAVSPYPTFNPSSDVAALHKAIMVKGVDEATIIDILTKRNNAQRQQIKAAYLQETGKPLDETLKKALTGHLEEVVLALLKTPAQFDADELRAAMKGLGTDEDTLIEILASRTNKEIRDINRVYREELKRDLAKDITSDTSGDFRNALLSLakGDRSEDFGVNEDLADSDARALYEAGERRKGTDVNVFNTILTTRSYPQLRRVFQKYTKYSKHDMNKVLDLELKGDIEKCLTAIVKCATSKPAFFAEKLHQAMKGVGTRHKALIRIMVSRSEIDMNDIKAFYQKMYGISLCQAILDETKGDYEKILVALCGGN

[0078] Refseq accession number NP_001002858.1 Annexin A2 isoform 1 [Homo sapiens](SEQ ID NO:2):MGRQLAGCGDAGKKASFKMSTVHEILCKLSLEGDHSTPPSAYGSVKAYTNFDAERDALNIETAIKTKGVDEVTIVNILTNRSNAQRQDIAFAYQRRTKKELASALKSALSGHLETVILGLLKTPAQYDASELKASMKGLGTDEDSLIEIICSRTNQELQEINRVYKEMYK TDLEKDIISDTSGDFRKLMVALAKGRRAEDGSVIDYELIDQDARDLYDAGVKRKGTDVPKWISIMTERSVPHLQKVFDRYKSYSPYDMLESIRKEVKGDLENAFLNLVQCIQNKPLYFADRLYDSMKGKGTRDKVLIRIMVSRSEVDMLKIRSEFKRKYGKSLYYYIQQDTKGDYQKALLYLCGGDD

[0079] Refseq accession number NP_001129487.1 Annexin A2 isoform 2 [Homo sapiens] (SEQ ID NO: 3): MSTVHEILCKLSLEGDHSTPPSAYGSVKAYTNFDAERDALNIETAIKTKGVDEVTIVNILTNRSNAQRQDIAFAYQRRTKKELASALKSALSGHLETVILGLLKTPAQYDASELKASMKGLGTDEDSLIEIICSRTNQELQEINRVYKEMYKTDLEKDIISDTSGDFRKLMVALAKGRRAEDGSVIDYELIDQDARDLYDAGVKRKGTDVPKWISIMTERSVPHLQKVFDRYKSYSPYDMLESIRKEVKGDLENAFLNLVQCIQNKPLYFADRLYDSMKGKGTRDKVLIRIMVSRSEVDMLKIRSEFKRKYGKSLYYYIQQDTKGDYQKALLYLCGGDD

[0080] Refseq accession number NP_005130.1 Annexin A3 [Homo sapiens] (sequence number 4): MASIWVGHRGTVRDYPDFSPSVDAEAIQKAIRGIGTDEKMLISILTERSNAQRQLIVKEYQAAYGKELKDDLKGDLSGHFEHLMVALVTPPAVFDAKQLKKSMKGAGTNEDALIEILTTRTSRQMKDISQAYYTVYKKSLGDDISSETSGDFRKALLTLADGRRDESLKVDEHLAKQDAQILYKAGENRWGTDEDKFTEILCLRSFPQLKLTFDEYRNISQKDIVDSIKGELSGHFEDLLLAIVNCVRNTPAFLAERLHRALKGIGTDEFTLNRIMVSRSEIDLLDIRTEFKKHYGYSLYSAIKSDTSGDYEITLLKICGGDD

[0081] Refseq accession number NP_001144.1 Annexin A4 isoform a [Homo sapiens] (sequence number 5): MAMATKGGTVKAASGFNAMEDAQTLRKAMKGLGTDEDAIISVLAYRNTAQRQEIRTAYKSTIGRDLIDDLKSELSGNFEQVIVGMMTPTVLYDVQELRRAMKGAGTDEGCLIEILASRTPEEIRRISQTYQQQYGRSLEDDIRSDTSFMFQRVLVSLSAGGRDEGNYLDDALVRQDAQDLYEAGEKKWGTDEVKFLTVLCSRNRNHLLHVFDEYKRISQKDIEQSIKSETSGSFEDALLAIVKCMRNKSAYFAEKLYKSMKGLGTDDNTLIRVMVSRAEIDMLDIRAHFKRLYGKSLYSFIKGDTSGDYRKVLLVLCGGDD

[0082] Refseq NP_001145.1 Annexin A5 [Homo sapiens] (sequence number 6): MAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLTLTSRSNAQRQISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD

[0083] Refseq accession number NP_001146.2 Annexin A6 isoform 1 [Homo sapiens] (sequence number 7): MAKPAQGAKYRGSIHDFPGFDPNQDAEALYTAMKGFGSDKEAILDIITSRSNRQRQEVCQSYKSLYGKDLIADLKYELTGKFERLIVGLMRPPAYCDAKEIKDAISGIGTDEKCLIEILASRTNEQMHQLVAAYKDAYERDLEADIIGDTSGHFQKMLVVLLQGTREEDDVVSEDLVQQDVQDLYEAGELKWGTDEAQFIYILGNRSKQHLRLVFDEYLKTTGKPIEASIRGELSGDFEKLMLAVVKCIRSTPEYFAERLFKAMKGLGTRDNTLIRIMVSRSELDMLDIREIFRTKYEKSLYSMIKNDTSGEYKKTLLKLSGGDDDAAGQFFPEAAQVAYQMWELSAVARVELKGTVRPANDFNPDADAKALRKAMKGLGTDEDTIIDIITHRSNVQRQQIRQTFKSHFGRDLMTDLKSEISGDLARLILGLMMPPAHYDAKQLKKAMEGAGTDEKALIEILATRTNAEIRAINEAYKEDYHKSLEDALSSDTSGHFRRILISLATGHREEGGENLDQAREDAQVAAEILEIADTPSGDKTSLETRFMTILCTRSYPHLRRVFQEFIKMTNYDVEHTIKKEMSGDVRDAFVAIVQSVKNKPLFFADKLYKSMKGAGTDEKTLTRIMVSRSEIDLLNIRREFIEKYDKSLHQAIEGDTSGDFLKALLALCGGED

[0084] Refseq accession number NP_001180473.1 Annexin A6 isoform 2 [Homo sapiens] (sequence number 8): MKGFGSDKEAILDIITSRSNRQRQEVCQSYKSLYGKDLIADLKYELTGKFERLIVGLMRPPAYCDAKEIKDAISGIGTDEKCLIEILASRTNEQMHQLVAAYKDAYERDLEADIIGDTSGHFQKMLVVLLQGTREEDDVVSEDLVQQDVQDLYEAGELKWGTDEAQFIYILGNRSKQHLRLVFDEYLKTTGKPIEASIRGELSGDFEKLMLAVVKCIRSTPEYFAERLFKAMKGLGTRDNTLIRIMVSRSELDMLDIREIFRTKYEKSLYSMIKNDTSGEYKKTLLKLSGGDDDAAGQFFPEAAQVAYQMWELSAVARVELKGTVRPANDFNPDADAKALRKAMKGLGTDEDTIIDIITHRSNVQRQQIRQTFKSHFGRDLMTDLKSEISGDLARLILGLMMPPAHYDAKQLKKAMEGAGTDEKALIEILATRTNAEIRAINEAYKEDYHKSLEDALSSDTSGHFRRILISLATGHREEGGENLDQAREDAQVAAEILEIADTPSGDKTSLETRFMTILCTRSYPHLRRVFQEFIKMTNYDVEHTIKKEMSGDVRDAFVAIVQSVKNKPLFFADKLYKSMKGAGTDEKTLTRIMVSRSEIDLLNIRREFIEKYDKSLHQAIEGDTSGDFLKALLALCGGED

[0085] Refseq accession number NP_001147.1 Annexin A7 isoform 1 [Homo sapiens] (SEQ ID NO: 9): MSYPGYPPTGYPPFPGYPPAGQESSFPPSGQYPYPSGFPPMGGGAYPQVPSSGYPGAGGYPAPGGYPAPGGYPGAPQPGGAPSYPGVPPGQGFGVPPGGAGFSGYPQPPSQSYGGGPAQVPLPGGFPGGQMPSQYPGGQPTYPSQPATVTQVTQGTIRPAANFDAIRDAEILRKAMKGFGTDEQAIVDVVANRSNDQRQKIKAAFKTSYGKDLIKDLKSELSGNM EELILALFMPPTYYDAWSLRKAMQGAGTQERVLIEILCTRTNQEIREIVRCYQSEFGRDLEKDIRSDTSGHFERLLVSMCQGNRDENQSINHQMAQEDAQRLYQAGEGRLGTDESCFNMI LATRSFPQLRATMEAYSRMANRDLLSSVSREFSGYVESGLKTILQCALNRPAFFAERLYYAMKGAGTDDSTLVRIVVTRSEIDLVQIKQMFAQMYQKTLGTMIAGDTSGDYRRLLLAIVGQ

[0086] Refseq accession number NP_004025.1 Annexin A7 isoform 2 [Homo sapiens] (SEQ ID NO: 10): MSYPGYPPTGYPPFPGYPPAGQESSFPPSGQYPYPSGFPPMGGGAYPQVPSSGYPGAGGYPAPGGYPAPGGYPGAPQPGGAPSYPGVPPGQGFGVPPGGAGFSGYPQPPSQSYGGGPAQVPLPGGFPGGQMPSQYPGGQPTYPSQINTDSFSSYPVFSPVSLDYSSEPATVTQVTQGTIRPAANFDAIRDAEILRKAMKGFGTDEQAIVDVVANRSNDQRQKIKAAFKTSYGKDL IKDLKSELSGNMEELILALFMPPTYYDAWSLRKAMQGAGTQERVLIEILCTRTNQEIREIVRCYQSEFGRDLEKDIRSDTSGHFERLLVSMCQGNRDENQSINHQMAQEDAQRLYQAGEGRLGTDE SCFNMILATRSFPQLRATMEAYSRMANRDLLSSVSREFSGYVESGLKTILQCALNRPAFFAERLYYAMKGAGTDDSTLVRIVVTRSEIDLVQIKQMFAQMYQKTLGTMIAGDTSGDYRRLLLAIVGQ

[0087] Refseq accession number NP_001258631.1 Annexin A8 isoform 1 [Homo sapiens] (SEQ ID NO: 11): MAWWKSWIEQEGVTVKSSSHFNPDPDAETLYKAMKGIGVGSQLLSHQAAAFAFPSSALTSVSPWGQQGHLCCNPAGTNEQAIIDVLTKRSNTQRQQIAKSFKAQFGKDLTETLKSELSGKFERLIVALMYPPYRYEAKELHDAMKGLGTKEGVIIEILASRTKNQLREIMKAYEEDYGSSLEEDIQADTSGYLERILVCLLQGSRDDVSSFVDPGLALQDAQDLYAAGEKIRGTDEMKFITILCTRSATHLLRVFEEYEKIANKSIEDSIKSETHGSLEEAMLTVVKCTQNLHSYFAERLYYAMKGAGTRDGTLIRNIVSRSEIDLNLIKCHFKKMYGKTLSSMIMEDTSGDYKNALLSLVGSDP

[0088] Refseq accession number NP_001035173.1 Annexin A8 isoform 2 [Homo sapiens] (SEQ ID NO: 12): MAWWKSWIEQEGVTVKSSSHFNPDPDAETLYKAMKGIGTNEQAIIDVLTKRSNTQRQQIAKSFKAQFGKDLTETLKSELSGKFERLIVALMYPPYRYEAKELHDAMKGLGTKEGVIIEILASRTKNQLREIMKAYEEDYGSSLEEDIQADTSGYLERILVCLLQGSRDDVSSFVDPGLALQDAQDLYAAGEKIRGTDEMKFITILCTRSATHLLRVFEEYEKIANKSIEDSIKSETHGSLEEAMLTVVKCTQNLHSYFAERLYYAMKGAGTRDGTLIRNIVSRSEIDLNLIKCHFKKMYGKTLSSMIMEDTSGDYKNALLSLVGSDP

[0089] Refseq accession number NP_003559.2 Annexin A9[Homo sapiens] (SEQ ID NO: 13):MSVTGGKMAPSLTQEILSHLGLASKTAAWGTLGTLRTFLNFSVDKDAQRLLRAITGQGVDRSAIVDVLTNRSREQRQLISRNFQERTQQDLMKSLQAALSGNLERIVMALLQPTAQFDAQELRTALKASDSAVDVAIEILATRTPPQLQECLAVYKHNFQVEAV DDITSETSGILQDLLLALAKGGRDSYSGIIDYNLAEQDVQALQRAEGPSREETWVPVFTQRNPEHLIRVFDQYQRSTGQELEEAVQNRFHGDAQVALLGLASVIKNTPLYFADKLHQALQETEPNYQVLIRILISRCETDLLSIRAEFRKKFGKSLYSSLQDAVKGDCQSALLALCRAEDM

[0090] Refseq accession number NP_009124.2 Annexin A10 [Homo sapiens] (SEQ ID NO: 14): MFCGDYVQGTIFPAPNFNPIMDAQMLGGALQGFDCDKDMLINILTQRCNAQRMMIAEAYQSMYGRDLIGDMREQLSDHFKDVMAGLMYPPPLYDAHELWHAMKGVGTDENCLIEILASRTNGEIFQMREAYCLQYSNNLQEDIYSETSGHFRDTLMNLVQGTREEGYTDPAMAAQDAMVLWEACQQKTGEHKTMLQMILCNKSYQQLRLVFQEFQNISGQDMVDAINECYDGYFQELLVAIVLCVRDKPAYFAYRLYSAIHDFGFHNKTVIRILIARSEIDLLTIRKRYKERYGKSLFHDIRNFASGHYKKALLAICAGDAEDY

[0091] Refseq accession number NP_665875.1 Annexin A11 isoform 1 [Homo sapiens] (SEQ ID NO: 15): MSYPGYPPPPGGYPPAAPGGGPWGGAAYPPPPSMPPIGLDNVATYAGQFNQDYLSGMAANMSGTFGGANMPNLYPGAPGAGYPPVPPGGFGQPPSAQQPVPPYGMYPPPGGNPPSRMPSYPPYPGAPVPGQPMPPPGQQPPGAYPGQPPVTYPGQPPVPLPGQQQPVPSYPGYPGSGTVTPAVPPTQFGSRGTITDAPGFDPLRDAEVLRKAMKGFGTDEQAIIDCLGSRSNKQRQQILLSFKT AYGKDLIKDLKSELSGNFEKTILALMKTPVLFDIYEIKEAIKGVGTDEACLIEILASRSNEHIRELNRAYKAEFKKTLEEAIRSDTSGHFQRLLISLSQGNRDESTNVDMSLAQRDAQELYAAGENRLGT DESKFNAVLCSRSRAHLVAVFNEYQRMTGRDIEKSICREMSGDLEEGMLAVVKCLKNTPAFFAERLNKAMRGAGTKDRTLIRIMVSRSETDLLDIRSEYKRMYGKSLYHDISGDTSGDYRKILLKICGGND

[0092] Refseq accession number NP_001265338.1 Annexin A11 isoform 2 [Homo sapiens] (SEQ ID NO: 16): MPPIGLDNVATYAGQFNQDYLSGMAANMSGTFGGANMPNLYPGAPGAGYPPVPPGGFGQPPSAQQPVPPYGMYPPPGGNPPSRMPSYPPYPGAPVPGQPMPPPGQQPPGAYPGQPPVTYPGQPPVPLPGQQQPVPSYPGYPGSGTVTPAVPPTQFGSRGTITDAPGFDPLRDAEVLRKAMKGFGTDEQAIIDCLGSRSNKQRQQILLSFKTAYGKDLIKDLKSELSG NFEKTILALMKTPVLFDIYEIKEAIKGVGTDEACLIEILASRSNEHIRELNRAYKAEFKKTLEEAIRSDTSGHFQRLLISLSQGNRDESTNVDMSLAQRDAQELYAAGENRLGTDESKFNAV LCSRSRAHLVAVFNEYQRMTGRDIEKSICREMSGDLEEGMLAVVKCLKNTPAFFAERLNKAMRGAGTKDRTLIRIMVSRSETDLLDIRSEYKRMYGKSLYHDISGDTSGDYRKILLKICGGND

[0093] Refseq accession number NP_004297.2 Annexin A13 isoform a [Homo sapiens] (SEQ ID NO: 17): MGNRHAKASSPQGFDVDRDAKKLNKACKGMGTNEAAIIEILSGRTSDERQQIKQKYKATYGKELEEVLKSELSGNFEKTALALLDRPSEYAARQLQKAMKGLGTDESVLIEVLCTRTNKEIIAIKEAYQRLFDRSLESDVKGDTSGNLKKILVSLLQANRNEGDDVDKDLAGQDAKDLYDAGEGRWGTDELAFNEVLAKRSYKQLRATFQAYQILIGKDIEEAIEEETSGDLQKAYLTLVRCAQDCEDYFAERLYKSMKGAGTDEETLIRIVVTRAEVDLQGIKAKFQEKYQKSLSDMVRSDTSGDFRKLLVALLH

[0094] Refseq accession number NP_001003954.1 Annexin A13 isoform b [Homo sapiens] (SEQ ID NO: 18):MGNRHSQSYTLSEGSQQLPKGDSQPSTVVQPLSHPSRNGEPEAPQPAKASSPQGFDVDRDAKKLNKACKGMGTNEAAIIEILSGRTSDERQQIKQKYKATYGKELEEVLKSELSGNFEKTALALLDRPSEYAARQLQKAMKGLGTDESVLIEVLCTRTNKEIIAIKEAYQRLFDRSLESDVKGDTSGNLKKILVSLLQANRNEGDDVDKDLAGQDAKDLYDAGEGRWGTDELAFNEVLAKRSYKQLRATFQAYQILIGKDIEEAIEEETSGDLQKAYLTLVRCAQDCEDYFAERLYKSMKGAGTDEETLIRIVVTRAEVDLQGIKAKFQEKYQKSLSDMVRSDTSGDFRKLLVALLH

[0095] Refseq accession number NP_001350043.1 Annexin A6 isoform 3 [Homo sapiens] (sequence number 45): MAKPAQGAKYRGSIHDFPGFDPNQDAEALYTAMKGFGSDKEAILDIITSRSNRQRQEVCQSYKSLYGKDLIADLKYELTGKFERLIVGLMRPPAYCDAKEIKDAISGIGTDEKCLIEILASRTNEQMHQLVAAYKDAYERDLEADIIGDTSGHFQKMLVVLLQGTREEDDVVSEDLVQQDVQDLYEAGELKWGTDEAQFIYILGNRSKQHLRLVFDEYLKTTGKPIEASIRGELSGDFEKLMLAVVKCIRSTPEYFAERLFKAMKGLGTRDNTLIRIMVSRSELDMLDIREIFRTKYEKSLYSMIKNDTSGEYKKTLLKLSGGDDDAAGQFFPEAAQVAYQMWELSAVARVELKGTVRPANDFNPDADAKALRKAMKGLGTDEDTIIDIITHRSNVQRQQIRQTFKSHFGRDLMTDLKSEISGDLARLILGLMMPPAHYDAKQLKKAMEGAGTDEKALIEILATRTNAEIRAINEAYKEDYHKSLEDALSSDTSGHFRRILISLATGHREEGGENLDQAREDAQEIADTPSGDKTSLETRFMTILCTRSYPHLRRVFQEFIKMTNYDVEHTIKKEMSGDVRDAFVAIVQSVKNKPLFFADKLYKSMKGAGTDEKTLTRIMVSRSEIDLLNIRREFIEKYDKSLHQAIEGDTSGDFLKALLALCGGED

[0096] The present disclosure also contemplates corresponding polynucleotides encoding each of the aforementioned annexin proteins. The following polynucleotides are contemplated for use with the present disclosure. Specifically, the following polynucleotides are messenger RNA (mRNA) sequences contemplated for use with the vectors of the present disclosure to increase the activity of annexin proteins. As noted above, when multiple sequence identifiers are used to identify mRNA sequences associated with the same annexin species herein (e.g., the mRNA sequence associated with annexin A2 is identified herein by SEQ ID NO:20 and SEQ ID NO:21), it is understood that different sequence identifiers serve to identify transcript variants that can be utilized with the vectors of the present disclosure that translate into specific annexin proteins, and that transcript variants can be used interchangeably or in combination in the methods and compositions of the present disclosure.

[0097] NM_000700.3 Homo sapiens annexin A1 (ANXA1), mRNA (SEQ ID NO: 19)

[0098] NM_001002858.2 Homo sapiens annexin A2 (ANXA2), transcript variant 1, mRNA (SEQ ID NO: 20)

[0099] NM_001136015.2 Homo sapiens annexin A2 (ANXA2), transcript variant 4, mRNA (SEQ ID NO: 21)

[0100] NM_005139.3 Homo sapiens annexin A3 (ANXA3), mRNA (SEQ ID NO: 22)

[0101] NM_001153.5 Homo sapiens annexin A4 (ANXA4), transcript variant 2, mRNA (SEQ ID NO: 23)

[0102] NM_001154.4 Homo sapiens annexin A5 (ANXA5), mRNA (SEQ ID NO: 24)

[0103] NM_001155.5 Homo sapiens annexin A6 (ANXA6), transcript variant 1, mRNA (SEQ ID NO: 25)

[0104] NM_001193544.1 Homo sapiens annexin A6 (ANXA6), transcript variant 2, mRNA (SEQ ID NO: 26)

[0105] NM_001156.5 Homo sapiens annexin A7 (ANXA7), transcript variant 1, mRNA (SEQ ID NO: 27)

[0106] NM_004034.3 Homo sapiens annexin A7 (ANXA7), transcript variant 2, mRNA (SEQ ID NO: 28)

[0107] NM_001271702.1 Homo sapiens annexin A8 (ANXA8), transcript variant 1, mRNA (SEQ ID NO: 29)

[0108] NM_001040084.2 Homo sapiens annexin A8 (ANXA8), transcript variant 2, mRNA (SEQ ID NO: 30)

[0109] NM_003568.3 Homo sapiens annexin A9 (ANXA9), mRNA (SEQ ID NO: 31)

[0110] NM_007193.4 Homo sapiens annexin A10 (ANXA10), mRNA (SEQ ID NO: 32)

[0111] NM_145868.2 Homo sapiens annexin A11 (ANXA11), transcript variant b, mRNA (SEQ ID NO: 33)

[0112] NM_001278409.1 Homo sapiens annexin A11 (ANXA11), transcript variant f, mRNA (SEQ ID NO: 34)

[0113] NM_004306.4 Homo sapiens annexin A13 (ANXA13), transcript variant 1, mRNA (SEQ ID NO: 35)

[0114] NM_001003954.2 Homo sapiens annexin A13 (ANXA13), transcript variant 2, mRNA (SEQ ID NO: 36)

[0115]

[0116] Treatment endpoints In various embodiments of the present disclosure, the use of an agent as described herein and any additional agent provides one or more benefits related to a particular therapeutic endpoint compared to patients not receiving the agent and / or additional agent.

