Methods for diagnosing and treating muscular dystrophy diseases

Abstract

A method is provided for diagnosing or prognosing a muscular dystrophy or a dystrophinopathy in a subject, the method comprising detecting the presence of a biomarker or a combination of biomarkers or detecting a change in level of a biomarker or a combination of biomarkers in a biological sample from the subject, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), or filamin-C (FLNC), and the combination is a combination of any two or more of MYOM3, PYGM, TTN, or FLNC. In some aspects, the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy. A method is provided for diagnosing Duchenne muscular dystrophy in a subject, the method comprising detecting the presence of a biomarker or a combination of biomarkers or detecting a change in level of a biomarker or a combination of biomarkers in a biological sample from the subject, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), filamin-C (FLNC), or myosin-2 (MYH2), and the combination is a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2. Methods for monitoring disease progression by measuring levels of one or more biomarkers over course of the disease, and methods for determining the efficacy of a therapeutic treatment for the muscular dystrophy in a subject by measuring levels of one or more biomarkers before and / or after treatment also are provided.

Description

28335 / 70816 / PC2023-045-02METHODS FOR DIAGNOSING AND TREATING MUSCULAR DYSTROPHY DISEASESCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority benefit of U. S. Provisional Application No. 63 / 705,640, filed on October 10, 2024, which is incorporated herein by reference in its entirety.FIELD

[0002] This disclosure relates to the field of diagnosing a subject having a dystrophinopathy or a muscular dystrophy including, but not limited to, Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In some aspects, the disclosure provides methods of using a biomarker, or a combination of biomarkers, to detect the muscular dystrophy. In some aspects, the disclosure provides methods for diagnosing and treating a subject after the muscular dystrophy is diagnosed. Additionally, the disclosure provides methods for monitoring disease progression by measuring levels of one or more biomarkers over course of the disease and methods for determining the efficacy of a therapeutic treatment for the muscular dystrophy in a subject by measuring levels of one or more biomarkers before and / or after treatment.BACKGROUND

[0003] Muscular dystrophies (MDs) are a group of genetic diseases. The group is characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD develop in infancy or childhood, while others may not appear until middle age or later. The disorders differ in terms of the distribution and extent of muscle weakness (some forms of MD also affect cardiac muscle), the age of onset, the rate of progression, and the pattern of inheritance.

[0004] The MDs are a group of diseases without identifiable efficient treatment that gravely impact individuals, families, and communities. The costs are incalculable. Individuals suffer emotional strain and reduced quality of life associated with loss of self-esteem. Extreme physical challenges resulting from loss of limb function creates hardships in activities of daily living. Family dynamics suffer through financial loss and challenges to interpersonal relationships. Siblings of the affected feel estranged, and strife between spouses often leads to divorce, especially if responsibility for the muscular dystrophy can be laid at the feet of one of the parental partners. The burden of the quest to find a cure often becomes a life-long, highly focused effort that detracts and challenges every aspect of life. Beyond the family, the community bears a financial burden through the need for added facilities to accommodate the handicaps of the muscular dystrophy population in special28335 / 70816 / PC2023-045-02 education, special transportation, and costs for recurrent hospitalizations to treat recurrent respiratory tract infections and cardiac complications. Financial responsibilities are shared by state and federal governmental agencies extending the responsibilities to the taxpaying community.

[0005] One form of MD is Duchenne muscular dystrophy (DMD). DMD is one of the severe forms of muscular dystrophy, affecting approximately one in every 3,600-5,200 newborn males and characterized by progressive muscle wasting and weakness, cardiovascular, and respiratory complications. These lead to loss of ambulation by age 12 and premature death by the 3rd or 4th decade of life. DMD is caused by mutations in the dystrophin gene leading to absence of dystrophin protein (427 kDa) in skeletal and cardiac muscles, as well as the gastrointestinal tract and retina. Dystrophin not only protects the sarcolemma from eccentric contractions, but also anchors a number of signaling proteins in close proximity to sarcolemma. Another form of MD is Becker muscular dystrophy (BMD). BMD, like DMD, is a genetic disorder that gradually makes the body's muscles weaker and smaller. BMD affects the muscles of the hips, pelvis, thighs, and shoulders, as well as the heart, but is known to cause less severe problems than DMD.

[0006] DMD and BMD are often diagnosed through a combination of clinical examination, genetic testing, and other tests, but there remains a need in the art for new methods for diagnosing, predicting disease progression (prognosis) and treating DMD and BMD and determining efficacy of treatment in these muscular dystrophies.SUMMARY

[0007] The disclosure provides methods of diagnosing a dystrophinopathy or a muscular dystrophy in a subject. In some aspects, the methods comprise detecting the presence or a change in level of a biomarker or a combination of biomarkers in a biological sample from the subject. The disclosure also provides methods for diagnosing and treating a subject after the muscular dystrophy is diagnosed. Additionally, the disclosure provides methods for determining efficacy of a therapeutic treatment for the muscular dystrophy in a subject by measuring levels of one or more biomarkers in a subject before and / or after treatment.

[0008] The disclosure provides a method of diagnosing a muscular dystrophy in a subject, the method comprising detecting the presence of a biomarker or a combination of biomarkers or detecting a change in level of a biomarker or a combination of biomarkers in a biological sample from the subject, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), or filamin-C (FLNC), and the combination is a combination of any two or more of MYOM3, PYGM, TTN, or FLNC, and28335 / 70816 / PC2023-045-02 wherein the presence or the change in level of the biomarker or the combination of biomarkers indicates the subject has a muscular dystrophy.