[0117] Creatine kinase (CK) is a clinically validated serum biomarker of skeletal muscle, heart, kidney, and brain injury. Lactate dehydrogenase (LDH) is a clinically validated serum biomarker of skeletal muscle, heart, kidney, liver, lung, and brain injury. Serum creatine kinase and lactate dehydrogenase levels are elevated with both acute and chronic tissue injury. Under theoretical or validated conditions with comparable muscle mass levels, a decrease in creatine kinase and / or lactate dehydrogenase may indicate enhanced injury repair or protection against injury. Aspartate aminotransferase (AST) is yet another clinically validated serum biomarker of skeletal muscle, heart, kidney, liver, and brain injury. Furthermore, an increase in serum troponin indicates cardiac injury, and an increase in alanine transaminase (ALT) is a biomarker of liver injury. A decrease in AST, ALT, or troponin during the acute phase after injury may indicate enhanced injury repair or protection against injury. Evans Blue dye is an important dye that binds to serum albumin and is normally excluded from healthy, intact muscle. Membrane disruption due to acute or chronic injury promotes dye influx into damaged cells. Evans Blue dye is commonly used to quantify cellular damage in experimental settings by measuring intrinsic dye fluorescence and / or by measuring radiolabeled dye uptake. Decreased dye uptake after acute injury or in models of chronic injury indicates enhanced protection against injury and / or repair. Indocyanine green (ICG) is a short-range dye that binds to plasma proteins and is used clinically to assess blood flow and tissue damage (ischemia, necrosis) in organs including the heart, liver, kidneys, skin, vasculature, lungs, muscle, and eyes. Improved blood flow and reduced ischemic areas indicate protection against injury and / or improved repair.

[0118] Increased membrane integrity and repair are believed to enhance function as measured through multiple modalities, including plethysmography, echocardiography, muscle strength, and the 6-minute walk test. Furthermore, histologic benefits may be observed, including decreased necrosis, decreased inflammation, decreased fibrosis, decreased fatty infiltration, and decreased edema. These beneficial effects can also be confirmed by MR and PET imaging.

[0119] Medication / Administration / Kit The specific dosing regimen for a particular subject will depend, in part, on the drug and any additional drugs used, the amounts of the drug and any additional drugs administered, the route of administration, the particular disease being treated, and the cause and extent of any side effects. The amounts of the drug and any additional drugs administered to a subject (e.g., a mammal such as a human) are sufficient to produce the desired response. The dosage typically depends on various factors, including the specific drug and / or additional drugs used, the age and weight of the subject, and the presence and severity of any disease or disorder in the subject. The size of the dose will also be determined by the route, timing, and frequency of administration. Thus, clinicians titrate the dosage and adjust the route of administration to obtain the optimal therapeutic effect. Conventional range-finding techniques are known to those of skill in the art. Purely by way of example, in some embodiments, the method involves administering, for example, from about 0.1 μg / kg to up to about 100 mg / kg or more of drug (e.g., protein), depending on the factors described above. In other embodiments, dosages can range from 1 μg / kg up to about 75 mg / kg, or from 5 μg / kg up to about 50 mg / kg, or from 10 μg / kg up to about 20 mg / kg. In certain embodiments, the dose comprises from about 0.5 mg / kg to about 20 mg / kg (e.g., about 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.3 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg) of the agent and any additional agent. In embodiments in which an agent and an additional agent are administered, it is intended that the dosages above represent the amount of each agent administered, or in further embodiments, the dosage represents the total dosage administered. In some embodiments in which a chronic condition is being treated, it is contemplated that the subject will receive the medication and / or additional medication over a course of treatment lasting weeks, months, or years, and may require one or more doses daily or weekly.In any of the aspects or embodiments of the present disclosure, the amount of annexin protein in the pharmaceutical composition can range from about 0.1 μg / kg up to about 100 mg / kg or more, depending on the factors described above. In other embodiments, dosages can range from 1 μg / kg up to about 75 mg / kg, or from 5 μg / kg up to about 50 mg / kg, or from 10 μg / kg up to about 20 mg / kg. In some embodiments, the dose comprises from about 0.5 mg / kg to about 20 mg / kg (e.g., about 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.3 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg) of annexin protein. Dosing is also contemplated for once-daily, twice-daily (BID), or three times-daily (TID) dosing. The unit dose can be formulated in the form of either a capsule or a tablet. In other embodiments, the agent and any additional agent are administered for a relatively short treatment period, for example, 1 to 14 days, to treat an acute condition (e.g., acute muscle injury or acute myocardial injury).

[0120] Suitable methods for administering physiologically acceptable compositions, such as pharmaceutical compositions comprising an agent (e.g., a recombinant protein) described herein and any additional agents, are well known in the art. More than one route can be used to administer a compound, although certain routes may provide a more rapid and effective means than others. Depending on the situation, pharmaceutical compositions may be applied or instilled into a body cavity, absorbed through the skin or mucous membranes, ingested, inhaled, and / or introduced into the circulation. In some embodiments, compositions comprising an agent and / or additional agents are administered intravenously, intraarterially, or intraperitoneally to introduce the agent and any additional agents into the circulation. Non-intravenous administration is also appropriate, particularly for low-molecular-weight therapeutics. In certain situations, it may be desirable to deliver a pharmaceutical composition containing a drug and / or additional agent orally, topically, sublingually, vaginally, rectally, intracerebrally (intraparenchymal), intracerebroventricularly, intramuscularly, intraocularly, intraportally, intralesionally, intramedullary, intrathecally, intraventricularly, transdermally, subcutaneously, intranasally, urethrally, or by enteral injection; by sustained release system; or by implantation device. If desired, the drug and / or additional agent may be administered locally via intraarterial or intravenous administration to the desired area, for example, via the femoral artery for delivery to the leg. In one embodiment, the composition is administered via implantation of a membrane, sponge, or another suitable material into which or onto which the desired drug and any additional agent are absorbed or encapsulated. When an implantation device is used, the device in one aspect is implanted into any suitable tissue, and delivery of the desired drug and / or additional agent may occur via diffusion, sustained release bolus, or continuous administration in various embodiments. In other embodiments, the agent and any additional agents are administered directly to exposed tissue during a surgical procedure or injury treatment, or via transfusion of a blood product. Therapeutic delivery approaches are well known to those skilled in the art, some of which are further described, for example, in U.S. Patent No. 5,399,363.

[0121] In some embodiments to facilitate administration, the drug and any additional drug are formulated into a physiologically acceptable composition comprising a carrier (i.e., a vehicle, adjuvant, buffer, or diluent). The particular carrier used is limited only by chemical and physical considerations, such as solubility and lack of reactivity with the drug and / or additional drug, the route of administration, and other requirements. Physiologically acceptable carriers are well known in the art. Exemplary pharmaceutical forms suitable for injectable use include, but are not limited to, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of injectable sterile solutions or dispersions (see, e.g., U.S. Pat. No. 5,466,468). Injectable formulations are further described, for example, in Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986), which are incorporated herein by reference.

[0122] Pharmaceutical compositions comprising an agent (e.g., a recombinant protein) provided herein and any additional agents are optionally disposed in a container / kit along with packaging materials that provide instructions for use of such pharmaceutical compositions. Generally, such instructions include specific wording describing reagent concentrations and, in certain embodiments, the relative amounts of excipient components or diluents that may be required to reconstitute the pharmaceutical composition.

[0123] Thus, the present disclosure includes administering to a subject one or more agents in combination with one or more additional agents, each administered according to a regimen suitable for that agent. In some embodiments, the agent is a recombinant protein such as an annexin protein (e.g., annexin A6). This aspect includes simultaneous administration (i.e., administration substantially simultaneously) and non-simultaneous administration (i.e., administration at different times, in any order, whether overlapping) of the agent and one or more additional agents. It will be understood that in certain aspects, the different components are optionally administered in the same or separate compositions, by the same or different routes of administration.

[0124] All publications, patents, and patent applications mentioned herein are incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of this document. For example, if protein therapy is described, embodiments including polynucleotide therapy (using a polynucleotide / vector encoding the protein) are specifically contemplated, and vice versa. It should be understood that with respect to elements described as one or more members of a set, all combinations within the set are contemplated. [Example]

[0125] The following example demonstrates annexin cap formation and Ca after laser-induced injury. 2+ To visualize the dynamics of intracellular Ca, we present the results of experiments in which muscle membrane repair was monitored by real-time, high-resolution imaging. Herein, we demonstrate that overexpression of annexin proteins significantly alters intracellular Ca dynamics. 2+We demonstrate that recombinant annexin A6 induced extracellular blebbing at sites of membrane damage, correlating with reduced accumulation of annexin A6. Pretreatment with recombinant annexin A6 prevented muscle damage in vitro and in vivo. Posttreatment with recombinant annexin A6 enhanced muscle repair in vivo. Overexpression of annexin A6 enhanced muscle fiber membrane repair, whereas overexpression of a dominant-negative annexin A6 mutant protein reduced membrane repair capacity. Furthermore, treatment with extracellular recombinant annexin A6 protein attenuated laser-induced muscle damage in both wild-type and dystrophic myofibers. Furthermore, administration of recombinant annexin A6, using multiple routes of administration, protected against toxin-induced muscle damage in vivo. Furthermore, administration of recombinant annexin A6 reduced circulating levels of creatine kinase and lactate dehydrogenase in dystrophic mice, indicating enhanced membrane repair. The data provided herein demonstrate that annexin proteins are important agonists of membrane repair and that recombinant annexin A6 is a therapeutic target for treating conditions resulting from membrane fragility, such as muscle injury and muscular dystrophies.

[0126] A polymorphism in Anxa6, the gene encoding annexin A6, was previously identified in several commonly used laboratory mouse strains that correlated with impaired muscle repair (Demonbreun et al., 2016a; Quattrocelli et al., 2017b; Swaggart et al., 2014). This polymorphism produced a truncated annexin A6 protein that interfered with sarcolemma repair. Further studies in mice have shown that loss of annexin A2 results in insufficient muscle fiber repair (Defour et al., 2017).

[0127] Annexin Ca during muscle membrane repair 2+ We investigated the binding of annexins A1, A2, and A6 to the nuclei of the repair cap after injury and assessed the kinetics of their formation. 2+Mutation of the coordination residues disrupted normal annexin repair cap formation. Overexpression of annexins promoted the formation of external blebs at the site of membrane damage, which were released from the repair cap. Overexpression of annexin A6 led to the formation of larger blebs, which were released from the repair cap. These vesicles contained Ca 2+ The binding protein GCaMP5G is abundant, which mediates intracellular Ca2+ binding at the injury site. 2+ Overexpression of annexin A6 reduced membrane damage, while the significant Ca 2+ Mutation of the coordinating residue E233 disrupted annexin repair complex formation and correlated with reduced repair capacity. Local and systemic treatment with recombinant annexin A6 reduced muscle damage and promoted membrane repair in vivo. The data provided herein identify a novel role for annexins in bleb release from muscle membrane lesions during membrane repair and identify annexin A6 as a therapeutic target for enhancing membrane repair capacity.

[0128] Example 1 material and method Animals. Wild-type mice on a 129T2 / SvEmsJ background were bred and housed in a specific pathogen-free facility with a 12-h light / dark cycle and fed ad libitum in accordance with the regulations of the Northwestern University Institutional Animal Care and Use Committee. 129T2 / SvEmsJ (129T2) mice were purchased from Jackson Laboratory (Ben Harbor, ME; stock number 002065). mdx / hLTBP4 mice were generated as described (Ceco et al., 2014; Quattrocelli et al., 2017b), and sgcg-deficient mice were generated as described (Hack et al., 1998). Male and female mice aged 2–3 months were used for all experiments.

[0129] Plasmids. Plasmids encoding annexins A1, A2, and A6 with a carboxyl-terminal turboGFP tag were obtained from Origene (Rockville, MD). Subcloning of annexins A1, A2, and A6 to replace the GFP tag with tdTomato (Addgene) was performed by Mutagenix (Suwanee, GA). Site-directed mutagenesis of annexins A1-GFP, A2-GFP, and A6-GFP was performed by Mutagenix to express Ca. 2+ The binding mutants A1-D171A-GFP, A2-D161A-GFP, A6-D149A-GFP, and A6-E233A-GFP were generated. To verify mutagenesis, the constructs were sequenced. Plasmid DNA was isolated using Qiagen's Endo-Free Maxi Prep Kit (Qiagen #12362). 2+ The sensor GCaMP5G was purchased from (Addgene #31788).

[0130] Sequence Comparison and Protein Schematic. Protein ribbon diagrams were generated using the Swiss-PdbViewer using the solved crystal structures of annexin A1 (1MCX), A2 (2HYW), and annexin A6 (1AVC) available at www.rcsb.org. Clustal Omega from the European Bioinformatics Institute (EMBL-EBI) was used to align the annexin mouse sequences annexin A1 (NM_010730, SEQ ID NO: 37), A2 (NM_007585, SEQ ID NO: 38), and A6 (NM_013472, SEQ ID NO: 39) from www.ncbi.nlm.nih.gov.

[0131] Electroporation, muscle fiber isolation, laser injury, cap and vesicle measurements. Flexor digitorum brevis (FDB) fibers were transfected with end-free plasmid DNA by in vivo electroporation. Methods have been previously described (Demonbreun and McNally, 2015; Demonbreun et al., 2016b; DiFranco et al., 2009). Z-stack projections were acquired after the last time-lapse frame, approximately 4 min after injury, from sequential acquisitions with a step size of 0.125 μM between slices. Z-stack renderings were constructed in FIJI. Cap area and ferret diameter measurements were performed from a single slice near the center of the z-stack using the FIJI imaging tool. Fibers expressing similar levels of tagged or GCaMP5G protein were compared. Ca of GCaMP5G was measured. 2+ Fluorescence was measured from acquired time-lapse images using a standard rectangular ROI placed within the myofiber below the injury site using FIJI. Fluorescence was expressed as F / F. External vesicle counts and GCaMP5G area were measured from endpoint z-stack and maximum projection images using FIJI. Vesicles were considered external if they were found outside the sarcolemma, as assessed in the brightfield and fluorescence channels. All measurements were taken from myofibers isolated from at least n ≥ 3 mice (n ≥ 3 myofibers per mouse).

[0132] For recombinant myofiber studies, myofibers were isolated from mdx / hLTBP4 mice as described above. Myofibers were incubated in Ringer's medium with or without 25 μg / ml recombinant annexin A6 (5186-A6-050, R&D Systems). FM4-64 (2.5 μM) was added to myofibers immediately prior to imaging. Images were acquired and quantified as described above. FM4-64 area was measured using FIJI at the imaging endpoint from a single slice near the center of the Z-stack. A Z-stack step size (0.125 μm) was acquired from the edge of the cap to the edge.

[0133] Quality control of myofibers was based on a number of characteristics, including the use of adherent myofibers with intact sarcomere structures detected by brightfield imaging. Myofibers appeared free of tears or ruptures induced during the isolation protocol. The region of the myofiber selected for injury was linear and not located at the nucleus or neuromuscular junction. Furthermore, the fluorescence intensity in both the red and green channels suggested similar expression levels before injury.

[0134] Multiphoton laser damage and imaging. Fibers were subjected to laser-induced damage at room temperature using a Nikon A1R-MP multiphoton microscope. Imaging was performed using a 25x 1.1 NA objective, guided by NIS-Elements AR imaging software. Green fluorescent protein (GFP) and FM4-64 were excited using a 920 nm laser, and emission wavelengths of 575 nm and 629 nm were collected, respectively. To induce laser damage in isolated myofibers, a diffraction-limited spot (approximately 410 nm in diameter) was created on the lateral membrane of the myofiber using a 920 nm laser for 1 second at 10-15% laser power. Time-lapse images were collected as follows: one before injury, one at the time of injury, and then every 8 seconds for 80 seconds (10 images), and every 30 seconds for 5 minutes (10 images). At the end of the time-lapse image series, z-stack images were collected at 250 nm intervals from the injury site of the myofiber as indicated by NIS-Elements AR imaging software.

[0135] Cardiotoxic Injury and Analysis. The tibialis anterior muscle of wild-type mice was injected with 25 μg / ml of recombinant annexin A6 (5186-A6-050, R&D Systems) or Ringer's (3% isoflurane, 0.8 L / min O2) in sedated mice. Additionally, mice were intraperitoneally injected with 10 mg / mL of Evans Blue dye (E-2129, Sigma-Aldrich, St. Louis, MO) at 5 μl / g body weight dissolved in phosphate-buffered saline (PBS). For systemic administration, wild-type mice were injected with 1 mg / kg of recombinant annexin A6 (5186-A6-050, R&D Systems) or PBS (5 μl / g body weight) diluted in Evans Blue (EBD) in PBS (3% isoflurane, 0.8 L / min O2) in the retroorbital space of sedated mice. Two hours after pretreatment, cardiotoxin injury was performed by injecting 20 μl of 10 μM cardiotoxin (discontinued, Sigma-Aldrich) in PBS (3% isoflurane, 0.8 l / min O2) into the tibialis anterior muscle of sedated animals. The cardiotoxin was released along the midline of the muscle, inducing a uniform injury area in the center of the muscle. Three hours after cardiotoxin injection, the muscle was harvested and frozen in liquid nitrogen.

[0136] Chronic injury and biomarker analysis. For systemic administration, sgcg-deficient or wild-type mice were injected with 1 mg / kg recombinant annexin A6 (5186-A6-050, R&D Systems) or PBS into the retroorbital space of sedated mice (3% isoflurane, 0.8 L / min O2). Mice were injected once every 3 days for 12 days. Serum was obtained as previously described (Demonbreun et al., 2016a). Serum CK was analyzed for each mouse using the EnzyChrom Creatine Kinase Assay (Catalog ECPK-100, BioAssay Systems) according to the manufacturer's instructions. Lactate dehydrogenase was analyzed for each mouse using the LDH Cytotoxicity Kit MK401 (Takara) according to the manufacturer's instructions. Results were acquired using a Synergy HTX multimode plate reader (BioTeK).

[0137] Immunofluorescence microscopy. Cryo-embedded muscle sections (10 μm thick) from the center were collected for immunostaining on a cryostat (chamber: -20°C, sample: -15°C, catalog no. CM1950, Leica, Wetzlar, Germany). Tissues were fixed with 4% paraformaldehyde on ice for 10 minutes. Blocking and permeabilization were performed for 60 minutes using 0.1% Triton (catalog no. X-100, Sigma-Aldrich), 10% fetal bovine serum, and PBS. For dystrophin detection, anti-dystrophin antibody (ab15277, Abcam, Cambridge, MA) was used at a dilution of 1:100 overnight at 4°C. Sections were rinsed with PBS, incubated with secondary antibody goat anti-rabbit (A11008, Invitrogen) for 1 hour, rinsed with PBS, and mounted in Vectashield with DAPI (H-1200, Vector Laboratories). Imaging was performed using a Zeiss Axio Observer A1 microscope (Zeiss, Oberkochen, Germany) with a 10x objective. Images were acquired using ZEN software (Zeiss, Jena, Germany). Fluorescence quantification and muscle area were performed using FIJI (NIH). Surface plots were created in FIJI (NIH). Fluorescence was quantified using Imaris Software v9.1.2.

[0138] Calcium dynamics. FDB muscles were electroporated and isolated as described above. Ca 2+ Muscle fibers were damaged in Ringer's solution at concentrations of 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.175 mM, 0.1 mM, 0.050 mM, and 0 mM. 2+ Only in Ringer's, Ca 2+ EDTA was added as a chelating agent. For these experiments, myofibers were directly isolated in 2 mM, 1 mM, and 0.5 mM Ringer's solution, respectively. Ca concentrations below 0.5 mM were used. 2+ In experiments using β-glucan, muscle fibers were treated with 0.5 mM Ca 2+ Isolated in Ringer's, 0 mM EDTA-free Ca 2+In the 0 mM experiment, muscle fibers were diluted with 0.5 mM Ca. 2+ Isolate in Ringer's and add 0 mM Ca immediately before imaging. 2+ + EDTA Ringer's. Co-electroporation of wild-type annexin + wild-type annexin construct was performed in one mouse paw, and wild-type annexin + mutant annexin was co-electroporated in the contralateral paw. All measurements were performed on at least n ≥ 2 mice (each Ca 2+ The area-Ca values ​​were obtained from muscle fibers isolated from mice (n ≥ 3 muscle fibers per mouse). 2+ The curves are for the range of 0–2 mM Ca. 2+ Concentration-dependent Hill curves were fitted, and kinetic parameters were calculated using Prism Graphpad.

[0139] Single-cell Ca 2+ and shortening measurements. Isolated FDB fibers were seeded onto laminin-coated glass-bottom 35-mm dishes for 1 h and cultured overnight in DMEM containing 10% FBS and 1% penicillin / streptomycin at 37°C in a 10% CO2 incubator. One hour before data collection, the medium was removed and the cells were incubated in Tyrode's buffer (119 mM NaCl, 5 mM KCl, 25 mM HEPES, 2 mM CaCl2, 2 mM MgCl2) containing 10 μM Indo-1 AM (TefLabs) for 1 h at 37°C in a 10% CO2 incubator. The dish was then filled with Tyrode's buffer, mounted on a custom stage, and a platinum pacing electrode was inserted into the dish. Stimulation was evoked using a 701C high-output stimulator controlled by 950A software (Aurora Scientific). Stimulation was performed at 40 Hz and 80 Hz with a 5 ms pulse width and 100 ms duration. 2+Signals were collected using two photomultiplier tubes and a FluoroDaq controller. Video sarcomere lengths were recorded with a high-speed camera and fast Fourier transform using an Aurora Scientific 900B-VSL system (Aurora, Ontario). Ten transitions were collected over 20 seconds and averaged per cell per frequency.

[0140] Statistical analysis. Statistical analysis was performed using Prism (Graphpad, La Jolla, CA). Comparisons relied on ANOVA (one-way ANOVA for single variables, and multivariate ANOVA for two variables (usually area and Ca). 2+ Two-way ANOVA was performed in the case of (concentration). Otherwise, unpaired t-tests were performed. Error bars represent the standard error of the mean (SEM).

[0141] Example 2 Ca 2+ localizes to the repair cap upon membrane damage. External Ca is required to activate muscle repair. 2+ The presence of Ca is required for repair cap repair (Bansal et al., 2003). It has previously been shown that annexin proteins aggregate into the repair cap at the site of injury adjacent to an annexin-free zone in the cytoplasm below the repair cap (Demonbreun et al., 2016a, Demonbreun et al., 2016b, Quattrocelli et al., 2017a, Swaggart et al., 2014). 2+ In vivo fluorescent Ca dynamics for real-time visualization 2+ We used the indicator protein GCaMP5G, which is a fusion protein consisting of green fluorescent protein (GFP), the calcium-binding protein calmodulin, and calmodulin M13-binding peptide. 2+ When not bound to Ca, fluorescence is minimal and 2+Upon binding, a conformational change occurs within the protein, resulting in an increase in GFP fluorescence intensity (Akerboom et al., 2012). Wild-type flexor digitorum brevis (FDB) muscles were electroporated with a GCaMP5G plasmid and the plasma membrane was injured using laser ablation (Demonbreun and McNally, 2015; Demonbreun et al., 2016b). Within 2 seconds of membrane injury, GCaMP5G fluorescence accumulated within the cytoplasm at the injury site. GCaMP5G fluorescence intensity gradually increased throughout 90 seconds of imaging (Figure 1A, arrowhead). In muscle fibers co-electroporated with plasmids expressing annexin A6 and GCaMP5G with a carboxyl-terminal tdTomato fluorescent tag, GCaMP5G fluorescence localized in a ring around the annexin A6-free zone (Figure 1B, arrowhead) and colocalized with annexin A6 in the repair cap (Figure 1B, arrow, merge). This time series is related to the increased Ca 2+ This is consistent with the finding that annexins may facilitate their translocation and assembly into the repair cap.

[0142] Annexin repair caps induce different Ca responses during repair cap mobilization 2+ Indicates sensitivity. Annexin proteins bind to Ca 2+ Annexin A6 is a Ca-dependent phospholipid- and actin-binding protein that contains four annexin repeat domains (eight in the case of annexin A6) (Figure 2). 2+ Although it binds to Ca, it does not contain the EF-hand and C2 domains found in other classes of repair proteins. 2+ Annexins bind Ca2+ and Ca3+, but not Ca4+. 2+ It binds to the membrane via its convex surface (Fig. 2) and binds to both type II and type III Ca 2+ The binding sites have been described in annexin proteins. Ca binding in annexin-mediated sarcolemmal repair in muscle fibers. 2+ To further define the requirements, we analyzed annexin A1, A2, or A6 repair cap formation using Ca 2+A range of concentrations was examined. Cap size was measured from the center of the z-stack, and the type of fluorescent tag (turboGFP or tdTomato) did not affect the parameter evaluation (Figures 3A and 3B). Annexin A1 and A6 repair cap size was significantly correlated with Ca. 2+ The largest repair cap was formed at 2 mM Ca, and a smaller repair cap at 0.1 mM, whereas the annexin A2 repair cap was formed at 0.05 mM Ca. 2+ The repair cap area was calculated using the modified Hill equation as the Ca 2+ The results were plotted as a function of Ca concentration. 2+ The repair cap was formed at the lowest concentration of 0.05 mM Ca, whereas annexins A1 and A6 were only formed at 0.1 mM Ca. 2+ Less than Ca 2+ did not form a discernible cap at concentrations of 0.12 mM and 0.17 mM K, respectively. m1 / 2 A K of 0.067 mM compared to A6 and A1 (Figure 1D) which show m1 / 2 The size and speed of the repair caps of annexins A1 and A6 are significantly different from those of annexin A2, which has a Ca 2+ The rate of annexin A2 cap formation and cap size were significantly dependent on the concentration (Figure 4). 2+ The rates of annexins A1 and A6 were similar at Ca 2+ The lower the concentration, the greater the decrease in Ca 2+This suggests that the formation of the repair cap is highly responsive to the laser damage (Figure 4). To confirm that the formation of the repair cap is not an artifact due to the type of laser damage, laser damage was induced with both a Nikon A1R GaSP confocal and a Nikon A1R MP+ multiphoton confocal. The damage induced by the multiphoton laser is theoretically more focused, resulting in less collateral membrane damage. The annexin A6 repair cap appeared comparable with both laser types (Figure 5). These data suggest that the formation of the annexin A1, A2, and A6 repair caps is due to the Ca2+ receptor present during myofiber repair. 2+ Annexin A2 was the most sensitive of the three annexins studied, affecting the levels of Ca 2+ This indicates sensitivity.

[0143] Overexpression of annexins promotes blebbing at sites of membrane damage. Membrane repair assays in Lytechinus pictus and Xenopus oocytes suggested that membrane structures emerge at the repair site through fusion (Bi et al., 1995; Davenport et al., 2016). Furthermore, extracellular recombinant annexins were shown to regulate Ca 2+GCaMP5G, a soluble ATP-dependent protein, promoted membrane folding and bleb formation in artificial membrane patches at membrane defect sites (Boye et al., 2018, Boye et al., 2017). Next, we investigated whether similar findings could be observed at muscle membrane injury sites, i.e., sites of membrane defects, in living skeletal muscle fibers. GCaMP5G was expressed alone or in combination with annexins A1, A2, or A6 in skeletal muscle fibers. We found that overexpression of annexins promoted the formation of extracellular blebs released from the annexin repair cap at the membrane lesion (Figure 6A). These blebs appeared after the formation of the annexin repair cap and were found at the extracellular tip of the repair cap (Figure 6A). Overexpression of annexin A6 and annexin A2 induced significantly more blebs than those observed after overexpression of annexin A1 or GCaMP5G alone (Figures 6A and 6B). Furthermore, annexin-induced blebs contained significant GCaMP5G signals, and annexin A6 induced the formation of significantly larger GCaMP5G-containing blebs compared with annexins A1, A2, or GCaMP5G alone (Figures 6A and 6C). These data suggest that annexins not only form repair caps at sites of membrane disruption, but that these caps also mediate Ca signaling. 2+ We have shown that binding proteins function as sites for the egress of abundant extracellular blebs.

[0144] Annexin overexpression induces intracellular Ca2+ upregulation at the injury site 2+ Decrease in fluorescence. Ca after laser damage 2+ Time-lapse imaging of the indicator GCaMP5G detects extracellular Ca 2+ These results suggest that intracellular Ca2+ decreases with the formation of extracellular blebs, and that these blebs are responsible for the formation of intracellular Ca2+. 2+ These findings suggest that annexins may help reduce the accumulation and / or release of cytoplasmic protein content from the injury site. 2+ The decrease in fluorescence was observed for all three annexins, A1, A2, and A6, but was most pronounced for annexins A2 and A6 (Figure 7A). Overexpression of annexin A6 significantly reduced intracellular Ca2+ levels during 240 s of imaging. 2+This decrease was due to the Ca release from the internal GCaMP5G. 2+ Detailed analysis of the first 20 seconds after injury revealed that Ca2+ was upregulated by annexins A2 and A6, but not by annexin A1, when compared with GCaMP5G alone (Figure 7B). 2+ A significant decrease in GCaMP5G fluorescence was observed (Figure 7C). Baseline GCaMP5G fluorescence intensity before injury was not significantly different between groups (Figure 7D). Therefore, annexin expression is associated with Ca uptake in injured myofibers. 2+ This induces a decrease in the Ca signal. 2+ The shedding of binding protein-filled blebs is enhanced, and annexin A6 was the most effective of the three annexins tested in maintaining this response.

[0145] Next, we investigated the Ca concentration in isolated myofibers overexpressing annexin A6 compared to controls. 2+ Handling and contractile properties were assessed. Isolated myofibers expressing annexin A6 were subjected to ratiometric Ca measurements. 2+ Ca concentrations at stimulation frequencies of 40 Hz or 80 Hz were measured between Annexin A6 or control fibers loaded with the indicator dye Indo-1. 2+ No differences in cycling were observed (Figures 8A, 8B, and 8C). Unloaded cell shortening was also unaffected by the presence of overexpressed annexin A6 (Figures 8D, 8E, and 8F). These results indicated that overexpression of annexin A6 was well tolerated by muscle fibers.

[0146] Annexin A6 Ca 2+ The binding is necessary for the formation of the repair cap and repair of muscle fibers. Mutations in annexin A1D171 and A2D161 have previously been shown to inhibit annexin membrane translocation in HEK cells (Jost et al., 1992; McNeil et al., 2006). We asked whether these mutations inhibit the translocation and formation of the polymeric annexin repair cap formed after muscle membrane injury. Alignment of annexin A1, A2, and A6 protein sequences revealed the presence of type II Ca2+ receptors in all three annexin proteins. 2+ Conserved residues within the consensus sequence of the binding site were identified (Fig. 2). 2+ To disrupt binding, site-directed mutagenesis was performed to identify the first type II Ca 2+ The aspartic acid residues in the binding site were converted to alanine residues (A1D171A, A2D161A, and A6D149A, respectively) (Fig. 9A). E233A was also generated in annexin A6, and the Ca2+ binding site in the second annexin repeat domain of annexin A6 was converted to alanine residues (A1D171A, A2D161A, and A6D149A, respectively). 2+ This resulted in similar changes in the binding site. Each construct also contained turboGFP or tdTomato at the C-terminus. To assess the impact of homotypic annexin interactions during repair cap formation, we co-electroporated muscle fibers with wild-type + wild-type (A6 + A6) or wild-type + mutant (A6 + A6E233A) annexin combinations. Mutation of E233 in annexin A6 acts as a dominant-negative and significantly reduced the cap size of co-expressed wild-type annexin A6 protein (Figure 10A). Previous structural studies have shown that D149 in the first annexin repeat domain in annexin A6 binds Ca. 2+ It has been suggested that annexin A6 does not bind to Ca (Avila-Sakar et al., 1998), and consistent with this, the D149A mutant in annexin A6 had little effect on cap size (Figure 9B, right panel). The diameter of the repaired cap ferret was calculated using a modified Hill equation: 2+The correlation coefficients were plotted as a function of annexin concentration. Expression of mutant annexin A6E233A was sufficient to significantly reduce the cap diameter (DMAX) of co-expressed wild-type annexin A6 protein (Figure 10B). To assess the impact of heterotypic annexin interactions on repair cap formation, various combinations of wild-type and mutant annexin constructs were co-electroporated into muscle fibers. Co-expression of mutant annexin A6E233A significantly reduced the cap size of annexins A1, A2, and A6 compared to the A1+A6, A2+A6, and A6+A6 controls, respectively (Figure 10C). Together, these data indicated that annexin proteins interact in a homotypic and heterotypic manner that affects annexin repair complex assembly, and that mutant annexin A6 protein is sufficient to negatively regulate annexin complex assembly during repair.

[0147] Both annexin A1 and A2 Ca 2+ Binding was also required for repair cap formation. The cap sizes of the A1D171A and A2D161A mutants were reduced compared to wild-type annexin A1 and A2 controls, respectively. Expression of mutant annexins A1D171A and A2D161A was sufficient to significantly reduce the repair cap diameter (DMAX) of co-expressed wild-type annexin proteins (Figure 9B, left and center panels). Despite the ability of mutant annexins A1D171A and A2D161A to significantly reduce the cap size of co-expressed wild-type annexins A1 and A2, respectively, A1D171A or A2D171A had minimal effect on wild-type annexin A6 cap size (Figure 9C). These data indicated that annexins A1 and A2 interact in a homotypic manner and affect self-cap assembly, while the localization of A6 to the repair cap is minimally regulated by the localization of annexins A1 and A2.

[0148] To determine the effect of dominant-negative annexin A6 on the assembly of annexins A1, A2, and A6 in the repair cap and membrane repair capacity, we similarly performed laser injury on isolated myofibers in the presence of FM4-64. FM4-64 is a membrane-impermeable dye that is nonfluorescent in aqueous solution and increases in fluorescence intensity upon binding to exposed membrane phospholipids during injury. It is commonly used as a membrane damage marker (Bansal et al., 2003; Cai et al., 2009; Demonbreun and McNally, 2015; Yeung et al., 2009; Zweifach, 2000). Myofibers expressing annexin A6E233A-GFP showed increased FM4-64 fluorescence area after laser injury compared with control myofibers expressing wild-type annexin A6-GFP (Figure 10D). These results indicated that a functional annexin repair complex is required for proper membrane repair and that annexin A6 is involved in regulating complex formation.

[0149] Annexin A6 protected against laser-induced muscle fiber damage in vitro. Annexin A6 promotes the formation of a macromolecular repair cap complex, which induces large Ca2+ release at the site of membrane damage. 2+ Because annexin A6 overexpression was most efficient in forming filled blebs, we evaluated whether it reduced membrane damage in wild-type myofibers. Wild-type myofibers were electroporated with annexin A6-GFP or mock-electroporated, and then laser-damaged in the presence of FM4-64 to mark the damaged area. In wild-type myofibers overexpressing annexin A6, FM4-64 dye uptake after laser-induced membrane damage was reduced compared to control myofibers (Figure 11A). These results suggested that annexin A6 overexpression was effective in improving membrane repair in isolated myofibers.

[0150] Next, we tested whether extracellular recombinant annexin A6 could also protect wild-type myofibers from membrane damage. Wild-type myofibers were isolated and incubated with recombinant annexin A6 (rANXA6) or vehicle control. Laser injury was performed in the presence of FM4-64. Pretreatment with extracellular recombinant annexin A6 reduced the FM4-64 fluorescence area compared to myofibers treated with the vehicle control (Figure 11B). These data indicated that recombinant annexin A6 protects against membrane damage due to extracellular exposure.

[0151] Recombinant annexin A6 protects against muscle fiber damage in a model of chronic muscle disease. Muscular dystrophies are progressive muscle-wasting diseases caused by loss-of-function mutations in key cytoskeletal or membrane-associated proteins, leading to membrane fragility. mdx mice lack the integral membrane protein dystrophin and are a model of Duchenne muscular dystrophy (DMD) (Hoffman et al., 1987; Petrof et al., 1993). mdx mice expressing human latent TGF-β binding protein 4 (mdx / hLTBP4) have more severe muscular dystrophy, similar to that seen in humans with DMD (Ceco et al., 2014; Flanigan et al., 2013; Quattrocelli et al., 2017b). Because recombinant annexin A6 protected wild-type muscle from laser-induced membrane damage, we next evaluated whether exposure to recombinant annexin A6 protects against membrane damage in the context of chronic muscle disease. Anti-HIS (green) immunostaining revealed the presence of recombinant annexin A6-HIS in the sarcolemma of mdx / hLTBP4 dystrophic muscles after systemic injection, but no anti-HIS staining was present in vehicle control muscles (Figure 11C). Similar to isolated wild-type myofibers, pretreatment with extracellular recombinant annexin A6 reduced FM4-64 fluorescence after laser-induced injury in mdx / hLTBP4 myofibers compared to myofibers treated with vehicle control (Figure 11D). These combined data indicated that extracellular annexin A6 targets damaged membranes, promoting more efficient repair and protecting healthy and dystrophic myofibers from injury.

[0152] Recombinant annexin A6 protected against muscle fiber damage in vivo. Next, we evaluated whether recombinant annexin A6 could protect against muscle damage in vivo. Recombinant annexin A6 or vehicle control was intramuscularly injected into the tibialis anterior (TA) muscles of wild-type mice. Mice were also intraperitoneally injected with Evans blue dye. This dye is an important tracer that is excluded by intact, healthy muscle fibers but readily taken up by damaged, permeabilized muscle fibers (Jennische and Hansson, 1986). Two hours after injection of recombinant annexin A6, TA muscles were injured with cardiotoxin. Three hours after cardiotoxin injury, muscles were harvested and Evans blue dye uptake was assessed (Figure 12A). Gross imaging demonstrated that pretreatment with recombinant annexin A6 reduced cardiotoxin-induced muscle damage in vivo, as evidenced by reduced dye uptake compared to controls (Figure 12B). Fluorescence imaging showed that pretreatment with recombinant annexin A6 reduced dye uptake by 50% compared to control muscles (Figures 12C and 12D). Surface plot profiles showed reduced dye fluorescence in tibialis anterior muscles pretreated with intramuscular recombinant annexin A6 (Figure 12C). These results indicate that intramuscular recombinant annexin A6 can reduce membrane damage and promote membrane repair in vivo.

[0153] Although intramuscular injection of annexin A6 was effective in reducing injury, this route of administration is not optimal for treating large muscle groups, internal tissues, or chronic diseases. Therefore, we investigated the efficacy of recombinant annexin A6 administered via systemic retro-orbital (RO) injection. Recombinant annexin A6 or a control solution was injected 2 hours before cardiotoxin-induced tibialis anterior muscle injury (pre-treatment). Alternatively, recombinant annexin A6 was administered immediately after cardiotoxin injury to the tibialis anterior (TA) muscle (post-treatment). Additionally, Evans blue dye was injected before injury. Muscles were harvested 3 hours after cardiotoxin injury, and dye uptake was assessed (Figure 13A). Fluorescence imaging demonstrated that dye uptake was significantly reduced with recombinant annexin A6 pre- and post-treatment compared to vehicle control (Figures 13B and 13C). Surface plot profiles demonstrate reduced dye fluorescence in TA muscles pre- and post-treated with systemic recombinant annexin A6 (Figure 13C). These results demonstrated that recombinant annexin A6 reduced membrane damage and promoted membrane repair through intravenous systemic administration in vivo both before and after muscle injury.

[0154] Recombinant annexin A6 protected against chronic muscle injury in vivo. Since recombinant annexin A6 protected wild-type muscles from acute membrane damage in vivo, we next evaluated whether exposure to recombinant annexin A6 protects against membrane damage in the context of chronic muscle disease. Mutations in the gene γ-sarcoglycan (SGCG) cause limb-girdle muscular dystrophy type 2C (LGMD2C) in both mice and humans. To determine whether recombinant annexin A6 can promote repair in a chronic injury setting, sgcg-deficient mice were injected with recombinant protein or a control solution every 3 days for 12 days, and serum biomarkers of muscle damage, creatine kinase (CK) and lactate dehydrogenase (LDH), were then quantified (Figure 14A). Systemic treatment with recombinant annexin A6 reduced both serum CK and LDH levels in sgcg-deficient mice (Figures 14B and 14C). These results demonstrated that systemic recombinant annexin A6 promotes membrane repair in a model of chronic muscle disease.

[0155] Consideration Annexins promote the formation of blebs at sites of membrane damage. Increased expression of annexins in muscle fibers increases the intramyocellular Ca2+ transport associated with damage. 2+ This decrease correlated with the formation of extracellular blebs at the site of the annexin repair cap. Annexins A2 and A6 act to release Ca released from the repair cap. 2+ We found that overexpression of annexins A1, A2, and A6 induced the formation of membranous blebs containing the Ca binding protein GCaMP5G. Furthermore, overexpression of annexins A1, A2, and A6 each increased the end-point Ca concentration in muscle fibers after injury. 2+ Annexin A6 overexpression reduced the damage-related Ca accumulation. 2+ Annexin A6 had the most sustained effect on reducing Ca accumulation and induced the formation of large GCaMP5G-containing blebs. 2+ A model has been proposed in which annexin A6 promotes the excretion of Ca and proteins into blebs, the formation of which is further induced by annexin A1 and annexin A2. In artificial membrane patches, the presence of annexin A1 or annexin A2 induced bleb formation at the site of membrane defects (Boye et al., 2018). In contrast, the presence of annexin A6 promotes Ca excretion. 2+ In a blebbing-dependent manner, contraction of the artificial membrane into large folds was induced (Boye et al., 2018). The difference in annexin A6 in inducing blebbing in live myofibers or folds in artificial membranes may be related to the presence of endogenously expressed annexins A1 and A2 in isolated myofibers compared to exposure to a single recombinant annexin protein in the artificial membrane studies. Without being bound by theory, it is possible that annexins, within macromolecular repair complexes, mediate wound closure, excision of damaged membranes, and Ca release at the injury site. 2+ It is hypothesized that IL-16 is actively involved in blebbing, acting to remove large membrane lesions that promote reduction of IL-16.

[0156] Similar to the data described herein, other data have shown that in injured Xenopus oocytes, GCaMP5G fluorescence rapidly localizes to a ring around the site of membrane disruption and fades approximately 5 minutes after injury as healing progresses (Davenport et al., 2016). Overexpression of annexin A1-GFP in injured Xenopus oocytes resulted in annexin A1-positive blebs originating from the injury site (Davenport et al., 2016). However, the effect of annexin overexpression on GCaMP5G fluorescence was not assessed. Combined, these data suggest that blebbing as a mechanism of membrane repair is conserved across species and tissue types and is facilitated by the presence of annexin proteins.

[0157] Annexin A6 protects against muscle membrane damage and enhances membrane repair. As shown herein, annexin proteins, including annexins A1, A2, and A6, localize to sites of membrane damage and promote membrane repair cap and bleb formation. Mutation of annexin A6 prevented repair cap formation, reduced repair capacity, and increased dye uptake. Pretreatment with recombinant annexin A6, on the other hand, reduced dye uptake after laser-induced and toxin-induced muscle injury in vivo. However, this data does not distinguish between annexin A6 enhancing membrane repair, reducing membrane damage, or a combination of both mechanisms. As a therapeutic tool, enhancing the cell's ability to repair and / or reduce damage by stabilizing the cell membrane is a potentially beneficial strategy, potentially leading to improved cell survival. Previous studies have shown that annexin A6 is upregulated in muscles from models of chronic muscular dystrophy (Demonbreun et al., 2016a, Demonbreun et al., 2014, Swaggart et al., 2014). Further proteomic approaches in mdx mouse models have shown that annexins A1, A2, and A6 are abundant in mdx muscle membranes, again suggesting a role for annexins in damaged cell membranes (Murphy et al., 2018). Annexins bind to membrane phospholipids containing phosphatidylserine that become exposed during membrane disruption. Phosphatidylserine rearrangements upon injury likely serve as binding targets for extracellular annexins, promoting membrane folding, blebbing, and rolling at sites of membrane damage and defects (Boye et al., 2018). Based on the data herein, upregulation of annexin A6 is a compensatory mechanism that facilitates the resection of defective membranes in chronically injured fibers. Furthermore, recombinant annexin A6 is believed to promote membrane repair and long-term reduction in susceptibility to injury in chronic disease models and in tissues beyond skeletal muscle.

[0158] Combinatorial Approaches to Improve Membrane Repair. As shown herein, recombinant annexin A6 protected normal and dystrophic muscles from laser-induced membrane damage. Furthermore, both intramuscular and systemic administration of recombinant annexin A6 protected against toxin-induced muscle membrane damage in vivo. Interestingly, glucocorticoid administration increased muscle annexin expression, which correlated with enhanced muscle repair in multiple mouse models of muscular dystrophy, including mdx (DMD), dysferlin-deficient (LGMD2B), and γ-sarcoglycan-deficient (LGMD2C) mice (Quattrocelli et al., 2017a, Quattrocelli et al., 2017c). Importantly, glucocorticoid treatment also increased the expression of mitsugumin53 (also known as MG53 or Trim72), a repair protein that localizes to sites of membrane damage and is considered a "molecular Band-Aid" that improves cellular wound healing. Like annexins, MG53 is upregulated in chronic muscle injury and enhances repair in dystrophic muscle as well as other tissues, such as the heart, lung, and kidney (Waddell et al., 2011) (Duann et al., 2015; He et al., 2012; Jia et al., 2014; Liu et al., 2015; Weisleder et al., 2012). MG53 is a component of the annexin-mediated repair complex and is localized juxtaposed to the annexin repair cap (Demonbreun et al., 2016b). Coadministration of recombinant annexin A6 with glucocorticoids and / or MG53 may further strengthen the clinical relevance of these therapies for conditions resulting from membrane pathology.

[0159] In summary, the data provided herein demonstrate that annexins promote the formation of blebs that are released at sites of muscle membrane injury, and that annexin A6 is most effective in promoting this process. Furthermore, recombinant annexin A6 protects against membrane damage in normal and dystrophic muscle. These data identify annexin A6 as a therapeutic target for enhancing membrane repair capacity in healthy and diseased muscle.

[0160] Example 3 This example details the results of further and updated experiments that were performed.