[0009] In some aspects, the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy. In some aspects, the method comprises detecting the presence of a biomarker or a combination of biomarkers or detecting a change in level of a biomarker or a combination of biomarkers in a biological sample from the subject, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), filamin-C (FLNC), or myosin-2 (MYH2), and the combination is a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy. In some aspects, the method comprises detecting the presence of the biomarker or the combination of biomarkers in the sample. In some aspects, the method comprises detecting a change in the level of the biomarker or the combination of biomarkers in the sample. In some aspects, wherein when the change in the level of the biomarker or the combination of biomarkers is an increase in the level of the biomarker or an increase in the levels of the biomarkers in the combination of biomarkers compared to a control or to a reference level, the subject is diagnosed as having a muscular dystrophy. In some aspects, wherein when the change in the level of the biomarker or the combination of biomarkers is an increase in the level of MYOM3, PYGM, TTN, or FLNC, or an increase in level of the biomarkers in the combination of any two or more of MYOM3, PYGM, TTN, or FLNC compared to a control or reference level, the subject is diagnosed as having a muscular dystrophy. In some aspects, the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy. In some aspects, wherein when the change in the level of the biomarker or the combination of biomarkers is an increase in the level of MYOM3, PYGM, TTN, FLNC, or MYH2 compared to a reference level or an increase in level of the biomarker in a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2 compared to a reference level, the subject is diagnosed as having a muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy. In some aspects, the change in the level of the biomarker is measured as being statistically significant compared to the reference level or to the control level. In some aspects, the sample is tissue or fluid isolated from the subject. In some aspects, the fluid is plasma or serum. In some aspects, the fluid is plasma. In some aspects, the sample is an extracellular vesicle (EV) isolated from plasma of the subject. In some aspects, the biomarker is a protein. In some aspects, the biomarker is a nucleic acid. In some aspects, the method further comprises treating the subject after diagnosing the subject with the muscular dystrophy, wherein treating comprises administering to the subject an effective amount of a) a steroid, b) eteplirsen (EXONDYS 51), c) golodirsen (VYONDYS 53), d) viltolarsen (VILTEPSO), e)28335 / 70816 / PC2023-045-02 casimersen (AMONDYS 45), f) micro-dystrophin gene therapy (ELEVIDYS), g) another not yet approved gene therapy or nucleic acid therapeutic, h) a creatine supplement, i) physical therapy, and / or j) mobility and / or breathing assistance, or a combination of any thereof. Thus, in some aspects, the disclosure provides a method of diagnosing and treating.

[0010] The disclosure also provides a method for determining efficacy of a therapeutic treatment for a muscular dystrophy in a subject, the method comprising measuring the level of a biomarker or the levels of biomarkers in a combination of biomarkers in a biological sample from the subject before and after the treatment, and determining that the treatment is effective in treating the muscular dystrophy if the level of the biomarker or the levels of the biomarkers in the combination of biomarkers is decreased relative to a reference level or to the level of the biomarker or the level of the biomarker in the combination of biomarkers in the sample from the subject before treatment. In some aspects, the biomarker is myomesin- 3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), or filamin-C (FLNC), and the combination of biomarkers is a combination of any two or more of MYOM3, PYGM, TTN, or FLNC. In some aspects, the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy. In some aspects, the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), filamin-C (FLNC), or myosin-2 (MYH2), and the combination of biomarkers is a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy. In some aspects, the muscular dystrophy is Becker muscular dystrophy. In some aspects, the sample is tissue or fluid isolated from the subject. In some aspects, the fluid is plasma or serum. In some aspects, the fluid is plasma. In some aspects, the sample is an extracellular vesicle (EV) isolated from plasma of the subject. In some aspects, the biomarker is a protein. In some aspects, the biomarker is a nucleic acid. In some aspects, the treatment comprises administering to the subject an effective amount of a treatment comprising any one of more of a) a steroid, b) eteplirsen (EXONDYS 51), c) golodirsen (VYONDYS 53), d) viltolarsen (VILTEPSO), e) casimersen (AMONDYS 45), f) microdystrophin gene therapy (ELEVIDYS), g) another not yet approved gene therapy or nucleic acid therapeutic, h) a creatine supplement, i) physical therapy, and / or j) mobility and / or breathing assistance, or a combination of any thereof.

[0011] The disclosure also provides a method for monitoring disease progression of a muscular dystrophy in a subject, the method comprising: measuring the level of a biomarker or the levels of the biomarkers in a combination of biomarkers in a biological sample from the subject at a first time point and at one or more subsequent time points to obtain biomarker level data across time points; comparing the level of the biomarker or the levels of the biomarkers in the combination of biomarkers across time points to determine if there is28335 / 70816 / PC2023-045-02 an increase or decrease in the level of the biomarker or the levels of the biomarkers in the combination of biomarkers; and determining that the muscular dystrophy is not progressing if the level of the biomarker or the levels of the biomarkers in the combination of biomarkers is decreased relative to a reference level or to the level of the biomarker or the level of the biomarker in the combination of biomarkers in the sample from the subject at the first time point or at an earlier time point. In some aspects, the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), or filamin-C (FLNC), and the combination of biomarkers is a combination of any two or more of MYOM3, PYGM, TTN, or FLNC. In some aspects, the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy. In some aspects, the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), filamin-C (FLNC), or myosin-2 (MYH2), and the combination of biomarkers is a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy. In some aspects, the muscular dystrophy is Becker muscular dystrophy. In some aspects, the sample is tissue or fluid isolated from the subject. In some aspects, the fluid is plasma or serum. In some aspects, the fluid is plasma. In some aspects, the sample is an extracellular vesicle (EV) isolated from plasma of the subject. In some aspects, the biomarker is a protein. In some aspects, the biomarker is a nucleic acid.