[0161] Membrane repair is essential for cell survival. In skeletal muscle, injury is often associated with disruption of the plasma membrane. Furthermore, muscular dystrophies are associated with genetic mutations that produce fragile membranes or reduce membrane repair. Methods that enhance repair and reduce susceptibility to injury could benefit muscles in both acute and chronic injury situations. Annexins are membrane-associated Ca2+ receptors involved in repair. 2+ Annexin A6, a family of binding proteins, was previously identified as a genetic modifier of muscle injury and disease. Annexin A6 forms a repair cap over the site of membrane disruption. To elucidate how annexins promote repair, we visualized the formation of the annexin cap during injury. The size of the annexin cap correlated with the Ca 2+ The overexpression of annexins was found to be positively correlated with increased Ca concentrations. 2+ Fluorescence-rich external blebbing promotes intracellular Ca at the injury site 2+ The results also correlated with a decrease in fluorescence. Overexpression of annexin A6 reduced membrane damage, consistent with enhanced repair. Treatment with recombinant annexin A6 protected against acute muscle injury in vitro and in vivo. Furthermore, administration of recombinant annexin A6 to a model of muscular dystrophy reduced serum creatine kinase, a biomarker of the disease. These data support the role of membrane-associated Ca during membrane repair. 2+ Annexins were identified as mediators of the release, and annexin A6 was identified as a therapeutic target to enhance membrane repair capacity.

[0162] Annexin A6 forms a repair cap at the site of muscle membrane disruption. The data herein demonstrate that the addition of exogenous recombinant annexin A6 (rANXA6) promotes membrane resealing and recovery from injury.

[0163] introduction Plasma membrane repair occurs after membrane disruption and is a highly conserved process. The active process required for membrane resealing is Ca 2+This is thought to depend on vascular-dependent vesicle fusion and local cytoskeletal remodeling (McNeil PL, and Khakee R. Disruptions of muscle fiber plasma membranes. Role in exercise-induced damage. Am J Pathol. 1992;140(5):1097-109; McNeil PL, and Kirchhausen T. An emergency response team for membrane repair. Nat Rev Mol Cell Biol. 2005;6(6):499-505). Other models suggest that membrane repair is mediated by the fusion of lysosomal vesicles, lateral diffusion of membranes to the site of injury, and extrusion of membrane blebs (Rodriguez A, Webster P, Ortego J, and Andrews NW. Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells. J Cell Biol. 1997;137(1):93-104; Reddy A, Caler EV, and Andrews NW. Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell. 2001;106(2):157-69; Demonbreun AR, Quattrocelli M, Barefield DY, Allen MV, Swanson KE, and McNally EM. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of Cell biology.2016;213(6):705-18, McDade JR, Archambeau A, and Michele DE.Rapid actin-cytoskeleton-dependent recruitment of plasma membrane-derived dysferlin at wounds is critical for muscle membrane repair. FASEB J. 2014;28(8):3660-70; Babiychuk EB, Monastyrskaya K, Potez S, and Draeger A. Blebbing confers resistance against cell lysis. Cell Death Differ. 2011;18(1):80-9). These models are not mutually exclusive and may vary depending on the type and severity of injury. Skeletal muscle is highly dependent on plasma membrane repair, as mutations in genes encoding repair proteins cause muscle disease (Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, et al. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature. 2003;423(6936):168-72, Bashir R, Britton S, Strachan T, Keers S, Vafiadaki E, Lako M, et al. A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B. Nat Genet. 1998;20(1):37-42, Demonbreun AR, Swanson KE, Rossi AE, Deveaux HK, Earley JU, Allen MV, et al. al.Eps 15 Homology Domain(EHD)-1 Remodels Transverse Tubules in Skeletal Muscle.PLoS One.2015;10(9):e0136679, Demonbreun AR, and McNally EM.Plasma Membrane Repair in Health and Disease.Curr Top Membr.2016;77:67-96, Defour A,Medikayala S,Van der Meulen JH,Hogarth MW,Holdreith N,Malatras A,et al.Annexin A2 links poor myofiber repair with inflammation and adipogenic replacement of the injured muscle.Human molecular genetics.2017;26(11):1979-91, Cai C, Masumiya H, Weisleder N, Matsuda N, Nishi M, Hwang M, et al.MG53 nucleates assembly of cell membrane repair machinery.Nat Cell Biol.2009;11(1):56-64). .

[0164] Annexins regulate lipid binding, cytoskeletal reorganization, and membrane folding, steps required for membrane repair. 2+Jimenez AJ, and Perez F. Plasma membrane repair: the adaptable cell life-insurance. Curr Opin Cell Biol.2017;47:99-107, Lauritzen SP, Boye TL, and Nylandsted J. Annexins are instrumental for efficient plasma membrane repair in cancer cells. Semin Cell Dev Biol.2015;45:32-8, Bizzarro V,Petrella A,and Parente L.Annexin A1:novel roles in skeletal muscle biology.J Cell Physiol.2012;227(8):3007-15,Grewal T,Hoque M,Conway JRW,Reverter M,Wahba M,Beevi SS,et al.Annexin A6-A multifunctional scaffold in cell motility.Cell Adh Migr. 2017;11(3):288-304, Boye TL, Jeppesen JC, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep. 2018;8(1):10309, Boye TL, Maeda K, Pezeshkian W, Sonder SL, Haeger SC, Gerke V, et al. Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair. Nat Commun. 2017;8(1):1623). Individual annexin repeat domains contain unique annexin-specific type II or type III binding sites and Ca binding sites. 2+ Coordinate binding of type II and type III binding sites to Ca2+ Due to their different affinities, each annexin binds to intracellular Ca 2+They have the unique ability to respond to a range of Ca2(+)-dependent phospholipid binding levels and phospholipid binding (Blackwood RA, and Ernst JD. Characterization of Ca2(+)-dependent phospholipid binding, vesicle aggregation, and membrane fusion by annexins. The Biochemical journal. 1990;266(1):195-200). Annexins have the ability to self-oligomerize and hetero-oligomerize (Zaks WJ, and Creutz CE. Ca(2+)-dependent annexin self-association on membrane surfaces. Biochemistry. 1991;30(40):9607-15). Typical annexins, such as A1 and A2, contain a single annexin core composed of four annexin repeat domains. In contrast, annexin A6 contains two annexin cores and therefore eight annexin repeat domains (Benz J, Bergner A, Hofmann A, Demange P, Gottig P, Liemann S, et al. The structure of recombinant human annexin VI in crystals and membrane-bound. J Mol Biol. 1996;260(5):638-43).The duplicated structure of annexin A6 allows the amino- and carboxyl-terminal annexin core domains to bind to one or two different membranes, making annexin A6 a primary target for promoting membrane coalescence and folding, which is required during membrane repair. (Boye TL, Jeppesen JC, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep 2018;8(1):10309; Boye TL, Maeda K, Pezeshkian W, Sonder SL, Haeger SC, Gerke V, et al. Annexins A4 and A6 induce membrane curvature and constriction during cell membrane repair. Nat Commun. 2017;8(1):1623; Buzhynskyy N, Golczak M, Lai-Kee-Him J, Lambert O, Tessier B, Gounou C, et al. Annexin-A6 presents two modes of association with phospholipid membranes. A combined QCM-D, AFM and cryo-TEM study. Journal of structural biology. 2009;168(1):107-16).

[0165] Annexins have a high affinity for phosphatidylserine, phosphatidylinositol, and cholesterol, which are highly abundant in the sarcolemma (Gerke V, Creutz CE, and Moss SE. Annexins: linking Ca 2+signaling to membrane dynamics.Nat Rev Mol Cell Biol.2005;6(6):449-61, Fiehn W, Peter JB, Mead JF, and Gan-Elepano M.Lipids and fatty acids of sarcolemma, sarcoplasmic reticulum, and mitochondria from rat skeletal muscle.The Journal of biological chemistry.1971;246(18):5617-20). Multiple annexins, including annexins A1, A2, and A6, are involved in membrane repair in skeletal muscle, Xenopus oocytes, human trophoblasts, and HeLa cancer cells, suggesting a conserved mechanism (Demonbreun AR, Quattrocelli M, Barefield DY, Allen MV, Swanson KE, and McNally EM. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology. 2016;213(6):705-18; Babbin BA, Laukoetter MG, Nava P, Koch S, Lee WY, Capaldo CT, et al. Annexin A1 regulates intestinal mucosal injury, inflammation, and repair. J Immunol. 2008;181(7):5035-44; Lennon NJ, Kho A, Bacskai BJ, Perlmutter SL, Hyman BT, and Brown RH, Jr. Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing.The Journal of biological chemistry.2003;278(50):50466-73, McNeil AK, Rescher U, Gerke V, and McNeil PL.Requirement for annexin A1 in plasma membrane repair.The Journal of biological chemistry.2006;281(46):35202-7, Roostalu U, and Strahle U.In vivo imaging of molecular interactions at damaged sarcolemma.Dev Cell.2012;22(3):515-29, Davenport NR,Sonnemann KJ,Eliceiri KW,and Bement WM.Membrane dynamics during cellular wound repair.Mol Biol Cell.2016;27(14):2272-85, Carmeille R, Degrelle SA, Plawinski L, Bouvet F, Gounou C, Evain-Brion D, et al. Annexin-A5 promotes membrane resealing in human trophoblasts. Biochimica et biophysica acta.2015;1853(9):2033-44, Bement WM, Mandato CA, and Kirsch MN. Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes. Curr Biol. 1999;9(11):579-87. Annexins are sequentially recruited to the damaged membrane, forming a macromolecular repair complex (called the repair cap) at the membrane lesion. (Demonbreun AR, Quattrocelli M, Barefield DY, Allen MV, Swanson KE, and McNally EM. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology.2016;213(6):705-18, Boye TL, Maeda K, Pezeshkian W, Sonder SL, Haeger SC, Gerke V, et al. Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair. Nat Commun.2017;8(1):1623, Roostalu U, and Strahle U. In vivo imaging of molecular interactions at damaged sarcolemma.Dev Cell.2012;22(3):515-29). Polymorphisms in Anxa6, the gene encoding annexin A6, have previously been identified in several commonly used laboratory mouse strains that correlate with impaired muscle repair (Demonbreun AR, Allen MV, Warner JL, Barefield DY, Krishnan S, Swanson KE, et al. Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption. Am J Pathol. 2016;186(6):1610-22, Quattrocelli M, Capote J, Ohiri JC, Warner JL, Vo AH, Earley JU, et al. Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury. PLoS Genet. 2017;13(10):e1007070, Swaggart KA, Demonbreun AR, Vo AH, Swanson KE, Kim EY, Fahrenbach JP, et al. Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.Proceedings of the National Academy of Sciences of the United States of America. 2014;111(16):6004-9). This polymorphism produces a truncated annexin A6 protein that acts in a dominant-negative manner to reduce repair cap formation and prevent sarcolemma repair. Further studies in mice have shown that loss of annexin A2 leads to poor myofiber repair (Defour A, Medikayala S, Van der Meulen JH, Hogarth MW, Holdreith N, Malatras A, et al. Annexin A2 links poor myofiber repair with inflammation and adipogenic replacement of the injured muscle. Human molecular genetics. 2017;26(11):1979-91). These data suggest that there is coordinated recruitment of annexin proteins to the repair cap, facilitated by dynamic protein-protein interactions.

[0166] Herein, we examine the dynamics of annexins A1, A2, and A6 in relation to multiple Ca 2+ The repair cap formed by annexins A1, A2, and A6 was assessed by Ca 2+ It increased with increasing concentration, but Ca 2+ Mutation of the coordination residues disrupted normal annexin repair cap formation. Overexpression of annexins promoted the formation of external blebs at the site of membrane damage that were released from the repair cap. Overexpression of annexin A6 led to the formation of larger blebs and the release of blebs from the repair cap. These vesicles contained Ca 2+ The Ca binding marker protein GCaMP5G is abundant and thus 2+ The abundance of Ca near the injury site indicates that 2+ The decrease in fluorescence correlated with the increase in annexin A6. Annexin A6 overexpression promoted membrane repair, while the important Ca 2+Mutation of the coordinating residue, E233, disrupted the formation of the annexin repair complex and reduced repair capacity. Local and systemic administration of recombinant annexin A6 attenuated muscle damage in vivo. These data identify a novel role for annexins in bleb release from muscle membrane lesions during membrane repair and identify annexin A6 as a therapeutic target for protecting against muscle damage.

[0167] method Wild-type mice on a 129T2 / SvEmsJ background were bred and housed in a specific pathogen-free facility with a 12-h light / dark cycle and fed ad libitum in accordance with Northwestern University's Animal Care and Use Committee regulations. 129T2 / SvEmsJ (129T2) mice were purchased from Jackson Laboratory (Ben Harbor, ME, stock number 002065). mdx / hLTBP4 mice were generated as described (Quattrocelli M, Capote J, Ohiri JC, Warner JL, Vo AH, Earley JU, et al. Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury. PLoS Genet. 2017;13(10):e1007070; Ceco E, Bogdanovich S, Gardner B, Miller T, DeJesus A, Earley JU, et al. Targeting latent TGFbeta release in muscular dystrophy. Science translational medicine. 2014;6(259):259ra144). Sgcg-deficient mice were generated as described (Hack AA, Cordier L, Shoturma DI, Lam MY, Sweeney HL, and McNally EM. Muscle degeneration without mechanical injury in sarcoglycan deficiency. Proceedings of the National Academy of Sciences of the United States of America. 1999;96(19):10723-8). All wild-type mice used for experiments were male and female mice aged 2-3 months. The Sgcg-deficient cohort was age- and sex-matched with 2- to 5-month-old mice.

[0168] Plasmids. Plasmids encoding annexins A1, A2, and A6 with a carboxyl-terminal turboGFP tag were obtained from Origene (Rockville, MD). Subcloning of annexins A1, A2, and A6 to replace the GFP tag with tdTomato (Addgene) was performed by Mutagenix (Suwanee, GA). Site-directed mutagenesis of annexins A1-GFP, A2-GFP, and A6-GFP was performed by Mutagenix to express Ca. 2+ The binding mutants A1-D171A-GFP, A2-D161A-GFP, A6-D149A-GFP, and A6-E233A-GFP were generated. To verify mutagenesis, the constructs were sequenced. Plasmid DNA was isolated using Qiagen's Endo-Free Maxi Prep Kit (Qiagen #12362). 2+ The sensor GCaMP5G was purchased from (Addgene #31788).

[0169] Sequence Comparison and Protein Schematics Protein ribbon diagrams were generated using Swiss-PdbViewer using the solved crystal structures of annexin A1 (1MCX), A2 (2HYW), and annexin A6 (1AVC) available at www.rcsb.org. Clustal Omega from the European Bioinformatics Institute (EMBL-EBI) was used to align the annexin sequences annexin A1 (NM_010730, SEQ ID NO: 37), annexin A2 (NM_007585, SEQ ID NO: 38), and annexin A6 (NM_013472, SEQ ID NO: 39) from www.ncbi.nlm.nih.gov, as well as annexin A6 coding sequences from several species: Homo sapiens (AAH17046.1, SEQ ID NO: 40), macaca (AFE65315.1, SEQ ID NO: 41), canis (XP_005619331.1, SEQ ID NO: 42), rattus (NP_077070.2, SEQ ID NO: 43), and mus (NP_038500.2, SEQ ID NO: 44).

[0170] Electroporation, muscle fiber isolation, laser injury, caps, and vesicle measurements. Flexor digitorum brevis (FDB) fibers were transfected with end-free plasmid DNA by in vivo electroporation. The method was previously described (Demonbreun AR, Quattrocelli M, Barefield DY, Allen MV, Swanson KE, and McNally EM. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology.2016;213(6):705-18, Demonbreun AR, and McNally EM. Myofibers.Journal of visualized experiments:JoVE.2015;106(106):e53551, DiFranco M, Quinonez M, Capote J, and Vergara J.DNA transfection of mammalian skeletal muscles using in vivo electroporation.Journal of Visualized Experiments:JoVE.2009;32(32)). Z-stack projections were acquired after the last time-lapse frame, approximately 4 min after injury, from successive acquisitions with a step size of 0.125 μM between slices. Z-stack renderings were constructed in FIJI. Cap area and ferret diameter measurements were performed from a single slice near the center of the z-stack using the FIJI imaging tool. Fibers expressing similar levels of tagged or GCaMP5G protein were compared. Ca of GCaMP5G was measured. 2+Fluorescence was measured from acquired time-lapse images using a standard rectangular ROI placed within the myofiber below the injury site using FIJI. Fluorescence was expressed as F / F. External vesicle counts and GCaMP5G area were measured from endpoint z-stack and maximum projection images using FIJI. Vesicles were considered external if they were found outside the sarcolemma, as assessed in the brightfield and fluorescence channels. All measurements were taken from myofibers isolated from at least n ≥ 3 mice (n ≥ 3 myofibers per mouse).

[0171] For recombinant myofiber studies, myofibers were isolated from mdx / hLTBP4 mice as described above. Myofibers were incubated in Ringer's medium with or without 25 μg / ml recombinant annexin A6 (5186-A6-050, R&D Systems). FM4-64 (2.5 μM) was added to myofibers immediately prior to imaging. Images were acquired and quantified as described above. FM4-64 area was measured using FIJI at the imaging endpoint from a single slice near the center of the Z-stack. A Z-stack step size (0.125 μm) was acquired from the edge of the cap to the edge.

[0172] Quality control of myofibers was based on a number of characteristics, including the use of adherent myofibers with intact sarcomere structures detected by brightfield imaging. Myofibers appeared free of tears or ruptures induced during the isolation protocol. The region of the myofiber selected for injury was linear and not located at the nucleus or neuromuscular junction. Furthermore, the fluorescence intensity in both the red and green channels suggested similar expression levels before injury.

[0173] Multiphoton laser damage and imaging. Fibers were subjected to laser-induced damage at room temperature using a Nikon A1R-MP multiphoton microscope. Imaging was performed using a 25x 1.1 NA objective, guided by NIS-Elements AR imaging software. Green fluorescent protein (GFP) and FM4-64 were excited using a 920 nm laser, and emission wavelengths of 575 nm and 629 nm were collected, respectively. To induce laser damage in isolated myofibers, a diffraction-limited spot (approximately 410 nm in diameter) was created on the lateral membrane of the myofiber using a 920 nm laser for 1 second at 10-15% laser power. Time-lapse images were collected as follows: one before injury, one at the time of injury, and then every 8 seconds for 80 seconds (10 images), and every 30 seconds for 5 minutes (10 images). At the end of the time-lapse series, z-stack images were collected at 250 nm intervals from the injury site of the muscle fiber, as indicated by the NIS-Elements AR imaging software. Multiphoton imaging was used to acquire the data presented in Figure 5, Figure 10D, and Figure 19.

[0174] For recombinant protein calcium studies, muscle fibers were isolated from wild-type mice as described above. Muscle fibers were then resuspended in 1 mM Ca. 2+ Ringer's or 0 mM Ca 2+ Myofibers were incubated in 20 μg / ml recombinant annexin A6 (5186-A6-050, R&D Systems) in +EGTA. FM1-43 (2.5 μM) was added to myofibers immediately prior to imaging. Images were acquired and quantified as described above. FM1-43 fluorescence over time was measured using FIJI and plotted as F / F over time.

[0175] Cardiotoxic injury and analysis. Wild-type mice were injected into the tibialis anterior muscle with 25 μg / ml recombinant annexin A6 (5186-A6-050, R&D Systems) or Ringer's solution (3% isoflurane, 0.8 L / min O2) in sedated mice. For systemic administration, wild-type mice were injected into the retroorbital space with 1 mg / kg recombinant annexin A6 (5186-A6-050, R&D Systems) or PBS (5 μl / g body weight) diluted in EBD (3% isoflurane, 0.8 L / min O2) in sedated mice. Additionally, Evans Blue dye (E-2129, Sigma-Aldrich, St. Louis, MO) was dissolved in phosphate-buffered saline (PBS) at 10 mg / mL and injected into mice at 5 μl / g body weight. Two hours after pretreatment, cardiotoxin injury was performed by injecting 20 μl of a 10 μM cardiotoxin solution in PBS into the tibialis anterior or gastrocnemius / soleus muscles (3% isoflurane, 0.8 L / min O2) of sedated animals. The cardiotoxin was released along the midline of the muscle, inducing a uniform injury area in the center of the muscle. Muscles were harvested 3 hours after cardiotoxin injection.

[0176] Evans Blue dye uptake. Sections (10 μm thick) from the center of cryoembedded muscles were collected on a cryostat (chamber: -20°C, sample: -15°C, catalog number CM1950, Leica, Wetzlar, Germany). Tissue sections were fixed in methanol for 2 minutes, rinsed, and mounted with Vectashield containing DAPI (H-1200, Vector Laboratories). Imaging was performed using a Zeiss Axio Observer A1 microscope (Zeiss, Oberkochen, Germany) with a 10x objective. Images were acquired using ZEN software (Zeiss, Jena, Germany). Fluorescence quantification, surface plots, and muscle area were performed using FIJI (NIH). To quantify total tissue dye, whole tissues were dissected, minced, weighed, and incubated in 1 mL of formamide at 55°C for 2 hours. Spectrophotometric absorbance was measured at 620 nm.

[0177] Serum collection and CK analysis. Mice were sedated (3% isoflurane, 0.8 l / min O2), and blood was collected by retro-orbital puncture using heparinized capillary tubes (20-362-566, Fisher Scientific, Waltham, MA) into Microtainer™ Gold Top Serum Separators (365967 Becton Dickinson, Franklin Lakes, NJ) and centrifuged at 8,000 × g for 10 min. Plasma fractions were frozen and stored at -80°C. Serum creatine kinase (CK) was analyzed in duplicate for each mouse using the EnzyChrom Creatine Kinase Assay (ECPK-100; BioAssay Systems, Hayward, CA) according to the manufacturer's instructions as described (Demonbreun AR, Allen MV, Warner JL, Barefield DY, Krishnan S, Swanson KE, et al. Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption. Am J Pathol. 2016;186(6):1610-22). Results were acquired using a Synergy HTX multimode plate reader (BioTek®, Winooski, VT).

[0178] Short-term chronic dosing regimen. Sgcg-deficient mice were sedated (3% isoflurane, 0.8 L / min O2) and bled as described above. While sedated, mice were injected with 1 mg / kg recombinant annexin A6 (5186-A6-050, R&D Systems) or PBS into the right retroorbital space once every three days for a total of five injections, and bled 14 days after the first injection. An additional cohort of Sgcg-deficient mice was bled as described above immediately before the first protein injection. Next, sedated mice were injected with 1 mg / kg recombinant annexin A6 or annexin A2 (both produced by Northwestern's Protein Production Core) into the right retroorbital space once every three days for a total of five injections, and bled 14 days after the first injection.

[0179] Exercise injury. Sgcg-deficient mice were sedated (3% isoflurane, 0.8 L / min O2), and pre-exercise blood was collected by left retro-orbital puncture as described above. While sedated, mice received five consecutive injections of 1 mg / kg recombinant annexin A6 (5186-A6-050, R&D Systems) or PBS + 1 mg / kg BSA into the right retro-orbital space at 9:00 AM and 5:00 PM over a 48-h period. Two hours after the fifth RO injection, mice were subjected to 60 min of treadmill running at 10 m / min with a 15° downward slope. Thirty min after exercise, blood was collected from the left retro-orbital space. Serum creatine kinase (CK) was analyzed as described above. Injections, exercise, and blood collection were performed blinded to the treatment groups.