[0012] Further aspects and advantages of the disclosure will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. It should be understood, however, that the detailed description (including the drawings and the specific examples), while indicating embodiments of the disclosed subject matter, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figs. 1 A-F show a characterization of plasma EVs of dystrophinopathy and healthy controls by Size Exclusion Chromatography (SEC). Fig. 1 A shows transmission electron microscopy (TEM) image showing a control EV and its lipid bilayer membrane. Figs. 1 B-C show size and concentration of EVs determined by Nanoparticle Tracking Analysis (NTA). Fig. 1 D is a Venn diagram of overlapping proteins between this study and the Exocarta Database for plasma EV proteins. Fig. 1 E shows enrichment analysis of proteins identified in this study. Enrichment analysis is conducted with DAVID software for Gene Ontology (GO) Terms with Cellular Compartment sub-analysis. Top five significantly (Bonferroni corrected pval<0.05) enriched terms are displayed. Fig. 1 F shows principal component analysis (PCA) of proteins identified by mass spectrometry. * pval < 0.05, ** pval < 0.01 by28335 / 70816 / PC2023-045-02 two-tailed unpaired t-test (N=5-9 for DMD, N=5-6 for BMD).

[0014] Figs. 2A-F show results from an analysis of differential protein levels in plasma EVs of dystrophinopathy and healthy control patients. Figs. 2A-B show volcano plots of the dystrophinopathy cohorts. For all cohorts, FDR corrected pval<0.1 and log2FC >1 (dashed lines) was used as a cutoff for significance. Labeled are proteins that met the cutoff criteria. Figs. 2C-D show corresponding heatmaps of relative levels (i.e., z-scores) displaying all proteins that are significantly changing using nominal pval<0.05. Figs. 2A and 2C show that FLNC, PYGM, MYOM3, and TTN are markers for BMD. Figs. 2B and 2D show that MYH2 in addition to FLNC, PYGM, MYOM3, and TTN are markers for DMD. Fig. 2E shows an enrichment analysis on the DMD cohort conducted with DAVID software for Gene Ontology (GO) Terms with Biological Processes as sub-analysis. The top five significantly enriched terms are displayed (Bonferroni corrected pval<0.05). Fig. 2F shows confirmation of the mass spectrometry results by western blot for MYOM3 and PYGM as biomarkers for DMD.

[0015] Figs. 3A-E show correlations of EV protein levels with confounding factors. Figs. 3A-B are dystrophinopathy cohorts combining DMD and BMD patients. Pearson correlations of PYGM and MYOM3 with age in dystrophinopathy patients are shown. Figs. 3C-E show DMD subsets of the dystrophinopathy cohorts, and PYGM and MYOM3 levels in patients with cardiomyopathy (Fig. 3C-D, respectively) and PYGM levels in patients with wheelchair use (Fig. 3E). The Pearson correlation coefficient (R) and statistical significance p was computed for each EV-protein. Confidence intervals (95%) for each correlation are indicated by gray shade. * pval < 0.05 by two-tailed unpaired t test or one-way ANOVA.

[0016] Figs. 4A-B show an illustration of study design (Fig. 4A) and the confirmation of packaging of potential biomarkers by EVs using western blot analysis (Fig. 4B). Western blots were carried out on EVs treated with 20 pg / mL Proteinase K in the presence of 20 pM PMSF or 1% Triton X-100. Proteinase K-resistant proteins (lane 3) are considered EV proteins. Fig. 4B shows the levels of biomarkers of dystrophinopathy, i.e., MYOM3 and PYGM, using classical markers of plasma lipoprotein (ApoB) and EV (CD9) as controls.

[0017] Figs. 5A-F show additional results from an analysis of differential protein levels in plasma EVs of dystrophinopathy and healthy control patients. Figs. 5A-B show volcano plots of the dystrophinopathy cohorts. For all cohorts, FDR corrected pval<0.1 and log2FC >1 (dashed lines) was used as a cutoff for significance. Labeled are proteins that met the cutoff criteria. Figs. 5C-D show corresponding heatmaps of relative levels (i.e., z-scores) displaying all proteins that are significantly changing using nominal pval<0.05. Figs. 5B and 5D show that MYH2 in addition to FLNC, PYGM, MYOM3, and TTN are markers for DMD. Fig. 5E shows an enrichment analysis on the DMD cohort conducted with DAVID software28335 / 70816 / PC2023-045-02 for Gene Ontology (GO) Terms with Biological Processes as sub-analysis. The top five significantly enriched terms are displayed (Bonferroni corrected pval<0.05). Fig. 5F shows confirmation of the mass spectrometry results by western blot for MYOM3 and PYGM as biomarkers for DMD. In Fig. 5F, the molecular weight of each detected protein is shown.

[0018] Figs. 6A-R show correlations of EV protein levels with confounding factors. Figs. 6A-E are dystrophinopathy cohorts showing biomarker levels. Figs. 6F-H are dystrophiopathy cohorts combining DMD and BMD patients. Pearson correlations of PYGM, MYOM3 and TTN with age in dystrophinopathy patients are shown. Figs. 6I-K show DMD subsets of the dystrophinopathy cohorts, and PYGM and MYOM3 levels in patients with cardiomyopathy (Figs. 6I-J) and PYGM levels in patients with wheelchair use (Fig. 6K). Figs. 6L-0 show these correlations losing significance upon adjusting for age. Figs. 6P-R show significant correlations of FLNC, MYH2, and TTN with various clinical outcome measures after adjusting for age. The Pearson correlation coefficient (R) and statistical significance (p) were computed for each EV protein. Confidence intervals (95%) for each correlation are indicated by gray shade. * pval < 0.05 by two-tailed unpaired t test or one-way ANOVA.

[0019] Figs. 7A-B show an illustration of study design (Fig. 7A) and the confirmation of packaging of potential biomarkers by EVs using western blot analysis (Fig. 7B). Western blots were carried out on EVs treated with 20 pg / mL Proteinase K in the presence of 20 pM PMSF or 1% Triton X-100. Proteinase K-resistant proteins (lane 3) are considered EV proteins. Fig. 7B show the levels of biomarkers of dystrophinopathy, i.e., MYOM3 and PYGM, using classical markers of plasma lipoprotein (ApoB) and EV (CD9) as controls. In Fig. 7B, the molecular weight of each detected protein is shown.DETAILED DESCRIPTION

[0020] The disclosure provides methods of diagnosing a dystrophinopathy or a muscular dystrophy in a subject. In some aspects, the methods comprise detecting the presence or a change in level of a biomarker or a combination of biomarkers in a biological sample from the subject. The disclosure also provides methods for diagnosing and treating a subject after the muscular dystrophy is diagnosed. Additionally, the disclosure provides methods for determining efficacy of a therapeutic treatment for the muscular dystrophy in a subject by measuring levels of one or more biomarkers in a subject before and / or after treatment.

[0021] More specifically, the disclosure provides a method of diagnosing a muscular dystrophy in a subject, the method comprising detecting the presence of a biomarker or a combination of biomarkers or detecting a change in level of a biomarker or a combination of biomarkers in a biological sample from the subject, wherein the biomarker is myomesin-328335 / 70816 / PC2023-045-02(MY0M3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), or filamin-C (FLNC), and the combination is a combination of any two or more of MY0M3, PYGM, TTN, or FLNC.

[0022] In some aspects, the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy. In some aspects, the method comprises detecting the presence of a biomarker or a combination of biomarkers or detecting a change in level of a biomarker or a combination of biomarkers in a biological sample from the subject, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), filamin-C (FLNC), or myosin-2 (MYH2), and the combination is a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy. In some aspects, the muscular dystrophy is Becker muscular dystrophy. In some aspects, the method comprises detecting the presence of the biomarker or the combination of biomarkers in the sample. In some aspects, the method comprises detecting a change in the level of the biomarker or the combination of biomarkers in the sample. In some aspects, wherein when the change in the level of the biomarker or the combination of biomarkers is an increase in the level of the biomarker or an increase in the levels of the biomarkers in the combination of biomarkers compared to a control or to a reference level, the subject is diagnosed as having a muscular dystrophy. In some aspects, wherein when the change in the level of the biomarker or the combination of biomarkers is an increase in the level of MYOM3, PYGM, TTN, or FLNC, or an increase in level of the biomarkers in the combination of any two or more of MYOM3, PYGM, TTN, or FLNC compared to a control or reference level, the subject is diagnosed as having a muscular dystrophy. In some aspects, the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy. In some aspects, wherein when the change in the level of the biomarker or the combination of biomarkers is an increase in the level of MYOM3, PYGM, TTN, FLNC, or MYH2 compared to a reference level or an increase in level of the biomarker in a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2 compared to a reference level, the subject is diagnosed as having a muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy. In some aspects, the muscular dystrophy is Becker muscular dystrophy. In some aspects, the change in the level of the biomarker is measured as being statistically significant compared to the reference level or to the control level. In some aspects, the sample is tissue or fluid isolated from the subject. In some aspects, the fluid is plasma or serum. In some aspects, the fluid is plasma. In some aspects, the sample is an extracellular vesicle (EV) isolated from plasma of the subject. In some aspects, the biomarker is a protein. In some aspects, the biomarker is a nucleic acid. In some aspects, the method further comprises treating the subject after diagnosing the subject with the muscular dystrophy, wherein treating comprises28335 / 70816 / PC2023-045-02 administering to the subject an effective amount of a) a steroid, b) eteplirsen (EXONDYS 51), c) golodirsen (VYONDYS 53), d) viltolarsen (VILTEPSO), e) casimersen (AMONDYS 45), f) micro-dystrophin gene therapy (ELEVIDYS), g) another not yet approved gene therapy or nucleic acid therapeutic, h) a creatine supplement, i) physical therapy, and / or j) mobility and / or breathing assistance, or a combination of any thereof. Thus, in some aspects, the disclosure provides a method of diagnosing and treating.