[0180] Protein Production. Recombinant mouse annexin A6 and mouse annexin A2 were produced and purified by the Northwestern University Recombinant Protein Production Core. Briefly, mouse annexin A6 (MG222645, Origene, Rockville, MD) and annexin A2 (MG205064, Origene, Rockville, MD) were subcloned into the pCMV6-AC-HIS backbone (PS100002, Origene, Rockville, MD). Plasmids were transfected and expressed in the ExpiCHO expression system (A29133, ThermoFisher Scientific, Waltham, MA). Carboxy-terminally tagged recombinant proteins were purified with Ni-charged MagBeads (L00295, GenScript, Piscataway, NJ) and purity was assessed by SDS-Page. Protein purity was further verified by immunoblotting using anti-HIS (MAB050, R&D Systems, Minneapolis, MN) and anti-annexin A6 (ab31026, Abcam) or anti-annexin A2 (ab154113, Abcam) antibodies. Proteins were diluted in phosphate-buffered saline and stored at -80°C.

[0181] Calcium dynamics. FDB muscles were electroporated and isolated as described above. Ca 2+ Muscle fibers were damaged in Ringer's solution at concentrations of 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.175 mM, 0.1 mM, 0.050 mM, and 0 mM. 2+ Only in Ringer's, Ca 2+ EDTA was added as a chelating agent. For these experiments, myofibers were directly isolated in 2 mM, 1 mM, and 0.5 mM Ringer's solution, respectively. Ca concentrations below 0.5 mM were used. 2+ In experiments using β-glucan, muscle fibers were treated with 0.5 mM Ca 2+ Isolated in Ringer's, 0 mM EDTA-free Ca 2+ In the 0 mM experiment, muscle fibers were diluted with 0.5 mM Ca. 2+ Isolate in Ringer's and add 0 mM Ca immediately before imaging.2+ + EDTA Ringer's. Co-electroporation of wild-type annexin + wild-type annexin construct was performed in one mouse paw, and wild-type annexin + mutant annexin was co-electroporated in the contralateral paw. All measurements were performed on at least n ≥ 2 mice (each Ca 2+ The area-Ca values ​​were obtained from muscle fibers isolated from mice (n ≥ 3 muscle fibers per mouse). 2+ Curves were fitted with a Hill curve over a Ca2+ concentration range of 0–2 mM. Kinetic parameters were calculated using Prism Graphpad.

[0182] Measurement of calcium and pH indicator dyes. Wild-type FDBs were isolated and plated in Ringer's on Matek glass-bottom dishes as described above. 20 minutes before imaging, myofibers were loaded with Fluo-4 AM (F10489, ThermoFisher Scientific, Waltham, MA) or pHrodo AM (P35373, ThermoFisher Scientific, Waltham, MA) at 37°C as described in the instruction manual. Fibers were rinsed once with Ringer's as described above, then injured and imaged on a Nikon A1R GaSP. Fluorescence intensity was measured using FIJI (NIH). pHrodo change in fluorescence intensity was calculated as F / F. Data were acquired from n = 3 mice per experiment from multiple myofibers per mouse. Additionally, wild-type myofibers were incubated in 0 mM Ca. 2+ The cells were incubated for 1 h in 0 mM external CaCl, preloaded with Fluo-4 AM for 20 min before imaging, rinsed, and then incubated in external Ringer's solution with or without EGTA and 0 mM external CaCl. 2+ In the presence of, MP+ caused damage.

[0183] Cardiotoxin injection and histology. Wild-type mice were sedated (3% isoflurane, 0.8 l / min O2) and injected with 1 mg / kg recombinant annexin A6 (5186-A6-050, R&D Systems) or PBS + 1 mg / kg BSA into the right retro-orbital cavity and allowed to recover. Two hours after injection, mice were sedated again. While sedated, the tibialis anterior muscle was injected along the midline with 10 μm cardiotoxin in 20 μl of PBS, as described above. Seven days after injection, the muscle was isolated and frozen. Muscle sections were taken every 100 μm from the tendon to the midbelly, fixed, and stained with hematoxylin and eosin. Imaging was performed using a Zeiss Axio Observer A1 microscope (Zeiss, Oberkochen, Germany) with a 10x objective. Tile images were acquired using ZEN software (Zeiss, Jena, Germany). The percentage of injury area was calculated as the average injury area (including internal myonuclei) divided by the total muscle area of ​​three sections per muscle. One muscle was excluded from each group due to technical errors in tissue processing. The injury area was measured using FIJI (NIH).

[0184] Single-cell Ca 2+ and shortening measurements. Isolated FDB fibers were seeded onto laminin-coated glass-bottom 35-mm dishes for 1 h and cultured overnight in DMEM containing 10% FBS and 1% penicillin / streptomycin at 37°C in a 10% CO2 incubator. One hour before data collection, the medium was removed and the cells were incubated in Tyrode's buffer (119 mM NaCl, 5 mM KCl, 25 mM HEPES, 2 mM CaCl2, 2 mM MgCl2) containing 10 μM Indo-1 AM (TefLabs) for 1 h at 37°C in a 10% CO2 incubator. The dish was then filled with Tyrode's buffer, mounted on a custom stage, and a platinum pacing electrode was inserted into the dish. Stimulation was evoked using a 701C high-output stimulator controlled by 950A software (Aurora Scientific). Stimulation was performed at 40 Hz and 80 Hz with a 5 ms pulse width and 100 ms duration. 2+Signals were collected using two photomultiplier tubes and a FluoroDaq controller. Video sarcomere lengths were recorded with a high-speed camera and fast Fourier transform using an Aurora Scientific 900B-VSL system (Aurora, Ontario). Ten transitions were collected over 20 seconds and averaged per cell per frequency.

[0185] Statistical analysis. Statistical analysis was performed using Prism (Graphpad, La Jolla, CA). Comparisons relied on ANOVA (one-way ANOVA for single variables, and multivariate ANOVA for two variables (usually area and Ca). 2+ Two-way ANOVA was performed in the case of (concentration). Otherwise, unpaired two-tailed t-tests were performed. P values ​​of 0.05 or less were considered significant. Error bars represent the standard error of the mean (SEM).

[0186] Study Approval. This study was conducted with the approval of the Northwestern University Animal Care and Use Committee (Chicago, IL).

[0187] result Ca 2+ Upon membrane damage, Ca localizes to the repair cap. Activation of muscle membrane repair requires external Ca 2+The presence of β-amyloid is required for dysferlin repair (Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, et al. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature. 2003;423(6936):168-72).Annexin proteins have previously been shown to aggregate into the repair cap at the site of injury, adjacent to an annexin-free zone in the cytoplasm beneath the repair cap. (Demonbreun AR, Quattrocelli M, Barefield DY, Allen MV, Swanson KE, and McNally EM. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology. 2016;213(6):705-18; Demonbreun AR, Allen MV, Warner JL, Barefield DY, Krishnan S, Swanson KE, et al. Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption. Am J Pathol. 2016;186(6):1610-22; Swaggart KA, Demonbreun AR, Vo AH, Swanson KE, Kim EY, Fahrenbach JP, et al. Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair. Proceedings of the National Academy of Sciences of the United States of America.2014;111(16):6004-9, Quattrocelli M, Barefield DY, Warner JL, Vo AH, Hadhazy M, Earley JU, et al. Intermittent Glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy.J Clin Invest.2017;127(6):2418-32). Ca at the injury site. 2+In vivo fluorescent Ca dynamics for real-time visualization 2+ We used the indicator protein GCaMP5G, which is a fusion protein consisting of green fluorescent protein (GFP), the calcium-binding protein calmodulin, and calmodulin M13-binding peptide. 2+ When not bound to Ca, fluorescence is minimal and 2+ Upon binding, a conformational change occurs within the protein, increasing the fluorescence intensity of GFP (Akerboom J, Chen TW, Wardill TJ, Tian L, Marvin JS, Mutlu S, et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J Neurosci. 2012;32(40):13819-40). Wild-type flexor digitorum brevis (FDB) muscles were electroporated with GCaMP5G plasmid and then laser ablation was used to damage the plasma membrane (Demonbreun AR, Quattrocelli M, Barefield DY, Allen MV, Swanson KE, and McNally EM. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology. 2016;213(6):705-18; Demonbreun AR, and McNally EM. DNA electroporation, isolation, and imaging of myofibers. Journal of Visualized Experiments: JoVE. 2015;106(106):e53551). Within 2 seconds of membrane damage (arrow), GCaMP5G fluorescence accumulated in the cytoplasm at the injury site. GCaMP5G fluorescence intensity gradually increased throughout 260 seconds of imaging (Figure 23A, upper panel).

[0188] To confirm that these results do not reflect protein aggregation of the GCaMP5G sensor, wild-type muscle fibers were injured in the presence of Fluo-4AM. 2+ When it binds to Ca, its fluorescence intensity increases. 2+ Non-protein Ca molecules routinely used to measure dynamics 2+ Similar to GCaMP5G fluorescence, Fluo-4 AM fluorescence intensity increased at the site of laser-induced membrane damage 2 seconds after injury (arrow) and continued to increase in intensity throughout 260 seconds of imaging (Figure 23A, lower panel). In myofibers co-electroporated with plasmids expressing annexin A6 and GCaMP5G with a carboxyl-terminal tdTomato fluorescent tag, GCaMP5G fluorescence localized in a ring around the annexin A6-free zone (Figure 23B, arrowhead) and co-localized with annexin A6 in the repair cap (Figure 23B, arrow, merge). We also evaluated whether injury altered pH using pHrodo fluorescence, a non-protein pH indicator dye whose fluorescence changes at different pH levels. 2+ When the indicator fluorescence was already increased at the injury site, no change from the pre-injury state (0 s) was observed compared to 10 s after injury (Figure 15). This time series indicates the Ca concentration at the injury site. 2+ This is consistent with their accumulation and promotes annexin translocation and assembly into the repair cap.

[0189] Annexin repair caps induce different Ca responses during repair cap mobilization 2+ Annexin proteins show sensitivity to Ca 2+ Annexin A6 is a Ca-dependent phospholipid- and actin-binding protein that contains four annexin repeat domains (eight in the case of annexin A6) (Figure 2). 2+ Although it binds to Ca, it does not contain the EF-hand and C2 domains found in other classes of repair proteins. 2+(Gerke V, and Moss SE. Annexins: from structure to function. Physiol Rev. 2002;82(2):331-71). Annexins bind Ca. 2+ It binds to the membrane via its convex surface (Fig. 2) and binds to both type II and type III Ca 2+ The binding sites have been described in annexin proteins. Ca binding in annexin-mediated sarcolemmal repair in muscle fibers. 2+ To further define the requirements, we analyzed annexin A1, A2, or A6 repair cap formation using multiple Ca2+ receptors. 2+ The cap size was measured from the center of the z-stack, and the type of fluorescent tag (turboGFP or tdTomato) did not affect the parameter evaluation (Figures 3A and 3B). The size of the Annexin A1 and A6 repair caps was determined by Ca concentration. 2+ The largest repair cap was formed at 2 mM Ca, and a smaller repair cap at 0.1 mM, whereas the annexin A2 repair cap was formed at 0.05 mM Ca. 2+ The repair cap area was calculated using a modified Hill equation based on the Ca 2+ The results were plotted as a function of Ca concentration. 2+ The repair cap was formed at the lowest concentration of 0.05 mM Ca, whereas annexins A1 and A6 were only formed at 0.1 mM Ca. 2+ Less than Ca 2+ The concentrations of 0.12 mM and 0.17 mM did not form a discernible cap, which is due to the Km 1 / 2 A Km of 0.067 mM compared to A6 and A1 (Figure 23D) which show 1 / 2 The size and speed of the repair caps of annexins A1 and A6 are significantly different from those of annexin A2, which has a Ca 2+ The rate of annexin A2 cap formation and cap size were significantly dependent on the concentration (Figure 4). 2+ The rates of annexins A1 and A6 were similar at Ca 2+The lower the concentration, the greater the decrease in Ca 2+ This suggests that the formation of the repair cap is highly responsive to the laser damage (Figure 4). To confirm that the formation of the repair cap is not an artifact due to the type of laser damage, laser damage was induced with both a Nikon A1R GaSP confocal and a Nikon A1R MP+ multiphoton confocal. The damage induced by the multiphoton laser was more focused and resulted in less collateral damage. The annexin A6 repair cap appeared comparable with both laser types (Figure 5). These data suggest that the formation of the annexin A1, A2, and A6 repair caps is due to the Ca2+ receptors present during myofiber repair. 2+ Annexin A2 was the most sensitive of the three annexins studied, affecting the levels of Ca 2+ This indicates sensitivity.

[0190] Overexpression of annexins promotes blebbing at sites of membrane damage. In membrane repair studies of Lytechinus pictus and Xenopus oocytes, membrane structures have been observed emerging and being released from membrane repair sites (Davenport NR, Sonnemann KJ, Eliceiri KW, and Bement WM. Membrane dynamics during cellular wound repair. Mol Biol Cell. 2016;27(14):2272-85; Bi GQ, Alderton JM, and Steinhardt RA. Calcium-regulated exocytosis is required for cell membrane resealing. The Journal of cell biology. 1995;131(6 Pt 2):1747-58). Furthermore, in artificial membrane preparations, the addition of recombinant annexins promotes calcium release at sites of membrane defects. 2+Annexins induce membrane curvature and constriction during cell membrane repair. Nat Commun. 2017;8(1):1623. We investigated whether similar findings could be observed in living skeletal muscle fibers at the site of muscle membrane injury. GCaMP5G was expressed alone or in combination with annexins A1, A2, or A6 in skeletal muscle fibers. Overexpression of annexins was found to promote the formation of extracellular blebs released from the annexin repair cap of membrane lesions (Figure 16A, red channel). These blebs appeared after repair cap formation and were found at the extracellular tip of the repair cap, which coincided with FM4-64 fluorescence (Figures 16A and 16B).FM4-64 is a membrane-impermeable dye that is non-fluorescent in aqueous solution and increases in fluorescence intensity when it binds to exposed membrane phospholipids during injury. FM4-64 is commonly used as a membrane damage marker. (Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, et al. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature. 2003;423(6936):168-72; Cai C, Masumiya H, Weisleder N, Matsuda N, Nishi M, Hwang M, et al. MG53 nucleates assembly of cell membrane repair machinery. Nat Cell Biol. 2009;11(1):56-64; Demonbreun AR, and McNally EM. DNA Electroporation, Isolation and Imaging of Myofibers. Journal of visualized experiments: JoVE. 2015;106(106):e53551; Yeung T, Heit B, Dubuisson JF, Fairn GD, Chiu B, Inman R, et al. Contribution of phosphatidylserine to membrane surface charge and protein targeting during phagosome maturation. The Journal of cell biology. 2009;185(5):917-28; Zweifach A. FM1-43 reports plasma membrane phospholipid scrambling in T-lymphocytes. The Biochemical journal. 2000;349(Pt 1):255-60). Overexpression of annexin A6 and annexin A2 induced significantly more blebs than observed after overexpression of annexin A1 or GCaMP5G alone (Figures 16C and 16D).Furthermore, annexin-induced blebs were enriched in GCaMP5G, and annexin A6 induced the formation of significantly larger GCaMP5G-containing blebs compared with annexin A1, A2, or GCaMP5G alone (Figure 16A, green channel, and Figure 16D). Z-stack compilation demonstrated large annexin A6-induced GCaMP5G-positive blebs releasing from the injury site. In contrast, annexin A2 extruded small blebs from the repair cap. These data demonstrate that annexins not only form repair caps at sites of membrane disruption, but also that these caps act as a Ca receptor. 2+ These results suggest that binding proteins function as sites for the export of abundant extracellular components.

[0191] Annexin overexpression induces intracellular Ca2+ upregulation at the injury site 2+ Decrease in fluorescence. Ca after laser damage 2+ Time-lapse imaging of the indicator GCaMP5G detects extracellular Ca 2+ These results suggest that intracellular Ca2+ is reduced with the formation of extracellular blebs, and that these blebs release intracellular Ca2+. 2+ These results suggest that annexin-induced intracellular Ca accumulation may be beneficial. 2+ The decrease in fluorescence was visible for all three annexins A1, A2, and A6, but was most pronounced for annexins A2 and A6 (Figure 7A). Overexpression of annexin A6 significantly reduced intracellular Ca2+ levels during 240 s of imaging. 2+ This decrease was due to the Ca release from the internal GCaMP5G. 2+ Detailed analysis of the first 20 seconds after injury revealed that Ca2+ was upregulated by annexins A2 and A6, but not by annexin A1, when compared with GCaMP5G alone (Figure 7B). 2+ A significant decrease in GCaMP5G fluorescence was observed (Fig. 7C). Baseline GCaMP5G fluorescence intensity before injury was not significantly different between groups (Fig. 7D). Annexin A6 expression was associated with the intracellular Ca2+ signaling in the lesion. 2+ The decrease in fluorescence was confirmed using Fluo-4 AM (Figure 17). Thus, annexin expression is a signaling factor for Ca2+ accumulation in injured muscle fibers. 2+This induces a decrease in the Ca signal. 2+ The shedding of binding protein-filled blebs was enhanced, and annexin A6 was the most effective of the three annexins tested in maintaining this response.

[0192] Annexin-like Ca 2+ Overexpression of binding proteins increases intracellular Ca 2+ This may have unexpected effects on signal transduction and cell function. Therefore, the Ca concentration in isolated myofibers overexpressing annexin A6 may be significantly increased. 2+ Treatment and contractile properties were assessed compared to controls. Isolated myofibers expressing annexin A6 were subjected to ratiometric Ca measurements. 2+ Ca concentrations at stimulation frequencies of 40 Hz or 80 Hz were measured between Annexin A6 or control fibers loaded with the indicator dye Indo-1. 2+ No differences in cycling were observed (Figures 8A, 8B, and 8C). Unloaded cell shortening was also unaffected by the presence of overexpressed annexin A6 (Figures 8D, 8E, and 8F). These results demonstrated that annexin A6 overexpression is well tolerated by muscle fibers.

[0193] Annexin A6 Ca 2+Binding is required for repair cap formation and muscle fiber repair. Mutations of annexin A1 residue D171 and annexin A2 residue D161 have previously been shown to inhibit annexin membrane translocation in HEK cells (McNeil AK, Rescher U, Gerke V, and McNeil PL. Requirement for annexin A1 in plasma membrane repair. The Journal of biological chemistry. 2006;281(46):35202-7; Jost M, Thiel C, Weber K, and Gerke V. Mapping of three unique Ca(2+)-binding sites in human annexin II. Eur J Biochem. 1992;207(3):923-30). We asked whether these mutations inhibit translocation and formation of the high molecular weight annexin repair cap formed after muscle membrane injury in living muscle fibers. Using an alignment of annexin A1, A2, and A6 protein sequences, we identified type II Ca binding sites in all three annexin proteins. 2+ We identified conserved residues within the consensus sequence of the binding site (Fig. 2). 2+ To disrupt binding, site-directed mutagenesis was performed to identify the first type II Ca 2+ The aspartic acid residues in the binding site were converted to alanine residues (A1D171A, A2D161A, and A6D149A, respectively) (Fig. 9A). E233A was also generated in annexin A6, and the Ca2+ binding site in the second annexin repeat domain of annexin A6 was converted to alanine residues (A1D171A, A2D161A, and A6D149A, respectively). 2+This resulted in similar changes in the binding site. Each construct also contained turboGFP or tdTomato at the C-terminus. To assess the impact of homotypic annexin interactions during repair cap formation, we co-electroporated muscle fibers with wild-type + wild-type (A6 + A6) or wild-type + mutant (A6 + A6E233A) annexin combinations. Mutation of E233 in annexin A6 acted as a dominant-negative and significantly reduced the cap size of co-expressed wild-type annexin A6 protein (Figure 10A). Previous structural studies have shown that D149 in the first annexin repeat domain of annexin A6 regulates Ca2+. 2+ (Avila-Sakar AJ, Creutz CE, and Kretsinger RH. Crystal structure of bovine annexin VI in a calcium-bound state. Biochimica et biophysica acta. 1998;1387(1-2):103-16). Consistent with this, the D149A mutant of annexin A6 had little effect on cap size (Figure 9B, right panel). The diameter of the repaired cap ferret was calculated using a modified Hill equation based on the Ca 2+ The results were plotted as a function of concentration. Expression of mutant annexin A6E233A significantly reduced the cap diameter (DMAX) of co-expressed wild-type annexin A6 protein (Figure 10B). To assess the impact of heterotypic annexin interactions on repair cap formation, various combinations of wild-type and mutant annexin constructs were co-electroporated into muscle fibers. Co-expression of mutant annexin A6E233A significantly reduced the cap size of annexins A1, A2, and A6 compared to the A1+A6, A2+A6, and A6+A6 controls, respectively (Figure 10C). Together, these data indicated that annexin proteins interact in a homotypic and heterotypic manner that affects annexin repair complex assembly, and that mutant annexin A6 protein is sufficient to negatively regulate annexin complex assembly during repair.

[0194] Both annexin A1 and A2 Ca2+ Binding was also required for repair cap formation. The cap sizes of the A1D171A and A2D161A mutants were reduced compared to the wild-type annexin A1 and A2 controls, respectively. Expression of mutant annexins A1D171A and A2D161A significantly reduced the repair cap diameter (DMAX) of the co-expressed wild-type annexin proteins (Figure 9B, left and center panels). Despite the ability of mutant annexins A1D171A and A2D161A to significantly reduce the cap size of co-expressed wild-type annexins A1 and A2, respectively, A1D171A or A2D171A had minimal effect on the cap size of wild-type annexin A6 (Figure 9C). These data indicated that annexins A1 and A2 interact in a homotypic manner and affect self-cap assembly, while the localization of A6 to the repair cap is minimally regulated by the localization of annexins A1 and A2.

[0195] To determine the effect of dominant-negative annexin A6 on the assembly of annexins A1, A2, and A6 in the repair cap and membrane repair capacity, we similarly performed laser injury on isolated myofibers in the presence of FM4-64. Myofibers expressing annexin A6E233A-GFP showed increased FM4-64 fluorescence after laser injury compared to control myofibers expressing wild-type annexin A6-GFP (Figure 10D). These results indicated that a functional annexin repair complex is required for proper membrane repair and that annexin A6 is involved in regulating complex formation.

[0196] Annexin A6 binds Ca 2+ Annexin A6 protected against laser-induced muscle fiber damage in vitro in a Ca-dependent manner. Annexin A6 promoted the formation of a macromolecular repair cap complex, resulting in a large Ca2+ release at the site of membrane damage. 2+Because annexin A6 was most efficient in forming filled blebs, we evaluated whether overexpression of annexin A6 attenuates membrane damage in wild-type myofibers. Wild-type myofibers were electroporated with annexin A6-GFP or mock-electroporated, followed by laser injury in the presence of FM4-64 to mark the damaged area. In wild-type myofibers overexpressing annexin A6, FM4-64 dye uptake after laser-induced membrane damage was reduced compared to control myofibers (Figure 18A). These results indicate that intracellular overexpression of annexin A6 is effective in improving membrane repair and / or protecting against laser-induced membrane damage in isolated myofibers.