[0023] The disclosure also provides a method for determining efficacy of a therapeutic treatment for a muscular dystrophy in a subject, the method comprising measuring the level of a biomarker or the levels of biomarkers in a combination of biomarkers in a biological sample from the subject before and after the treatment, and determining that the treatment is effective in treating the muscular dystrophy if the level of the biomarker or the levels of the biomarkers in the combination of biomarkers is decreased relative to a reference level or to the level of the biomarker or the level of the biomarker in the combination of biomarkers in the sample from the subject before treatment. In some aspects, the biomarker is myomesin- 3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), or filamin-C (FLNC), and the combination of biomarkers is a combination of any two or more of MYOM3, PYGM, TTN, or FLNC. In some aspects, the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy. In some aspects, the muscular dystrophy is Becker muscular dystrophy. In some aspects, the sample is tissue or fluid isolated from the subject. In some aspects, the fluid is plasma or serum. In some aspects, the fluid is plasma. In some aspects, the sample is an extracellular vesicle (EV) isolated from plasma of the subject. In some aspects, the biomarker is a protein. In some aspects, the biomarker is a nucleic acid. In some aspects, the treatment comprises administering to the subject an effective amount of a treatment comprising any one of more of a) a steroid, b) eteplirsen (EXONDYS 51), c) golodirsen (VYONDYS 53), d) viltolarsen (VILTEPSO), e) casimersen (AMONDYS 45), f) micro-dystrophin gene therapy (ELEVIDYS), g) another not yet approved gene therapy or nucleic acid therapeutic, h) a creatine supplement, i) physical therapy, and / or j) mobility and / or breathing assistance, or a combination of any thereof.

[0024] The disclosure also provides a method for monitoring disease progression of a muscular dystrophy in a subject, the method comprising: measuring the level of a biomarker or the levels of the biomarkers in a combination of biomarkers in a biological sample from the subject at a first time point and at one or more subsequent time points to obtain biomarker level data across time points; comparing the level of the biomarker or the levels of the biomarkers in the combination of biomarkers across time points to determine if there is an increase or decrease in the level of the biomarker or the levels of the biomarkers in the combination of biomarkers; and determining that the muscular dystrophy is not progressing if28335 / 70816 / PC2023-045-02 the level of the biomarker or the levels of the biomarkers in the combination of biomarkers is decreased relative to a reference level or to the level of the biomarker or the level of the biomarker in the combination of biomarkers in the sample from the subject at the first time point or at an earlier time point. In some aspects, the first time point is at diagnosis, shortly after diagnosis, or possibly a period of time after diagnosis. In some aspects, the one or more subsequent time points are after the first time point, and they may occur monthly, bimonthly, yearly, or biyearly to monitor disease progression to obtain biomarker level data across time points throughout the subject’s life to monitor progression of the disease. In some aspects, the biomarker is myomesin-3 (MY0M3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), or filamin-C (FLNC), and the combination of biomarkers is a combination of any two or more of MY0M3, PYGM, TTN, or FLNC. In some aspects, the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy. In some aspects, the biomarker is myomesin-3 (MY0M3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), filamin-C (FLNC), or myosin-2 (MYH2), and the combination of biomarkers is a combination of any two or more of MY0M3, PYGM, TTN, FLNC, or MYH2. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy. In some aspects, the muscular dystrophy is Becker muscular dystrophy. In some aspects, the sample is tissue or fluid isolated from the subject. In some aspects, the fluid is plasma or serum. In some aspects, the fluid is plasma. In some aspects, the sample is an extracellular vesicle (EV) isolated from plasma of the subject. In some aspects, the biomarker is a protein. In some aspects, the biomarker is a nucleic acid.

[0025] Before any embodiments of the subject matter of the disclosure are explained in detail, it is to be understood that the disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the figures and examples. Accordingly, the disclosure embraces other embodiments and is practiced or carried out in various ways.

[0026] It is noted here that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. The terms "including," "comprising," "containing," or "having" and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0027] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues linked via peptide bonds. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides. The term "protein", as used herein, includes a fragment of a protein or any portion of the protein smaller than the full-length protein or protein expression product.28335 / 70816 / PC2023-045-02Fragments are deletion analogs of the full-length protein wherein one or more amino acid residues have been removed from the amino terminus (protein) and / or the carboxy terminus of the full-length protein.

[0028] The term "nucleic acid" or "nucleic acid sequence" or "nucleic acid molecule" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or doublestranded form. The term nucleic acid is used interchangeably with gene, deoxyribonucleic acid, complementary DNA (cDNA), ribonucleic acid, messenger RNA (mRNA), oligonucleotide, and polynucleotide. The term "nucleic acid", as used herein, includes a fragment of a nucleic acid or any portion of the nucleic acid smaller than the full-length nucleic acid. Fragments include deletion analogs of the full-length nucleic acid wherein one or more nucleotides have been removed from the 5' end and / or the 3' end of the full-length nucleic acid.

[0029] A "biomarker" in the context of the disclosure encompasses, without limitation, proteins, nucleic acids, and metabolites, together with their polymorphisms, mutations, variants, modifications, subunits, fragments, protein-ligand complexes, and degradation products, protein-ligand complexes, elements, related metabolites, and other analytes or sample-derived measures. In some aspects, therefore, a biomarker includes a protein or a fragment thereof or a nucleic acid or a fragment thereof. In additional aspects, one or more biomarkers are measured together to provide an array for the prediction that the subject will positively respond to a treatment regimen. In exemplary aspects of the disclosure, a biomarker is a protein.

[0030] In some aspects, the methods, kits, and uses of the disclosure comprise a biomarker or two or more biomarkers in combination. In some aspects, a kit of the disclosure comprises reagents, such as antibodies or nucleic acids, for detecting the presence of the biomarker or the combination of any two or more biomarkers.

[0031] A biomarker, e.g., protein or nucleic acid, of the disclosure is any one or more of the following: myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), filamin-C (FLNC), and myosin-2 (MYH2). In some aspects, a biomarker may be a combination of any two or more of the biomarkers of the disclosure.

[0032] In aspects of the disclosure, the term "MYOM3" as used herein refers to a MYOM3 protein or nucleic acid. In aspects of the disclosure, the term "PYGM" as used herein refers to a PYGM protein or nucleic acid. In aspects of the disclosure, the term "TTN" as used herein refers to a TTN protein or nucleic acid. In aspects of the disclosure, the term "FLNC" as used herein refers to an FLNC protein or nucleic acid. In aspects of the disclosure, the term “MYH2” as used herein refers to an MYH2 protein or nucleic acid.28335 / 70816 / PC2023-045-02

[0033] MYOM3 (Uniprot Q5Vtt5) is also referred to as myomesin-3 or myomesin family member 3. MYOM3 belongs to a family of structural proteins that localize to the M-band of the sarcomere in striated muscle. MYOM proteins are involved in sarcomere stability and resistance during intense or sustained stretch. MYOM3 is believed to link the intermediate filament cytoskeleton to the M-disk of the myofibrils in striated muscle.

[0034] PYGM (Uniprot P11217) is also referred to as glycogen phosphorylase, muscle form. PYGM is an allosteric enzyme that catalyzes the rate-limiting step in glycogen catabolism, the phosphorolytic cleavage of glycogen to produce glucose-1 -phosphate, and plays a central role in maintaining cellular and organismal glucose homeostasis.

[0035] TTN (Uniprot Q8WZ42) is also referred to as titin. TTN is a key component in the assembly and functioning of vertebrate striated muscles. By providing connections at the level of individual microfilaments, TTN contributes to the fine balance of forces between the two halves of the sarcomere. The size and extensibility of the cross-links are the main determinants of sarcomere extensibility properties of muscle. In non-muscle cells, TTN seems to play a role in chromosome condensation and chromosome segregation during mitosis.

[0036] FLNC (Uniprot Q14315) is also referred to as filamin-C. FLNC is a muscle-specific filamin, which plays a central role in sarcomere assembly and organization. FLNC is critical for normal myogenesis, and appears to function as a large actin-cross-linking protein with structural functions at the Z lines in muscle cells.

[0037] MYH2 (Uniprot Q9UKX2) is also referred to as myosin-2 (also referred to as myosin heavy chain 2; myosin heavy chain 2a; myosin heavy chain Ila; and myosin heavy chain, skeletal muscle, adult 2). MYH2 is a gene that encodes MyHClIa, a myosin heavy chain found in fast type 2A muscle fibers. Mutations in the MYH2 gene can cause a rare group of neuromuscular diseases called MYH2-related myopathies.

[0038] The disclosure includes the use of any biomarker or combination of biomarkers described herein in any of the disclosed methods, kits, uses and the like.

[0039] In some aspects, an increase in the level of the biomarker compared to the level of the biomarker at an initial measurement is indicative of progression of the disease. In some aspects, a decrease in the level of the biomarker compared to the level of the biomarker at an initial measurement is indicative of regression of the disease, or possible response to a therapeutic treatment regimen.

[0040] Thus, in some aspects, an increase in the level of MYOM3, PYGM, TTN, FLNC, or MYH2, or any combination thereof, compared to an initial level or a reference level are28335 / 70816 / PC2023-045-02 indicative of progression of a dystrophinopathy or a muscular dystrophy.

[0041] In some aspects of the disclosure, the level of a biomarker or the combination of biomarkers is measured in a sample from a subject and compared to a reference level. A “reference level”, as used herein, provides a known comparator for measuring the level or amount of biomarker, e.g., protein or nucleic acid, present in a biological sample from a subject.

[0042] In some aspects of the disclosure, the reference level is a mean level of the biomarker in a biological sample from a population of subjects determined to be healthy or not to have a dystrophinopathy.

[0043] In some aspects of the disclosure, a control or control level is used. A control or control level, in various aspects, is a level of the biomarker in a biological sample from a subject, or the mean level from a population of subjects, determined to be healthy or not to have a dystrophinopathy.

[0044] In some aspects, the population of subjects is, optionally, matched to the subject in other parameters, such as one or more of the following: age, sex, body mass index (BM I) and the like. In various aspects, the level of the biomarker is a relative level. In some aspects, the level of the biomarker is an absolute level. In some aspects, the level of the biomarker is within the range of the mean level of the biomarker in a population of healthy subjects or subjects determined not to have a dystrophinopathy or a muscular dystrophy.

[0045] In some aspects of the disclosure, the level of a biomarker or the levels of a combination of biomarkers is measured in a sample from a subject at a first time point and at one or more subsequent time points to obtain biomarker level data across time points. Thus, biomarker level or levels can be measured over the course of time. Thus, a first level or levels and one or more subsequent level or levels are measured. In such aspects, the first level is the level of the biomarker or the levels of each of the biomarkers in the sample from the subject at a first time point. In such aspects, the one or more subsequent level or levels is the level of the biomarker or the levels of each of the biomarkers in the sample from the subject at the one or more subsequent time points. In some aspects, the level of the biomarker or the levels of the multiple biomarkers in a combination of biomarkers is measured at multiple times throughout the course of the subject’s lifetime to monitor disease progression. In some aspects, the level of the biomarker in the subject at a first or initial time point is determined to be a baseline by which the level or levels of the biomarker over time are compared. In some aspects, the reference level is a mean level of the biomarker in a biological sample from a population of subjects determined to be healthy or not to have a dystrophinopathy.28335 / 70816 / PC2023-045-02

[0046] In some aspects of the disclosure, the level of a biomarker or the levels of a combination of biomarkers is measured in a sample from a subject before and after a therapeutic treatment. Thus, a first level and a second level is measured. In such aspects, the first level is the level of the biomarker or the levels of each of the biomarkers in the sample from the subject before the therapeutic treatment. In such aspects, the second level is the level of the biomarker or the levels of each of the biomarkers in the sample from the subject after the therapeutic treatment. In some aspects, the level of a biomarker or the levels of multiple biomarkers is measured at multiple times throughout the course of a therapeutic treatment. In such aspects, the reference level is the level of the biomarker in the subject before the therapeutic treatment or agent is administered (i.e., baseline).