[0197] Because intracellular annexin A6 targets exposed phospholipids at sites of membrane damage and enhances membrane repair, we hypothesized that extracellular recombinant annexin A6 (rANXA6) would also localize to sites of damage and protect against membrane damage. Muscular dystrophies are progressive muscle-wasting diseases resulting from loss-of-function mutations in key cytoskeletal or membrane-associated proteins, often resulting in membrane weakening. To determine whether recombinant annexin A6 could protect against membrane damage in both healthy and dystrophic muscles, wild-type and dystrophic muscle fibers were isolated from a model representing Duchenne muscular dystrophy and incubated with recombinant annexin A6 or vehicle control. To visualize the extent of damage, laser injury was performed in the presence of FM4-64. Treatment with extracellular recombinant annexin A6 reduced the FM4-64 fluorescent area compared to muscle fibers treated with the vehicle control, indicating enhanced repair in both healthy and dystrophic muscle fibers (Figures 18B and 18C).

[0198] The protective effect of recombinant annexin A6 on external Ca 2+ To assess whether FM4-64 requires FM1-43 as a fluorescent marker of membrane damage, wild-type myofibers were pretreated with recombinant annexin A6 loaded with FM1-43, a fluorescent marker of membrane damage similar to FM4-64, and then treated with 1 mM Ca. 2+ or 0 mM Ca 2+ +EGTA(Ca 2+The time course of FM1-43 fluorescence accumulation (F / F0) in the lesion was measured with a solution containing 1 mM Ca. 2+ Compared with the presence of Ca 2+ These data suggest that extracellular recombinant annexin A6 protects against membrane damage and / or inhibits Ca 2+ We demonstrated that the repair was enhanced through extracellular exposure in a cytotoxic manner.

[0199] Recombinant annexin A6 protected against acute muscle injury in vivo. To determine the therapeutic potential of recombinant annexin A6 for protecting against muscle injury in vivo, recombinant annexin A6 was utilized as a tool compound. Recombinant annexin A6 or vehicle control was intramuscularly injected into the tibialis anterior (TA) muscle of wild-type mice 2 hours before toxin-induced injury. Mice were intraperitoneally injected with Evans blue dye, a key tracer that is excluded by intact, healthy muscle fibers but readily taken up by injured, permeabilized muscle fibers (Jennische E, and Hansson HA. Postischemic skeletal muscle injury: patterns of injury in relation to adequacy of reperfusion. Exp Mol Pathol. 1986;44(3):272-80). Three hours after cardiotoxin injury, muscles were assessed for Evans blue dye uptake (Figure 12A). Gross imaging showed that pretreatment with recombinant annexin A6 reduced cardiotoxin-induced muscle damage in vivo, as evidenced by reduced dye (blue) uptake compared to control (Figure 12B). Fluorescence imaging showed that pretreatment with recombinant annexin A6 reduced dye (red) uptake by 50% compared to control muscle (Figure 12C). Surface plot profiles showed reduced dye fluorescence in tibialis anterior muscles pretreated with intramuscular recombinant annexin A6 (Figure 12D).

[0200] Although intramuscular injection of annexin A6 was effective in reducing acute injury, this route of application is not optimal for treating large muscle groups, internal tissues, or chronic diseases. Therefore, we investigated the efficacy of recombinant annexin A6 administered systemically via retroorbital (RO) injection in protecting against acute muscle injury. Recombinant annexin A6 or vehicle was injected into the retroorbital space 2 hours before toxin-induced injury. Evans blue dye was simultaneously injected into mice. Three hours after cardiotoxin injury, muscles were assessed for Evans blue dye uptake (Figure 20A). Fluorescence imaging showed that pretreatment with recombinant annexin A6 reduced dye (green) uptake by 38% compared to vehicle controls (Figures 20B and 20C). Surface plot profiles demonstrate reduced dye fluorescence in muscles pretreated with systemic recombinant annexin A6 (Figure 20C). Furthermore, whole-tissue spectroscopy of injured gastrocnemius / soleus muscles revealed that pretreatment with rANXA6 reduced dye uptake by 58% compared with vehicle treatment (Figure ​(Figure20D). 20D). These results demonstrated that local and systemic administration of recombinant annexin A6 protects against acute muscle injury in vivo.

[0201] To determine whether recombinant annexin A6 administration enhances myocyte survival and / or recovery from injury, mice were administered recombinant annexin A6 or a BSA control at 1 mg / kg via retro-orbital systemic injection. Two hours after protein administration, cardiotoxin was injected into the tibialis anterior muscle to induce focal muscle injury. Muscles were harvested 7 days after injury and evaluated histologically for the injury area (Figure 20E). Pretreatment with recombinant annexin A6 reduced the percentage of damaged muscle marked by internal myonuclei (black dotted outline) 7 days after the insult (Figures 20F and 20G). These data demonstrate that systemic administration of recombinant annexin A6 protects against acute muscle injury and promotes myocyte survival and recovery from injury.

[0202] Recombinant annexin A6 protected against chronic muscle damage in vivo. We next evaluated the ability of systemically administered recombinant annexin A6 to protect against muscle damage in the Sgcg-deficient mouse model of limb-girdle muscular dystrophy type 2C (LGMD2C) (Hack AA, Cordier L, Shoturma DI, Lam MY, Sweeney HL, and McNally EM. Muscle degeneration without mechanical injury in sarcoglycan deficiency. Proceedings of the National Academy of Sciences of the United States of America. 1999;96(19):10723-8). Sgcg-deficient mice lack γ-sarcoglycan, an integral membrane component of the dystrophin glycoprotein complex required for membrane stability and function. Humans and mice lacking γ-sarcoglycan exhibit progressive muscle disease, decreased muscle function, and elevated serum creatine kinase (CK), a serum biomarker of muscle damage and membrane leakage. To determine whether recombinant annexin A6 protects in dystrophic muscle, Sgcg-deficient mice were systemically treated with recombinant annexin A6 or a BSA control for 48 hours (Figure 22A). Next, mice were subjected to 60 minutes of treadmill running to induce physiological muscle damage and CK release. Recombinant annexin A6 reduced the fold change in CK kinase activity after exercise compared to pretreatment, consistent with improved membrane resealing (Figures 22A and 22B). Recombinant annexin A6 was also injected into Sgcg-deficient mice over a 2-week period. 1 mg / kg of recombinant annexin A6 was injected once every 3 days for 14 days (Figure 22C). Administration of recombinant annexin A6 significantly reduced serum creatine kinase (CK) levels compared to control treatment on day 14 (Figure 22D). Annexin A6 significantly reduced intracellular Ca after injury. 2+It was more effective than annexin A2 in reducing CK levels (Figure 16D). Using the same 14-day dosing regimen in Sgcg-deficient mice, short-term systemic administration of recombinant annexin A6 significantly reduced serum CK levels compared with recombinant annexin A2 (Figure 22E). Gastrocnemius / soleus muscles from recombinant annexin-treated mice showed qualitatively less damage (Figure 22F). These data suggest that extracellular recombinant annexin A6 prevents muscle damage and enhances repair in vivo in chronically injured dystrophic mice.

[0203] Consideration Annexins promote the formation of calcium-filled blebs at sites of membrane damage. Plasma membrane instability is inherent in many forms of muscular dystrophy, and Ca 2+ It is believed to contribute to dysregulation of homeostasis and disease pathogenesis. Molkentin et al. found that transgenic overexpression of TRPC3 significantly increased Ca transport into muscle fibers. 2+ We have shown that SERCA1 increases cytosolic Ca influx and is sufficient to result in a dystrophic-like phenotype (Millay DP, Goonasekera SA, Sargent MA, Maillet M, Aronow BJ, and Molkentin JD. Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(45):19023-8). Similarly, transgenic overexpression of SERCA1 increases cytosolic Ca 2+ levels and Ca 2+(Goonasekera SA, Lam CK, Millay DP, Sargent MA, Hajjar RJ, Kranias EG, et al. Mitigation of muscular dystrophy in mice by SERCA overexpression in skeletal muscle. J Clin Invest. 2011;121(3):1044-52). As shown and described herein, increased expression of annexins in muscle fibers reduces damage-related Ca2+ accumulation within muscle fibers. 2+ The fluorescence decreased. 2+ This decrease in associated fluorescence occurred at the site of injury and correlated with the formation of extracellular blebs released from the annexin repair cap. 2+ Furthermore, overexpression of annexins A1, A2, and A6, respectively, increased the end-point Ca2+ levels in injured muscle fibers. 2+ Of the three annexins tested, overexpression of annexin A6 significantly reduced injury-related Ca accumulation. 2+ In HEK293 cells injured with streptolysin O (SLO), the presence of extracellular membrane-like blebs was associated with increased cell survival and cytosolic Ca accumulation. 2+This process correlated with a decrease in membrane repair levels, and this process was promoted by annexin A1 (Babiychuk EB, Monastyrskaya K, Potez S, and Draeger A. Blebbing confers resistance against cell lysis. Cell Death Differ. 2011;18(1):80-9). Davenport et al. showed that overexpression of annexin A1-GFP in injured Xenopus oocytes resulted in annexin A1-positive blebs originating from the injury site (Davenport NR, Sonnemann KJ, Eliceiri KW, and Bement WM. Membrane dynamics during cellular wound repair. Mol Biol Cell. 2016;27(14):2272-85). However, the effects of annexin A2 or A6 overexpression were not evaluated in either study. Combined, these data suggest that blebbing as a mechanism of membrane repair is conserved across species and tissue types and is promoted by the presence of annexin proteins.

[0204] Without being bound by theory, annexin A6 acts as a cytosolic Ca 2+ and promotes protein export into extracellular blebs, the formation of which is thought to be further induced by annexin A1 and annexin A2. In artificial membrane patches, the presence of annexin A1 or annexin A2 induced bleb formation at the site of membrane defects (Boye TL, Jeppesen JC, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep. 2018;8(1):10309). In contrast, the presence of annexin A6 prevented Ca transport into large folds of the artificial membrane. 2+(Boye TL, Jeppesen JC, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep. 2018;8(1):10309). The difference in annexin A6 in inducing blebs in live myofibers or folds in artificial membranes may reflect the presence of endogenously expressed annexins A1 and A2 in isolated myofibers compared to exposure to a single recombinant annexin protein in artificial membrane studies. Without being bound by theory, multiple annexins within the macromolecular repair complex may be involved in wound closure, resection of damaged membranes, and Ca2+ uptake at the injury site. 2+ It is contemplated that the vasoconstrictor is actively involved in blebbing, which acts to remove large membrane lesions, promoting the reduction of vasoconstriction.

[0205] Annexin A6 protects against muscle membrane damage and enhances membrane repair. Annexin proteins, including annexins A1, A2, and A6, have been shown to localize to sites of membrane damage and promote membrane repair cap and bleb formation. Mutation of annexin A6 prevented repair cap formation, reduced repair capacity, and increased dye uptake. Pretreatment with recombinant annexin A6, on the other hand, reduced dye uptake after laser-induced and toxin-induced muscle injury in vivo. As a therapeutic tool, enhancing the cells' ability to repair and / or reduce damage by stabilizing the cell membrane is potentially beneficial, potentially leading to improved cell survival.Previous studies have shown that annexin A6 is upregulated in muscles from models of chronic muscular dystrophy (Demonbreun AR, Allen MV, Warner JL, Barefield DY, Krishnan S, Swanson KE, et al. Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption. Am J Pathol. 2016;186(6):1610-22, Swaggart KA, Demonbreun AR, Vo AH, Swanson KE, Kim EY, Fahrenbach JP, et al. Annexin A6 Modifies Muscular Dystrophy by Mediating Sarcolemmal Repair. Proceedings of the National Academy of Sciences of the United States of America. 2014;111(16):6004-9, Demonbreun AR, Rossi AE, Alvarez MG, Swanson KE, Deveaux HK, Earley JU, et al. Dysferlin and myoferlin regulate transverse tubule formation and glycerol sensitivity. Am J Pathol. 2014;184(1):248-59). Proteomic profiling of mdx mouse muscle showed that annexins A1 and A2 are abundant in mdx muscle membranes, consistent with a role for annexins in the membranes of injured muscle cells (Murphy S, Zweyer M, Henry M, Meleady P, Mundegar RR, Swandulla D, et al. Proteomic analysis of the sarcolemma-enriched fraction from dystrophic mdx-4cv skeletal muscle. J Proteomics. 2018).Annexins bind to membrane phospholipids, including phosphatidylserine, which becomes exposed during membrane disruption. The rearrangement of phosphatidylserine after injury provides optimal binding targets for extracellular annexins, promoting membrane folding, blebbing, and rolling at sites of membrane damage and defects (Boye TL, Jeppesen JC, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep. 2018;8(1):10309). Annexin upregulation is thought to be a compensatory mechanism facilitating the resection of defective membranes in chronically injured fibers.

[0206] Further studies of myocardial injury further suggest a role for annexin proteins in regulating repair responses. Administration of recombinant annexin A1 or the N-terminal annexin A1 peptide (AC2-26) induced a cardioprotective response in a rat model of myocardial ischemia-reperfusion-induced injury (La M, D'Amico M, Bandiera S, Di Filippo C, Oliani SM, Gavins FN, et al. Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action. FASEB J. 2001;15(12):2247-56). Meng et al. demonstrated that downregulation of annexin A3 protects the heart and reduces myocardial infarct size in rats through activation of AKT signaling (Meng H, Zhang Y, An ST, and Chen Y. Annexin A3 gene silencing promotes myocardial cell repair through activation of the PI3K / Akt signaling pathway in rats with acute myocardial infarction. J Cell Physiol. 2019;234(7):10535-46). In addition to membrane reorganization, annexins function as scaffolds that regulate multiple downstream intracellular signaling cascades that are important for orchestrating repair from injury. Both intracellular and extracellular functions of annexin proteins must be considered when evaluating their therapeutic potential.

[0207] The research presented herein demonstrates that human recombinant annexin A6 protein is a suitable biologic for protecting against acute muscle injury. It is shown herein that human recombinant annexin A6 can reseal damaged membranes in a mouse model, confirming the functional activity of the human recombinant protein in a mouse preclinical model. Human and mouse annexin A6 proteins are 94.35% identical at the amino acid level, with increasing amino acid conservation between humans and rats (94.65%), dogs (95.54%), and monkeys (98.96%). This high similarity is consistent with the human recombinant protein having efficacy in mouse models (Figure 21). Current research is limited by the available recombinant protein, and further studies are needed to determine whether recombinant annexin A6 can promote membrane repair and long-term reduce susceptibility to injury in chronic models of muscle disease and in tissues beyond skeletal muscle. Annexin A6 was first identified as a genetic modifier of muscle membrane leakage in a mouse model of muscular dystrophy. Annexin A6 was subsequently shown to modify the injury response in healthy mouse muscle (Swaggart KA, Demonbreun AR, Vo AH, Swanson KE, Kim EY, Fahrenbach JP, et al. Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair. Proceedings of the National Academy of Sciences of the United States of America. 2014;111(16):6004-9). Herein, we contemplate that enhanced repair through administration of recombinant annexin A6 protein will provide similar protection in dystrophic muscle, but require long-term intermittent dosing. Future studies will require optimizing mammalian recombinant annexin A6 protein production to generate sufficient amounts of purified protein.

[0208] A combinatorial approach to improve membrane repair. Herein, we describe and demonstrate recombinant annexin A6 and its ability to protect normal and dystrophic muscle from laser-induced membrane damage. Furthermore, both intramuscular and systemic administration of recombinant annexin A6 protected against toxin-induced muscle membrane damage in vivo. Glucocorticoid administration was previously found to increase annexin expression in muscle, which correlated with enhanced muscle repair in multiple mouse models of muscular dystrophy, including mdx (DMD), dysferlin-deficient (limb-girdle muscular dystrophy 2B), and gamma-sarcoglycan-deficient (limb-girdle muscular dystrophy 2C) mice. (Quattrocelli M, Barefield DY, Warner JL, Vo AH, Hadhazy M, Earley JU, et al. Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy. J Clin Invest. 2017;127(6):2418-32; Quattrocelli M, Salamone IM, Page PG, Warner JL, Demonbreun AR, and McNally EM. Intermittent glucocorticoid dosing improves muscle repair and function in mice with Limb-Girdle Muscular Dystrophy. Am J Pathol. 2017;187(11):2520-35). Glucocorticoid treatment also increased expression of the Trim72 gene, which encodes the repair protein Mitsugumin 53 (known as MG53), which localizes to sites of membrane damage and is considered a "molecular Band-Aid" for improving cellular wound healing. Similar to annexins, MG53 is upregulated in chronic muscle injury and enhances repair of dystrophic muscle and other tissues, such as the heart, lung, and kidney (Waddell LB, Lemckert FA, Zheng XF, Tran J, Evesson FJ, Hawkes JM, et al.).Dysferlin,annexin A1,and mitsugumin 53 are upregulated in muscular dystrophy and localize to longitudinal tubules of the T-system with stretch.J Neuropathol Exp Neurol.2011;70(4):302-13、Duann P,Li H,Lin P,Tan T,Wang Z,Chen K,et al.MG53-mediated cell membrane repair protects against acute kidney injury.Science translational medicine.2015;7(279):279ra36、He B,Tang RH,Weisleder N,Xiao B,Yuan Z,Cai C,et al.Enhancing muscle membrane repair by gene delivery of MG53 ameliorates muscular dystrophy and heart failure in delta-Sarcoglycan-deficient hamsters.Molecular therapy:the journal of the American Society of Gene Therapy.2012;20(4):727-35、Jia Y,Chen K,Lin P,Lieber G,Nishi M,Yan R,et al.Treatment of acute lung injury by targeting MG53-mediated cell membrane repair.Nat Commun.2014;5:4387、Liu J,Zhu H,Zheng Y,Xu Z,Li L,Tan T,et al.Cardioprotection of recombinant human MG53 protein in a porcine model of ischemia and reperfusion injury.Journal of molecular and cellular cardiology.2015;80:10-9, Weisleder N, Takizawa N, Lin P, Wang X, Cao C, Zhang Y, et al. Recombinant MG53 protein modulates therapeutic cell membrane repair in treatment of muscular dystrophy. Science translational medicine. 2012;4(139):139ra85). MG53 is a component of the annexin-mediated repair complex and is localized adjacent to the annexin repair cap (Demonbreun AR, Quattrocelli M, Barefield DY, Allen MV, Swanson KE, and McNally EM. An actin-dependent annexin complex mediates plasma membrane repair in muscle. Journal of cell biology. 2016;213(6):705-18).

[0209] Example 4 Further experiments were designed and performed to evaluate the role of endogenous annexin A6, annexin A6 in the heart, and annexin A6 in additional models of muscular dystrophy.

[0210] method Animals. Genetically encoded annexin A6 GFP mice were generated and backcrossed onto the 129T2 / SvEmsJ background. Dysferlin-deficient mice on the 129T2 / SvEmsJ background were generated previously (Demonbreun et al., HMG 2011). Sgcg-deficient mice were generated as described (Hack et al., PNAS 1999). Mice were housed in a specific pathogen-free facility with a 12-hour light / dark cycle and fed ad libitum in accordance with Northwestern University's Animal Care and Use Committee regulations. 129T2 / SvEmsJ (129T2) mice were originally purchased from Jackson Laboratory (Ben Harbor, ME, stock number 002065). Male and female mice aged 2–3 months were used for all experiments. All animal experiments were approved in accordance with Northwestern University's Animal Care and Use Committee regulations.

[0211] Schematic representation of the genome structure. The Annexin A6 genome structure was visualized using the UCSC genome browser.

[0212] Muscle fiber isolation. Flexor digitorum brevis (FDB) fibers were isolated as previously described (Demonbreun and McNally, 2015; Demonbreun et al., 2016b; DiFranco et al., 2009). Briefly, fibers were dissociated in 0.2% BSA + collagenase type II (catalog no. 17101, Thermo Fisher Scientific, Waltham, MA) for 60 minutes at 37°C in 10% CO2. Fibers were then transferred to Ringer's solution and placed in a MatTek confocal microscope dish (catalog no. P35G-1.5-14-C, MatTek, Ashland, MA).

[0213] Immunofluorescence microscopy. Mice were systemically injected with recombinant annexin A6 bearing a C-terminal HIS tag (R&D). Muscles were harvested and flash-frozen. Cryo-embedded muscle sections (10 μm thick) from the center were collected on a cryostat (chamber: -20°C, sample: -15°C, catalog no. CM1950, Leica, Wetzlar, Germany) for immunostaining. Tissues were fixed with 4% paraformaldehyde on ice for 10 minutes. Blocking and permeabilization were performed for 60 minutes using 0.1% Triton (catalog no. X-100, Sigma-Aldrich), 10% fetal bovine serum, and PBS. For laminin detection, anti-laminin antibody was used at a dilution of 1:100, and for HIS detection, anti-HIS antibody was used at a dilution of 1:100 overnight at 4°C. Hoechst-labeled nuclei. Sections were rinsed in PBS, incubated with secondary antibodies for 1 h, rinsed in PBS, and mounted. Imaging was performed using a Zeiss Axio Observer A1 microscope (Zeiss, Oberkochen, Germany).

[0214] Cardiomyocyte isolation. Twenty minutes before sacrifice, mice were intraperitoneally treated with 50 U of heparin. Mice were anesthetized under 5% vaporized isoflurane mixed with 100% oxygen. A thoracotomy was performed, and the heart and lungs were rapidly removed and immersed in ice-cold calcium-free Tyrode's solution (143 mM NaCl, 2.5 mM KCl, 16 mM MgCl, 11 mM glucose, 25 mM NaHCO, pH adjusted to 7.4). The ascending aorta was dissected free from surrounding tissue, cannulated with an animal feeding needle (7900, Cadence Science, Stanton, VA), and secured with 6-0 silk suture. The heart was first perfused with 1 ml of ice-cold calcium-free Tyrode's solution and then transferred to a Langendorff apparatus (Radnoti, Covina, CA). The heart was perfused with calcium-free Tyrode's solution at 37°C for 1–2 min using constant pressure (vertical distance between the buffer reservoir and the cannula tip: 65 cm), followed by 5.5 min with digestion solution (0.15% collagenase type 2 [Worthington Biochemical, Lakewood, NJ], 0.1% 2,3-butanedione monoxime, 0.1% glucose, 100 U / ml penicillin / streptomycin, 112 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 40 μM CaCl2, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 30 μM phenol, 21.4 mM NaHCO3, 10 mM HEPES, and 30 mM taurine; pH adjusted to 7.4). The heart was removed from the cannula, triturated with a transfer pipette, and filtered through a 100 μm cell strainer. Cardiomyocytes were allowed to pellet by gravity for 7 minutes. The digestion medium was then aspirated and washed with stop buffer (formulated identically to the digestion solution, except without collagenase and containing 1% bovine serum albumin). The cells were again gravity-pelleted and washed with stop buffer without bovine serum albumin. Cardiomyocytes were made calcium-tolerant by adding Tyrode's buffer containing 0.3 mM CaCl2 dropwise. Cell culture dishes were coated with 20 μg / ml laminin (23017-015, Gibco, Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature.After aspirating the laminin solution, cardiomyocytes were seeded for 1 h to allow cells to adhere before the experiment.