[0047] In various aspects, an increased level of biomarker is a level greater than the reference level. In various aspects, an increase in the level of the biomarker in a subject is at least or about 1% greater, at least or about 2% greater, at least or about 3% greater, at least or about 4% greater, at least or about 5% greater, at least or about 6% greater, at least or about 7% greater, at least or about 8% greater, at least or about 9% greater, at least or about 10% greater, at least or about 11% greater, at least or about 12% greater, at least or about 13% greater, at least or about 14% greater, at least or about 15% greater, at least or about 16% greater, at least or about 17% greater, at least or about 18% greater, at least or about 19% greater, at least or about 20% greater, at least or about 21% greater, at least or about 22% greater, at least or about 23% greater, at least or about 24% greater, at least or about 25% greater, at least or about 26% greater, at least or about 27% greater, at least or about 28% greater, at least or about 29% greater, at least or about 30% greater, at least or about 35% greater, at least or about 40% greater, at least or about 45% greater, at least or about 50% greater, at least or about 55% greater, at least or about 60% greater, at least or about 65% greater, at least or about 70% greater, at least or about 75% greater, at least or about 80% greater, at least or about 85% greater, at least or about 90% greater, at least or about 95% greater, at least or about 100% greater, at least or about greater than 100% greater than the reference level, or the level of the biomarker before treatment.

[0048] In additional aspects, an increase in the level of the biomarker in a subject is at least or about 1 / 10 greater, at least or about 1 / 9 greater at least or about 1 / 8 greater, at least or about 1 / 7 greater, at least or about 1 / 6 greater, at least or about 1 / 5 greater, at least or about 1 / 4 greater, at least or about 1 / 3 greater, at least or about 1 / 2 greater, at least or about 1 times greater, at least or about 1 .5 times greater, at least or about 2.0 times greater, at least or about 2.5 times greater, at least or about 3.0 times greater, at least or about 3.5 times greater, at least or about 4.0 times greater, at least or about 4.5 times greater, at least or about 5 times greater, at least or about 10 times greater, at least or about 15 times28335 / 70816 / PC2023-045-02 greater, at least or about 20 times greater, at least or about 25 times greater, at least or about 30 times greater, at least or about 35 times greater, at least or about 40 times greater, at least or about 45 times greater, at least or about 50 times greater, at least or about 55 times greater, at least or about 60 times greater, at least or about 65 times greater, at least or about 70 times greater, at least or about 75 times greater, at least or about 80 times greater, at least or about 85 times greater, at least or about 90 times greater, at least or about 100 times greater, or at least or about greater than about 100 times than the reference level, or the level of the biomarker before treatment.

[0049] In some aspects, this increase in the level of the biomarker is referred to as “fold greater”, or at least or about 1 / 10 fold greater, at least or about 1 / 9 fold greater, at least or about 1 / 8 fold greater, at least or about 1 / 7 fold greater, at least or about 1 / 6 fold greater, at least or about 1 / 5 fold greater, at least or about 1 / 4 fold greater, at least or about 1 / 3 fold greater, at least or about 1 / 2 fold greater, at least or about 1 fold greater, at least or about 1 .5 fold greater, at least or about 2.0 fold greater, at least or about 2.5 fold greater, at least or about 3.0 fold greater, at least or about 3.5 fold greater, at least or about 4.0 fold greater, at least or about 4.5 fold greater, at least or about 5 fold greater, at least or about 10 fold greater, at least or about 15 fold greater, at least or about 20 fold greater, at least or about 25 fold greater, at least or about 30 fold greater, at least or about 35 fold greater, at least or about 40 fold greater, at least or about 45 fold greater, at least or about 50 fold greater, at least or about 55 fold greater, at least or about 60 fold greater, at least or about 65 fold greater, at least or about 70 fold greater, at least or about 75 fold greater, at least or about 80 fold greater, at least or about 85 fold greater, at least or about 90 fold greater, at least or about 100 fold greater, or at least or about greater than about 100 fold than the reference level, or the level of the biomarker before treatment.

[0050] In various aspects, a decreased level of a biomarker is a level lesser than the reference level. In various aspects, a decrease in the level of the biomarker is at least or about 1% lesser, at least or about 2% lesser, at least or about 3% lesser, at least or about 4% lesser, at least or about 5% lesser, at least or about 6% lesser, at least or about 7% lesser, at least or about 8% lesser, at least or about 9% lesser, at least or about 10% lesser, at least or about 11% lesser, at least or about 12% lesser, at least or about 13% lesser, at least or about 14% lesser, at least or about 15% lesser, at least or about 16% lesser, at least or about 17% lesser, at least or about 18% lesser, at least or about 19% lesser, at least or about 20% lesser, at least or about 21% lesser, at least or about 22% lesser, at least or about 23% lesser, at least or about 24% lesser, at least or about 25% lesser, at least or about 26% lesser, at least or about 27% lesser, at least or about 28% lesser, at least or about 29% lesser, at least or about 30% lesser, at least or about 35% lesser, at least or28335 / 70816 / PC2023-045-02 about 40% lesser, at least or about 45% lesser, at least or about 50% lesser, at least or about 55% lesser, at least or about 60% lesser, at least or about 65% lesser, at least or about 70% lesser, at least or about 75% lesser, at least or about 80% lesser, at least or about 85% lesser, at least or about 90% lesser, at least or about 95% lesser, at least or about 100% lesser, or about lesser than 100% lesser than the reference level, or the level of the biomarker before treatment.