[0215] Multiphoton laser damage and imaging. Isolated fibers were subjected to laser-induced damage at room temperature using a Nikon A1R-MP multiphoton microscope. Imaging was performed using a 25x 1.1 NA objective, guided by NIS-Elements AR imaging software. Green fluorescent protein (GFP) and FM4-64 were excited using a 920 nm laser, and emission wavelengths of 575 nm and 629 nm were collected, respectively. To induce laser damage in isolated myofibers, a diffraction-limited spot (approximately 410 nm in diameter) was created on the lateral membrane of the myofiber using a 920 nm laser for 1 second at 10-15% laser power. Time-lapse images were collected as follows: one before injury, one at the time of injury, and then every 8 seconds for 80 seconds (10 images), and every 30 seconds for 5 minutes (10 images). At the end of the time-lapse series, z-stack images were collected at 250 nm intervals from the injury site of the myofiber, as indicated by the NIS-Elements AR imaging software. Fluorescence intensity and cap area were measured using Fiji (NIH).

[0216] For recombinant protein studies, muscle fibers were isolated from mice as described above. Muscle fibers were cultured in 1 mM Ca. 2+ Myofibers were incubated in 10–40 μg / ml recombinant annexin A6 (5186-A6-050, R&D Systems) in Ringer's or BSA control. Cap size was assessed from images acquired with FIJI. FM4-64 (2.5 μM) was added to myofibers immediately before imaging. Images were acquired and quantified as described above. FM4-64 fluorescence at the endpoint was measured using FIJI.

[0217] For prednisone studies, prednisone (catalog P6254) was resuspended at 5 mg / ml in DMSO (catalog D2650, Sigma-Aldrich). Dosing was based on pretreatment weight (1 mg / kg body weight) in 30 μl total PBS. Mice were injected at 7:00 AM the day before imaging. On the day of injection, stock solutions stored at -20°C were diluted into sterile Eppendorf tubes containing sterile PBS (catalog 14190, Life Technologies). A sterile BD Micro-Fine IV Insulin Syringe (catalog 14-829-1A, Fisher Scientific) was used to inject into the IP cavity of unsedated animals. Muscle fibers were harvested 24 hours after injection and imaged the same day.

[0218] Statistical analysis. Statistical analysis was performed using Prism (Graphpad, La Jolla, CA). Comparisons relied on ANOVA (one-way ANOVA in the case of one variable). Otherwise, unpaired two-tailed t-tests were performed. P values ​​of 0.05 or less were considered significant. Data were presented as single values ​​where appropriate. Error bars represent the standard error of the mean (SEM).

[0219] The experimental results are shown in Figures 24 to 28.

[0220] Glucocorticoid steroids regulate the localization of endogenous annexin A6 (GFP) and protect against injury. Using gene editing, we introduced a GFP tag into the carboxyl terminus of the Anxa6 gene locus in mice, and isolated Anxa6. em1(GFP) We have generated mice that offer the advantage of examining genomically encoded annexin A6 under the control of the Anxa6 promoter and regulatory sequences. em1(GFP)Mice express annexin A6 protein under the control of the Anxa6 gene, so expression levels are normally lower than those of plasmid-encoded annexin A6-GFP. This animal model allows for real-time examination of the expression of various annexin A6 proteins. Glucocorticoid steroids, including prednisone, have been shown to upregulate the expression of annexins A1 and A6. Furthermore, prednisone administration protected against membrane damage and reduced cap formation of plasmid-overexpressed annexin GFP after laser injury. To determine whether prednisone modulated the capping dynamics of genomically encoded annexin A6-GFP, we investigated the effects of Anxa6 on the expression of various annexin A6 proteins. em1(GFP) Mice were intraperitoneally injected with 1 mg / kg prednisone or control DMSO. 24 hours after injection, muscle fibers were isolated and subjected to laser injury. Annexin A6 GFP cap size was reduced in prednisone-treated muscle fibers compared to controls (Figure 24), demonstrating the responsiveness of annexin A6 GFP protein to steroid administration. This demonstrates the potential for external agents to modulate endogenous annexin A6 expression to elicit protection against injury.

[0221] Recombinant annexin A6 promotes skeletal muscle repair. Recombinant annexin A6 was shown to protect against laser-induced injury by reducing FM dye uptake in healthy and dystrophic muscle fibers. To further elucidate the mechanism by which recombinant annexin A6 confers protection, we investigated the effects of annexin A6 on skeletal muscle repair. em1(GFP) Myofibers were pretreated with recombinant annexin A6 protein or control. Subsequently, the myofibers were incubated in FM4-64 dye and subjected to laser injury. Compared to control myofibers, myofibers pretreated with recombinant annexin A6 exhibited smaller annexin A6GFP repair caps and concomitantly reduced FM dye uptake (Figure 25). These data indicate that recombinant annexin A6 is involved in regulating endogenous repair complexes, enhancing membrane protection and repair and reducing membrane leakage.

[0222] Annexin A6GFP is expressed in the heart and forms a repair cap in damaged cardiomyocytes. Annexin A6 was identified as a modulator of membrane leakage in the heart, but there was no previous method to assess cardiomyocyte repair. When endogenous annexin A6GFP was expressed in the heart, this novel Anxa6 em1(GFP) It was hypothesized that the mouse model could be used as a tool to assess endogenous cardiomyocyte membrane repair. To determine whether endogenous annexin A6GFP plays a role in membrane repair in tissues beyond skeletal muscle, we analyzed Anxa6. em1(GFP) Cardiomyocytes were isolated and subjected to laser injury. Endogenous annexin A6 GFP localized in a sarcomeric pattern in live, isolated cardiomyocytes, similar to skeletal muscle fibers (Figure 26). Within seconds of laser-induced injury, annexin A6 GFP localized to the membrane lesion and organized into a repair cap in cardiomyocytes (Figure 26, white arrow). A magnified image of the area enclosed by the white dotted line depicting the bright annexin A6 GFP repair cap is shown in the image on the right (Figure 26). Time-lapse images showing the progression of annexin A6 GFP localization to the repair cap (arrow) in isolated cardiomyocytes are shown up to 50 seconds after injury (Figure 26). These data demonstrate that endogenous annexin A6 GFP localizes to the site of membrane injury and forms a repair cap at the membrane lesion in live, adult cardiomyocytes, suggesting a broad role for annexin A6 in membrane repair.

[0223] Annexin A6 enhances repair in a model of limb-girdle muscular dystrophy type 2B (LGMD2B). Dysferlin is a Ca2+-binding membrane-associated protein involved in membrane repair. Loss-of-function mutations in dysferlin reduce membrane repair capacity, causing LGMD2B. It was hypothesized that agents that enhance repair or reduce susceptibility to injury may offer therapeutic benefit in treating LGMD2B. To test this hypothesis, mice lacking dysferlin on a 129 background (Dysf129) were electroporated with an annexin A6 plasmid and subjected to laser injury in the presence of FM4-64. Myofibers overexpressing annexin A6 showed significantly reduced FM4-64 dye uptake after injury (Figure 27A). Because overexpression of the plasmid protected dysferlin-deficient myofibers from injury, we next tested the efficacy of recombinant annexin A6. To determine whether recombinant annexin A6 could protect against membrane damage in dystrophic dysferlin-deficient muscles, myofibers from Dysf129 mice were isolated and incubated with recombinant annexin A6 or vehicle control. To visualize the extent of damage, laser injury was performed in the presence of FM4-64. Treatment with extracellular recombinant annexin A6 reduced the FM4-64 fluorescent area compared to myofibers treated with vehicle control (Figure 27B). These data indicate that annexin A6 enhances repair and protects against damage in dystrophic LGMD2B myofibers.

[0224] Recombinant annexin A6 localizes to the sarcolemma in a limb-girdle muscular dystrophy 2C (LGMD2C) model. Because recombinant annexin A6 protects against membrane damage, the localization ability of systemically administered recombinant annexin A6 was evaluated in an Sgcg-deficient mouse model of LGMD2C compared with wild-type controls. Sgcg-deficient mice lack γ-sarcoglycan, an integral membrane component of the dystrophin glycoprotein complex required for membrane stability and function. Recombinant annexin A6 localized to the sarcolemma of chronically injured Sgcg-deficient mice is visualized by a strong anti-HIS immunofluorescence signal that colocalizes with anti-laminin membrane staining (Figure 28). In comparison, minimal sarcolemmal anti-HIS fluorescence was present in intact wild-type sarcolemma (Figure 28). These data demonstrated that recombinant annexin A6 localizes to the membrane of chronically injured muscles.

[0225] Example 5 This example details the production of recombinant annexin A6 protein (rANXA6) expressed from a plasmid in both mammalian and prokaryotic cells.

[0226] Production of rANXA6 protein in mammalian cells Production of rANXA6 protein was carried out in a total volume of 4 liters using two mammalian cell lines (Expi293F™ Cells and FreeStyle™ CHO-S Cells, ThermoFisher Scientific). Estimated yields, as measured by Coomassie staining (Figure 29), were 15 mg / L from Expi293F™ cells and 2.3 mg / L from FreeStyle™ CHO-S cells. Recombinant protein was also assessed by immunoblot analysis to detect annexin A6 and an anti-HIS antibody to detect the C-terminal HIS tag found on recombinantly produced rANXA6 protein (Figure 29).

[0227] The buffers and immobilized metal affinity chromatography (IMAC) protocol are shown in Table 1 below. [Table 1]

[0228] Purification of annexin 6 (ANXA6) from Expi293F™ cells. Protein lysate was loaded onto an IMAC column and eluted with 500 mM imidazole. The yield at this stage was determined to be approximately 270 mg. Relevant fractions were pooled and loaded onto an ion exchange Q (IExQ) column, and the indicated fractions were pooled. The yield at this stage was approximately 208 mg. These pools were loaded onto an S200 26 / 60 column in 1X PBS. The final yield was approximately 77 mg. The purity of the recombinant protein was determined to be greater than 95%.

[0229] Absolute size exclusion chromatography (aSEC) showed that the protein was in a monomeric state, and mass spectrometry (MS) showed multiple modifications.

[0230] Purification of annexin 6 (ANXA6) from FreeStyle™ CHO-S cells. Protein lysate was loaded onto an IMAC column and eluted with 500 mM imidazole. Yield at this point was approximately 78 mg. Relevant fractions were pooled and loaded onto an ion exchange Q (IExQ) column, and the indicated fractions were combined into two separate pools. Yield at this stage was approximately 24 mg (approximately 12 mg for each pool). These pools were run on an S200 26 / 60 column (SEC was performed on Pool 1 and Pool 2). Final yields were determined to be Pool 1 = 2.25 mg and Pool 2 = 1.9 mg.

[0231] Absolute size exclusion chromatography (aSEC) and MS showed no differences between Pool 1 and Pool 2, nor were any differences detected between the recombinant proteins expressed in Expi293F™ and FreeStyle™ CHO-S cells.

[0232] Production of rANXA6 protein in prokaryotic cells rANXA6 protein was also purified from prokaryotic cells. Production of rANXA6 protein was carried out using a 4-liter low-endotoxin purification method in E. coli cells (Rosetta (DE3) competent cells) in TB medium. Cells were grown at 30°C for 4 and 18 hours. Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer was used at 0.4 mM. The compositions of the various buffers used are listed in Table 2. The total yield obtained using this method was 308 mg (approximately 2.5 mg / mL). [Table 2]

[0233] The recombinant proteins were analyzed by Coomassie staining and immunoblot analysis, as shown in FIG.

[0234] Purification of annexin 6 (ANXA6) from E. coli cells. Protein lysate was loaded onto an IMAC column and eluted with 500 mM imidazole. The yield at this stage was determined to be approximately 546 mg. Relevant fractions were pooled and loaded onto an ion exchange Q (IExQ) column, and the indicated fractions were pooled. The yield at this stage was approximately 408 mg. These pools were loaded onto an S200 26 / 60 column in 1X PBS. After three runs, the final yield was approximately 306 mg. Final endotoxin levels were approximately 4 EU / mg / 10.5 EU / mL. The purity of the recombinant protein was determined to be greater than 95%.

[0235] Absolute size exclusion chromatography (aSEC) showed that the protein was in a monomeric state, and mass spectrometry (MS) showed multiple modifications.

[0236] Example 6 This example details experiments comparing the function of rANXA6 produced in mammalian cells with that produced in prokaryotic cells. These experiments were performed to test whether the activity of an annexin protein (in this case, rANXA6) differs based on whether it is produced in mammalian or prokaryotic cells. The predicted post-translational modification signature of annexin A6 was determined using PTMcode (http: / / ptmcode.embl.de) and is presented in Table 3. The various forms of post-translational modifications (PTMs) (e.g., acetylation, nitrosylation, phosphorylation) identified by mass spectrometry (MS) are presented in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0237] As shown in Table 3, the post-translational modification signature of annexin A6 differs depending on whether the protein is expressed in prokaryotic or mammalian cells. Therefore, it is expected that the altered post-translational modification signature will result in changes in activity between annexin A6 protein produced in prokaryotic cells and annexin A6 protein produced in mammalian cells. Unexpectedly, however, we found that annexin A6 protein produced in prokaryotic cells functions similarly to the protein produced in mammalian cells in the muscle fiber injury experiments described below.

[0238] Myofiber isolation, laser injury, and dye measurement. For recombinant myofiber studies, myofibers were isolated from wild-type mice. They were incubated in Ringer's medium containing 13 μg / ml or 130 μg / ml of human recombinant annexin, E. coli, or HEK cells. Bovine serum albumin (BSA) was used as a negative control. FM4-64 (2.5 μM) was added to myofibers immediately before imaging. Fibers were subjected to laser-induced injury at room temperature using a Nikon A1R-MP multiphoton microscope. Imaging was performed using a 25x 1.1 NA objective guided by NIS-Elements AR imaging software. FM4-64 was excited using a 920 nm laser, and emission wavelengths of 575 nm and 629 nm were collected, respectively. To induce laser injury in isolated myofibers, a 920 nm wavelength laser was used for 1 second at 10-15% laser power to create a diffraction-limited spot (approximately 410 nm in diameter) on the lateral membrane of the myofiber. FM4-64 area was measured using FIJI from a single slice near the center of the Z-stack. Quality control of myofibers was based on a number of characteristics, including the use of adherent myofibers with intact sarcomere structure. Myofibers appeared free of tears or ruptures induced during the isolation protocol. The region of the myofiber selected for injury was linear and not located at the nucleus or neuromuscular junction.

[0239] Statistical analysis was performed using Prism (Graphpad, La Jolla, CA). Comparisons relied on ANOVA (one-way ANOVA in the case of one variable). A P value of 0.05 or less was considered significant. Error bars represent the standard error of the mean (SEM). The results are shown in Figure 31.

[0240] These experiments demonstrated that myofibers injured in the presence of extracellular recombinant annexin A6 (rANXA6) produced in E. coli or mammalian cells had similar beneficial effects on enhancing membrane repair and protecting against injury. This was demonstrated across multiple doses (13 mg / ml and 130 mg / ml), measured as reduced FM4-64 dye uptake after laser injury compared to myofibers treated with a BSA control.

[0241] Example 7 This example was designed to examine the necessity of the amino acid sequence VAAEIL (exon 21 of annexin A6) (SEQ ID NO: 47) in promoting the localization and organization of the annexin repair cap at sites of membrane damage.

[0242] method Animals. Wild-type mice on a 129T2 / SvEmsJ background were bred and housed in a specific pathogen-free facility with a 12-h light / dark cycle and fed ad libitum in accordance with Northwestern University's Animal Care and Use Committee regulations. 129T2 / SvEmsJ (129T2) mice were purchased from Jackson Laboratory (Ben Harbor, ME, stock number 002065). All wild-type mice used in experiments were male and female, 2–3 months of age.

[0243] Plasmids. A plasmid encoding annexin A6 with a carboxyl-terminal turboGFP tag was obtained from Origene (Rockville, MD). Subcloning of annexin A6 to replace the GFP tag with tdTomato (Addgene) was performed by Mutagenix (Suwanee, GA). Site-directed mutagenesis was performed by Mutagenix on annexin A6-GFP to generate a construct lacking the VAAEIL (SEQ ID NO: 47) sequence. Plasmid DNA was isolated using Qiagen's Endo-Free Maxi Prep Kit (Qiagen #12362).

[0244] Electroporation, muscle fiber isolation, laser injury, capping, and vesicle measurement. Flexor digitorum brevis (FDB) fibers were transfected with endotoxin-free plasmid DNA by in vivo electroporation. The method was previously described in Demonbreun et al. (JoVE 2015). Briefly, 10 μl of hyaluronidase (8 units) (H4272, Sigma, St. Louis, MO) was injected into the hind paw footpad. Two hours after injection, up to 20 μl of 2 μg / μl endotoxin-free plasmid was injected into the footpad. Electroporation was performed by applying 20 pulses of 20 ms duration at 1 Hz and 100 V / cm. To avoid examination of damaged muscles and allow sufficient time for plasmid expression, animals were allowed to recover for a minimum of 7 days, but not more than 10 days.

[0245] The FDB muscle was removed, and individual myofibers were isolated and imaged (Demonbreun et al., JoVE, 2015). Fibers were dissected and laser-injured as described (Demonbreun et al., JoVE 2015; Swaggart et al., PNAS 2014). Briefly, fibers were dissociated in 0.2% BSA + collagenase type II (catalog #17101, Life Technologies, Grand Island, NY) for up to 90 minutes with intermittent trituration at 37°C in 10% CO2. Fibers were then transferred to Ringer's solution and placed in a MatTek confocal microscope dish (catalog #P35G-1.5-14-C, MatTek, Ashland, MA). After 1 hour, fibers were allowed to adhere, and the Ringer's solution was replaced with 1 ml of fresh Ringer's solution. Imaging and ablation were performed on a Nikon A1R laser scanning confocal equipped with a GaSP detector through a 60x Apo lambda 1.4NA objective driven by Nikon Elements AR software. 2A single pixel set as ) was ablated using a 405 nm laser at 100% power for up to 5 seconds. Z-stack projections were acquired after the last time-lapse frame, approximately 4 minutes after injury, from successive acquisitions with a step size of 0.125 μM between slices. Z-stack renderings were constructed in FIJI. Fibers expressing similar levels of tagged protein were compared.

[0246] result Next, we investigated whether the presence of the VAAEIL (SEQ ID NO: 47) sequence in annexin A6 affects its localization during muscle membrane injury. Muscle fibers were electroporated with full-length annexin A6-tdTomato and A6-GFP (SEQ ID NO: 47) lacking the VAAEIL sequence, followed by laser injury. Annexin A6 isoforms with and without the VAAEIL (SEQ ID NO: 47) sequence localized to muscle membrane injury sites, forming a repair cap (Figure 32). Annexin A6 lacking the VAAEIL (SEQ ID NO: 47) partially colocalized with full-length annexin A6, forming a flatter repair cap at the membrane lesion. These data indicate that multiple annexin A6 isoforms, regardless of the presence or absence of the amino acid VAAEIL, are involved in muscle injury responses. Additional References Akerboom,J.,TWChen,TJWardill,L.Tian,JSMarvin,S.Mutlu,NCCalderon,F.Esposti,BGBorghuis,XRSun,A.Gordus,MBOrger,R.Portugues,F.Engert,JJMacklin,A.Filos a,A.Aggarwal,RAKerr,R.Takagi,S.Kracun,E.Shigetomi,BSKhakh,H.Baier,L.Lagnado,SSWang,CIBargmann,BEKimmel,V.Jayaraman,K.Svoboda,DSKim,ERSchreiter,and LLLooger.2012.Optimization of a GCaMP calcium indicator for neural activity imaging.J Neurosci.32:13819-13840. Avila-Sakar,AJ,CECreutz,and RHKretsinger.1998.Crystal structure of bovine annexin VI in a calcium-bound state.Biochimica et biophysica acta.1387:103-116. Babbin,BA,MGLaukoetter,P.Nava,S.Koch,WYLee,CTCapaldo,E.Peatman,EASeverson,RJFlower,M.Perretti,CAParkos,and A.Nusrat.2008.Annexin A1 regulates intestinal mucosal injury, inflammation, and repair.J Immunol.181:5035-5044. Bansal,D.,K.Miyake,S.S.Vogel,S.Groh,C.C.Chen,R.Williamson,P.L.McNeil,and K.P.Campbell.2003.Defective membrane repair in dysferlin-deficient muscular dystrophy.Nature.423:168-172. Bashir,R.,S.Britton,T.Strachan,S.Keers,E.Vafiadaki,M.Lako,I.Richard,S.Marchand,N.Bourg,Z.Argov,M.Sadeh,I.Mahjneh,G.Marconi,M.R.Passos-Bueno,S.Moreira Ede,M.Zatz,J.S.Beckmann,and K.Bushby.1998.A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.Nat Genet.20:37-42. Bement,W.M.,C.A.Mandato,and M.N.Kirsch.1999.Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes.Curr Biol.9:579-587. Benz,J.,A.Bergner,A.Hofmann,P.Demange,P.Gottig,S.Liemann,R.Huber,and D.Voges.1996.The structure of recombinant human annexin VI in crystals and membrane-bound.J Mol Biol.260:638-643. Bi,G.Q.,J.M.Alderton,and R.A.Steinhardt.1995.Calcium-regulated exocytosis is required for cell membrane resealing.The Journal of cell biology.131:1747-1758. Bizzarro,V.,A.Petrella,and L.Parente.2012.Annexin A1:novel roles in skeletal muscle biology.J Cell Physiol.227:3007-3015. Blackwood,R.A.,and J.D.Ernst.1990.Characterization of Ca2(+)-dependent phospholipid binding,vesicle aggregation and membrane fusion by annexins.The Biochemical journal.266:195-200. Boye,T.L.,J.C.Jeppesen,K.Maeda,W.Pezeshkian,V.Solovyeva,J.Nylandsted,and A.C.Simonsen.2018.Annexins induce curvature on free-edge membranes displaying distinct morphologies.Sci Rep.8:10309. Boye,T.L.,K.Maeda,W.Pezeshkian,S.L.Sonder,S.C.Haeger,V.Gerke,A.C.Simonsen,and J.Nylandsted.2017.Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair.Nat Commun.8:1623. Buzhynskyy,N.,M.Golczak,J.Lai-Kee-Him,O.Lambert,B.Tessier,C.Gounou,R.Berat,A.Simon,T.Granier,J.M.Chevalier,S.Mazeres,J.Bandorowicz-Pikula,S.Pikula,and A.R.Brisson.2009.Annexin-A6 presents two modes of association with phospholipid membranes.A combined QCM-D,AFM and cryo-TEM study.Journal of structural biology.168:107-116. Cai,C.,H.Masumiya,N.Weisleder,N.Matsuda,M.Nishi,M.Hwang,J.K.Ko,P.Lin,A.Thornton,X.Zhao,Z.Pan,S.Komazaki,M.Brotto,H.Takeshima,and J.Ma.2009.MG53 nucleates assembly of cell membrane repair machinery.Nat Cell Biol.11:56-64. Carmeille,R.,S.A.Degrelle,L.Plawinski,F.Bouvet,C.Gounou,D.Evain-Brion,A.R.Brisson,and A.Bouter.2015.Annexin-A5 promotes membrane resealing in human trophoblasts.Biochimica et biophysica acta.1853:2033-2044. Cagliani,R.,F.Magri,A.Toscano,L.Merlini,F.Fortunato,C.Lamperti,C.Rodolico,A.Prelle,M.Sironi,M.Aguennouz,P.Ciscato,A.Uncini,M.Moggio,N.Bresolin,and G.P.Comi.2005.Mutation finding in patients with dysferlin deficiency and role of the dysferlin interacting proteins annexin A1 and A2 in muscular dystrophies.Human mutation.26:283. Ceco,E.,S.Bogdanovich,B.Gardner,T.Miller,A.DeJesus,J.U.Earley,M.Hadhazy,L.R.Smith,E.R.Barton,J.D.Molkentin,and E.M.McNally.2014.Targeting latent TGFbeta release in muscular dystrophy.Science translational medicine.6:259ra144. Christmas,P.,J.Callaway,J.Fallon,J.Jones,and H.T.Haigler.1991.Selective secretion of annexin 1,a protein without a signal sequence,by the human prostate gland.The Journal of biological chemistry.266:2499-2507. Davenport,N.R.,K.J.Sonnemann,K.W.Eliceiri,and W.M.Bement.2016.Membrane dynamics during cellular wound repair.Mol Biol Cell.27:2272-2285. Defour,A.,S.Medikayala,J.H.Van der Meulen,M.W.Hogarth,N.Holdreith,A.Malatras,W.Duddy,J.Boehler,K.Nagaraju,and J.K.Jaiswal.2017.Annexin A2 links poor myofiber repair with inflammation and adipogenic replacement of the injured muscle.Human molecular genetics.26:1979-1991. de Laat,B.,R.H.Derksen,I.J.Mackie,M.Roest,S.Schoormans,B.J.Woodhams,P.G.de Groot,and W.L.van Heerde.2006.Annexin A5 polymorphism (-1C-->T) and the presence of anti-annexin A5 antibodies in the antiphospholipid syndrome.Annals of the rheumatic diseases.65:1468-1472. Demonbreun,A.R.,M.V.Allen,J.L.Warner,D.Y.Barefield,S.Krishnan,K.E.Swanson,J.U.Earley,and E.M.McNally.2016a.Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption.Am J Pathol.186:1610-1622. Demonbreun,A.R.,and E.M.McNally.2015.DNA Electroporation,Isolation and Imaging of Myofibers.Journal of visualized experiments:JoVE.106:e53551. Demonbreun,A.R.,and E.M.McNally.2016.Plasma Membrane Repair in Health and Disease.Curr Top Membr.77:67-96. Demonbreun,A.R.,M.Quattrocelli,D.Y.Barefield,M.V.Allen,K.E.Swanson,and E.M.McNally.2016b.An actin-dependent annexin complex mediates plasma membrane repair in muscle.The Journal of cell biology.213:705-718. Demonbreun,A.R.,A.E.Rossi,M.G.Alvarez,K.E.Swanson,H.K.Deveaux,J.U.Earley,M.Hadhazy,R.Vohra,G.A.Walter,P.Pytel,and E.M.McNally.2014.Dysferlin and myoferlin regulate transverse tubule formation and glycerol sensitivity.Am J Pathol.184:248-259. Demonbreun,A.R.,K.E.Swanson,A.E.Rossi,H.K.Deveaux,J.U.Earley,M.V.Allen,P.Arya,S.Bhattacharyya,H.Band,P.Pytel,and E.M.McNally.2015.Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle.PLoS One.10:e0136679. Deora,A.B.,G.Kreitzer,A.T.Jacovina,and K.A.Hajjar.2004.An annexin 2 phosphorylation switch mediates p11-dependent translocation of annexin 2 to the cell surface.The Journal of biological chemistry.279:43411-43418. DiFranco,M.,M.Quinonez,J.Capote,and J.Vergara.2009.DNA transfection of mammalian skeletal muscles using in vivo electroporation.Journal of visualized experiments:JoVE.32. Duann,P.,H.Li,P.Lin,T.Tan,Z.Wang,K.Chen,X.Zhou,K.Gumpper,H.Zhu,T.Ludwig,P.J.Mohler,B.Rovin,W.T.Abraham,C.Zeng,and J.Ma.2015.MG53-mediated cell membrane repair protects against acute kidney injury.Science translational medicine.7:279ra236. Echigoya Y,Lim KRQ,Nakamura A,and Yokota T.Multiple Exon Skipping in the Duchenne Muscular Dystrophy Hot Spots:Prospects and Challenges.J Pers Med 8,2018. Fiehn,W.,J.B.Peter,J.F.Mead,and M.Gan-Elepano.1971.Lipids and fatty acids of sarcolemma,sarcoplasmic reticulum,and mitochondria from rat skeletal muscle.The Journal of biological chemistry.246:5617-5620. Flanigan,K.M.,E.Ceco,K.M.Lamar,Y.Kaminoh,D.M.Dunn,J.R.Mendell,W.M.King,A.Pestronk,J.M.Florence,K.D.Mathews,R.S.Finkel,K.J.Swoboda,E.Gappmaier,M.T.Howard,J.W.Day,C.McDonald,E.M.McNally,and R.B.Weiss.2013.LTBP4 genotype predicts age of ambulatory loss in duchenne muscular dystrophy.Ann Neurol.73:481-488. Gerke,V.,C.E.Creutz,and S.E.Moss.2005.Annexins:linking Ca2+ signalling to membrane dynamics.Nat Rev Mol Cell Biol.6:449-461. Gerke,V.,and S.E.Moss.2002.Annexins:from structure to function.Physiol Rev.82:331-371. Grewal,T.,M.Hoque,J.R.W.Conway,M.Reverter,M.Wahba,S.S.Beevi,P.Timpson,C.Enrich,and C.Rentero.2017.Annexin A6-A multifunctional scaffold in cell motility.Cell Adh Migr.11:288-304. Goulet,F.,K.G.Moore,and A.C.Sartorelli.1992.Glycosylation of annexin I and annexin II.Biochemical and biophysical research communications.188:554-558. Hack AA,et al.Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin.J Cell Biol.1998;142(5):1279-1287. Hannon,R.,J.D.Croxtall,S.J.Getting,F.Roviezzo,S.Yona,M.J.Paul-Clark,F.N.Gavins,M.Perretti,J.F.Morris,J.C.Buckingham,and R.J.Flower.2003.Aberrant inflammation and resistance to glucocorticoids in annexin 1- / - mouse.FASEB J.17:253-255. He,B.,R.H.Tang,N.Weisleder,B.Xiao,Z.Yuan,C.Cai,H.Zhu,P.Lin,C.Qiao,J.Li,C.Mayer,J.Li,J.Ma,and X.Xiao.2012.Enhancing muscle membrane repair by gene delivery of MG53 ameliorates muscular dystrophy and heart failure in delta-Sarcoglycan-deficient hamsters.Molecular therapy:the journal of the American Society of Gene Therapy.20:727-735. Hoffman,E.P.,R.H.Brown,Jr.,and L.M.Kunkel.1987.Dystrophin:the protein product of the Duchenne muscular dystrophy locus.Cell.51:919-928. Jennische,E.,and H.A.Hansson.1986.Postischemic skeletal muscle injury:patterns of injury in relation to adequacy of reperfusion.Exp Mol Pathol.44:272-280. Jia,Y.,K.Chen,P.Lin,G.Lieber,M.Nishi,R.Yan,Z.Wang,Y.Yao,Y.Li,B.A.Whitson,P.Duann,H.Li,X.Zhou,H.Zhu,H.Takeshima,J.C.Hunter,R.L.McLeod,N.Weisleder,C.Zeng,and J.Ma.2014.Treatment of acute lung injury by targeting MG53-mediated cell membrane repair.Nat Commun.5:4387. Jimenez,A.J.,and F.Perez.2017.Plasma membrane repair:the adaptable cell life-insurance.Curr Opin Cell Biol.47:99-107. Jost,M.,C.Thiel,K.Weber,and V.Gerke.1992.Mapping of three unique Ca(2+)-binding sites in human annexin II.Eur J Biochem.207:923-930. Kaetzel,M.A.,Y.D.Mo,T.R.Mealy,B.Campos,W.Bergsma-Schutter,A.Brisson,J.R.Dedman,and B.A.Seaton.2001.Phosphorylation mutants elucidate the mechanism of annexin IV-mediated membrane aggregation.Biochemistry.40:4192-4199. Lauritzen,S.P.,T.L.Boye,and J.Nylandsted.2015.Annexins are instrumental for efficient plasma membrane repair in cancer cells.Semin Cell Dev Biol.45:32-38. Lennon,N.J.,A.Kho,B.J.Bacskai,S.L.Perlmutter,B.T.Hyman,and R.H.Brown,Jr.2003.Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing.The Journal of biological chemistry.278:50466-50473. Li D,Mastaglia FL,Fletcher S,and Wilton SD.Precision Medicine through Antisense Oligonucleotide-Mediated Exon Skipping.Trends Pharmacol Sci 39:982-994,2018. Ling,Q.,A.T.Jacovina,A.Deora,M.Febbraio,R.Simantov,R.L.Silverstein,B.Hempstead,W.H.Mark,and K.A.Hajjar.2004.Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo.The Journal of clinical investigation.113:38-48. Liu,J.,H.Zhu,Y.Zheng,Z.Xu,L.Li,T.Tan,K.H.Park,J.Hou,C.Zhang,D.Li,R.Li,Z.Liu,N.Weisleder,D.Zhu,P.Lin,and J.Ma.2015.Cardioprotection of recombinant human MG53 protein in a porcine model of ischemia and reperfusion injury.Journal of molecular and cellular cardiology.80:10-19. McDade,J.R.,A.Archambeau,and D.E.Michele.2014.Rapid actin-cytoskeleton-dependent recruitment of plasma membrane-derived dysferlin at wounds is critical for muscle membrane repair.FASEB J.28:3660-3670. McNeil,A.K.,U.Rescher,V.Gerke,and P.L.McNeil.2006.Requirement for annexin A1 in plasma membrane repair.The Journal of biological chemistry.281:35202-35207. McNeil,P.L.,and R.Khakee.1992.Disruptions of muscle fiber plasma membranes.Role in exercise-induced damage.Am J Pathol.140:1097-1109. McNeil,P.L.,and T.Kirchhausen.2005.An emergency response team for membrane repair.Nat Rev Mol Cell Biol.6:499-505. Min YL,Bassel-Duby R,and Olson EN.CRISPR Correction of Duchenne Muscular Dystrophy.Annu Rev Med,2018. Murphy,S.,M.Zweyer,M.Henry,P.Meleady,R.R.Mundegar,D.Swandulla,and K.Ohlendieck.2018.Proteomic analysis of the sarcolemma-enriched fraction from dystrophic mdx-4cv skeletal muscle.J Proteomics. Petrof,B.J.,J.B.Shrager,H.H.Stedman,A.M.Kelly,and H.L.Sweeney.1993.Dystrophin protects the sarcolemma from stresses developed during muscle contraction.Proceedings of the National Academy of Sciences of the United States of America.90:3710-3714. Quattrocelli,M.,D.Y.Barefield,J.L.Warner,A.H.Vo,M.Hadhazy,J.U.Earley,A.R.Demonbreun,and E.M.McNally.2017a.Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy.J Clin Invest.127:2418-2432. Quattrocelli,M.,J.Capote,J.C.Ohiri,J.L.Warner,A.H.Vo,J.U.Earley,M.Hadhazy,A.R.Demonbreun,M.J.Spencer,and E.M.McNally.2017b.Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury.PLoS Genet.13:e1007070. Quattrocelli,M.,I.M.Salamone,P.G.Page,J.L.Warner,A.R.Demonbreun,and E.M.McNally.2017c.Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy.Am J Pathol.187:2520-2535. Reddy,A.,E.V.Caler,and N.W.Andrews.2001.Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes.Cell.106:157-169. Rodriguez,A.,P.Webster,J.Ortego,and N.W.Andrews.1997.Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells.J Cell Biol.137:93-104. Roostalu,U.,and U.Strahle.2012.In vivo imaging of molecular interactions at damaged sarcolemma.Dev Cell.22:515-529. Swaggart,K.A.,A.R.Demonbreun,A.H.Vo,K.E.Swanson,E.Y.Kim,J.P.Fahrenbach,J.Holley-Cuthrell,A.Eskin,Z.Chen,K.Squire,A.Heydemann,A.A.Palmer,S.F.Nelson,and E.M.McNally.2014.Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.Proceedings of the National Academy of Sciences of the United States of America.111:6004-6009. Waddell,L.B.,F.A.Lemckert,X.F.Zheng,J.Tran,F.J.Evesson,J.M.Hawkes,A.Lek,N.E.Street,P.Lin,N.F.Clarke,A.P.Landstrom,M.J.Ackerman,N.Weisleder,J.Ma,K.N.North,and S.T.Cooper.2011.Dysferlin,annexin A1,and mitsugumin 53 are upregulated in muscular dystrophy and localize to longitudinal tubules of the T-system with stretch.J Neuropathol Exp Neurol.70:302-313. Wallner,B.P.,R.J.Mattaliano,C.Hession,R.L.Cate,R.Tizard,L.K.Sinclair,C.Foeller,E.P.Chow,J.L.Browing,K.L.Ramachandran,and et al.1986.Cloning and expression of human lipocortin,a phospholipase A2 inhibitor with potential anti-inflammatory activity.Nature.320:77-81. Weisleder,N.,N.Takizawa,P.Lin,X.Wang,C.Cao,Y.Zhang,T.Tan,C.Ferrante,H.Zhu,P.J.Chen,R.Yan,M.Sterling,X.Zhao,M.Hwang,M.Takeshima,C.Cai,H.Cheng,H.Takeshima,R.P.Xiao,and J.Ma.2012.Recombinant MG53 protein modulates therapeutic cell membrane repair in treatment of muscular dystrophy.Science translational medicine.4:139ra185. Yeung,T.,B.Heit,J.F.Dubuisson,G.D.Fairn,B.Chiu,R.Inman,A.Kapus,M.Swanson,and S.Grinstein.2009.Contribution of phosphatidylserine to membrane surface charge and protein targeting during phagosome maturation.The Journal of cell biology.185:917-928. Zaks,W.J.,and C.E.Creutz.1991.Ca(2+)-dependent annexin self-association on membrane surfaces.Biochemistry.30:9607-9615. Zhang Y,Long C,Bassel-Duby R,and Olson EN.Myoediting:Toward Prevention of Muscular Dystrophy by Therapeutic Genome Editing.Physiol Rev 98:1205-1240,2018. Zweifach,A.2000.FM1-43 reports plasma membrane phospholipid scrambling in T-lymphocytes.The Biochemical journal.349:255-260.

Claims

1. A method for treating cell membrane damage, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein.

2. A method for delaying the onset of, enhancing recovery from, or preventing cell membrane damage, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein.

3. 3. The method of claim 1 or claim 2, wherein the agent is selected from the group consisting of a recombinant protein, a steroid, and a polynucleotide capable of expressing an annexin protein.

4. 4. The method of claim 3, wherein the steroid is a corticosteroid or a glucocorticoid.

5. The method of claim 3 , wherein the recombinant protein is an annexin protein.

6. The method of claim 5, wherein the annexin protein is annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof).

7. The method of any one of claims 1 to 6, wherein the patient is suffering from an acute injury.

8. 8. The method of claim 7, wherein the acute injury results from surgery, burns, toxins, chemicals, radiation induced injury, acute myocardial injury, acute muscle injury, acute lung injury, acute epithelial injury, acute epidermal injury, acute kidney injury, acute liver injury, vascular injury, excessive mechanical force, or trauma.

9. The method of any one of claims 1 to 6, wherein the patient is suffering from a chronic disorder.

10. 10. The method of claim 9, wherein the chronic disorder is Becker muscular dystrophy (BMD), Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), myotonic dystrophy, facioscapulohumeral dystrophy (FSHD), oculopharyngeal muscular dystrophy, distal muscular dystrophy, cystic fibrosis, pulmonary fibrosis, muscle atrophy, cerebral palsy, epithelial disorders, epidermal disorders, renal disorders, hepatic disorders, sarcopenia, or cardiomyopathy (hypertrophic, dilated, congenital, arrhythmogenic, restrictive, ischemic, heart failure).

11. 11. The method of claim 10, wherein the cardiomyopathy is hypertrophic, dilated, congenital, arrhythmogenic, restrictive, ischemic, or heart failure.

12. 12. The method of any one of claims 1 to 11, further comprising administering an effective amount of a second agent, wherein the second agent is selected from the group consisting of mitsugumin 53 (MG53), a modulator of latent TGF-beta binding protein 4 (LTBP4), a modulator of transforming growth factor beta (TGF-beta) activity, a modulator of androgen response, a modulator of inflammatory response, a promoter of muscle growth, a chemotherapeutic agent, a modulator of fibrosis, and combinations thereof.

13. The method of any one of claims 3 to 12, wherein the polynucleotide is associated with a nanoparticle.

14. The method of any one of claims 3 to 13, wherein the polynucleotide is contained in a vector.

15. The method of claim 14 , wherein the vector is in a chloroplast.

16. The method of claim 14 , wherein the vector is a viral vector.

17. 17. The method of claim 16, wherein the viral vector is selected from the group consisting of a herpes virus vector, an adeno-associated virus (AAV) vector, an adenovirus vector, and a lentivirus vector.

18. 18. The method of claim 17, wherein the AAV vector is a recombinant AAV5, AAV6, AAV8, AAV9, or AAV74.

19. 19. The method of claim 18, wherein the AAV74 vector is AAVrh74.

20. 20. The method of any one of claims 1 to 19, wherein the composition increases the activity of annexin A1 (SEQ ID NO:1), annexin A2 (SEQ ID NO:2 or SEQ ID NO:3), annexin A3 (SEQ ID NO:4), annexin A4 (SEQ ID NO:5), annexin A5 (SEQ ID NO:6), annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof), annexin A7 (SEQ ID NO:9 or SEQ ID NO:10), annexin A8 (SEQ ID NO:11 or SEQ ID NO:12), annexin A9 (SEQ ID NO:13), annexin A10 (SEQ ID NO:14), annexin A11 (SEQ ID NO:15 or SEQ ID NO:16), annexin A13 (SEQ ID NO:17 or SEQ ID NO:18), or a combination thereof.

21. The method of claim 20, wherein the composition increases the activity of annexin A1 (sequence number 1), annexin A2 (sequence number 2 or sequence number 3), and annexin A6 (sequence number 7, sequence number 8, sequence number 45, or a combination thereof).

22. The method of claim 20, wherein the composition increases the activity of annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3) and annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof).

23. The method of claim 20, wherein the composition increases the activity of annexin A1 (SEQ ID NO: 1) and annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof).

24. The method of claim 20, wherein the composition increases the activity of Annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof).

25. The method according to any one of claims 1 to 24, wherein the composition is a pharmaceutical composition according to any one of claims 24 to 45.

26. A pharmaceutical composition comprising a modified annexin protein and a pharma- ceutically acceptable carrier, buffer, and / or diluent.

27. 27. The pharmaceutical composition of claim 26, wherein the annexin protein is annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof), annexin A7 (SEQ ID NO: 9 or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 or SEQ ID NO: 16), annexin A13 (SEQ ID NO: 17 or SEQ ID NO: 18), or a combination thereof.

28. The pharmaceutical composition of claim 26 or claim 27, wherein the annexin protein is annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof).

29. The pharmaceutical composition of claim 26 or claim 27, wherein the pharmaceutical composition comprises annexin A1 (sequence number 1), annexin A2 (sequence number 2 or sequence number 3), and annexin A6 (sequence number 7, sequence number 8, sequence number 45, or a combination thereof).

30. The pharmaceutical composition of claim 26 or claim 27, wherein the pharmaceutical composition comprises annexin A2 (sequence number 2 or sequence number 3) and annexin A6 (sequence number 7, sequence number 8, sequence number 45, or a combination thereof).

31. The pharmaceutical composition of claim 26 or claim 27, wherein the pharmaceutical composition comprises annexin A1 (sequence number 1) and annexin A6 (sequence number 7, sequence number 8, sequence number 45, or a combination thereof).

32. 32. The pharmaceutical composition of any one of claims 26 to 31, further comprising a steroid.

33. 33. The pharmaceutical composition of claim 32, wherein the steroid is a corticosteroid or a glucocorticoid.

34. 34. The pharmaceutical composition of any one of claims 26-33, further comprising an effective amount of a second agent, said second agent being selected from the group consisting of mitochondrial 53 (MG53), a modulator of latent TGF-beta binding protein 4 (LTBP4), a modulator of transforming growth factor beta (TGF-beta) activity, a modulator of androgen response, a modulator of inflammatory response, a promoter of muscle growth, a chemotherapeutic agent, a modulator of fibrosis, and combinations thereof.

35. 35. The pharmaceutical composition of any one of claims 26 to 34, wherein the purity of the annexin protein in the composition is about 90% or greater as measured by a standard release assay.

36. 36. The pharmaceutical composition of any one of claims 26-35, wherein the composition has an endotoxin level that is less than about 0.50000 endotoxin units per milligram (EU / mg).

37. A pharmaceutical composition comprising a modified annexin A6 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:45, or a combination thereof) and a pharma- ceutically acceptable carrier, buffer, and / or diluent.

38. 38. The pharmaceutical composition of claim 37, further comprising annexin A1 (sequence number 1) and annexin A2 (sequence number 2 or sequence number 3).

39. 38. The pharmaceutical composition of claim 37, further comprising annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3).

40. 38. The pharmaceutical composition of claim 37, further comprising annexin A1 (SEQ ID NO: 1).

41. 41. The pharmaceutical composition of any one of claims 37 to 40, further comprising a steroid.

42. 42. The pharmaceutical composition of claim 41, wherein the steroid is a corticosteroid or a glucocorticoid.

43. 43. The pharmaceutical composition of any one of claims 37-42, further comprising an effective amount of a second agent, said second agent being selected from the group consisting of mitugumin 53 (MG53), a modulator of latent TGF-beta binding protein 4 (LTBP4), a modulator of transforming growth factor beta (TGF-beta) activity, a modulator of androgen response, a modulator of inflammatory response, a promoter of muscle growth, a chemotherapeutic agent, a modulator of fibrosis, and combinations thereof.

44. 44. The pharmaceutical composition of any one of claims 37 to 43, wherein the purity of the annexin protein in the composition is about 90% or greater as measured by a standard release assay.

45. 45. The pharmaceutical composition of any one of claims 37-44, wherein the composition has an endotoxin level that is less than about 0.50000 endotoxin units per milligram (EU / mg).

46. The pharmaceutical composition of any one of claims 37 to 45, wherein the modified annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof) is produced in a prokaryotic cell.

Images