[0051] In additional aspects, a decrease in the level of the biomarker in a subject is at least or about 1 / 10 lesser, at least or about 1 / 9 lesser at least or about 1 / 8 lesser, at least or about 1 / 7 lesser, at least or about 1 / 6 lesser, at least or about 1 / 5 lesser, at least or about 1 / 4 lesser, at least or about 1 / 3 lesser, at least or about 1 / 2 lesser, at least or about 1 times lesser, at least or about 1 .5 times lesser, at least or about 2.0 times lesser, at least or about 2.5 times lesser, at least or about 3.0 times lesser, at least or about 3.5 times lesser, at least or about 4.0 times lesser, at least or about 4.5 times lesser, or at least or about 5 times lesser, at least or about 10 times lesser, at least or about 15 times lesser, at least or about 20 times lesser, at least or about 25 times lesser, at least or about 30 times lesser, at least or about 35 times lesser, at least or about 40 times lesser, at least or about 45 times lesser, at least or about 50 times lesser, at least or about 55 times lesser, at least or about 60 times lesser, at least or about 65 times lesser, at least or about 70 times lesser, at least or about 75 times lesser, at least or about 80 times lesser, at least or about 85 times lesser, at least or about 90 times lesser, at least or about 100 times lesser, or at least or about lesser than about 100 times lesser than the reference level, or the level of the biomarker before treatment.

[0052] In additional aspects, a decrease in the level of the biomarker in a subject is at least or about 1 / 10 fold lesser, at least or about 1 / 9 fold lesser at least or about 1 / 8 fold lesser, at least or about 1 / 7 fold lesser, at least or about 1 / 6 fold lesser, at least or about 1 / 5 fold lesser, at least or about 1 / 4 fold lesser, at least or about 1 / 3 fold lesser, at least or about 1 / 2 fold lesser, at least or about 1 fold lesser, at least or about 1 .5 fold lesser, at least or about 2.0 fold lesser, at least or about 2.5 fold lesser, at least or about 3.0 fold lesser, at least or about 3.5 fold lesser, at least or about 4.0 fold lesser, at least or about 4.5 fold lesser, or at least or about 5 fold lesser, at least or about 10 fold lesser, at least or about 15 fold lesser, at least or about 20 fold lesser, at least or about 25 fold lesser, at least or about 30 fold lesser, at least or about 35 fold lesser, at least or about 40 fold lesser, at least or about 45 fold lesser, at least or about 50 fold lesser, at least or about 55 fold lesser, at least or about 60 fold lesser, at least or about 65 fold lesser, at least or about 70 fold lesser, at least or about 75 fold lesser, at least or about 80 fold lesser, at least or about 85 fold lesser, at least or about 90 fold lesser, at least or about 100 fold lesser, or at least or about lesser28335 / 70816 / PC2023-045-02 than about 100 fold lesser than the reference level, or the level of the biomarker before treatment.

[0053] In some aspects, the level is a spectral count level. In some aspects, spectral counting simply counts the number of spectra identified for a given peptide in different biological samples and then integrates the results for all measured peptides of the protein(s) that are quantified.

[0054] In various aspects of the disclosure, level of the protein biomarker is detected or quantitatively measured in a biological sample by any suitable means known in the art for quantifying protein including, but not limited to, an immunoassay (e.g., ELISA, RIA), immunoturbidimetry, rapid immunodiffusion, laser nephelometry, visual agglutination, quantitative Western blot analysis, multiple reaction monitoring-mass spectrometry (MRM Proteomics), Lowry assay, Bradford assay, BCA assay, UV spectroscopic assays, such as a UV spectroscopic assay, surface plasmon resonance, or SOMAscan® assay. In various aspects, therefore, the level measured is an absolute level or is a relative level.

[0055] In some aspects, the protein biomarker is simply measured for the presence or absence of the biomarker.

[0056] In various aspects of the disclosure, the level of the nucleic acid biomarker is detected or quantitatively measured in a biological sample by any suitable means known in the art for quantifying nucleic acid including, but not limited to, RNA sequencing (RNA-seq), high-throughput sequencing (HT-seq), PCR, quantitative PCR, qT-PCR, RT-qPCR, digital PCR, real-time PCR, direct digital quantification, serial analysis of gene expression (SAGE), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), branched DNA (bDNA) assays, and / or Northern or Southern blotting. In various aspects, therefore, the level measured is an absolute level or is a relative level.

[0057] In some aspects, the nucleic acid biomarker is simply measured for the presence or absence of the biomarker.

[0058] In some aspects, the level of the biomarker is a relative level, for example, a Z- score. In some aspects, such relative levels are assessed with consideration of a false discovery rate (FDR), which is the metric for global confidence assessment of a large-scale proteomics dataset (PMID: 26519173; Aggarwal et al., Methods Mol Biol . 2016:1362:119- 28. doi: 10.1007 / 978-1 -4939-3106-4_7).

[0059] In some aspects, the level of the biomarker is measured in male versus female subjects. Thus, in some exemplary aspects, the biomarker or the level of the biomarker is relevant for one sex versus the other sex. Thus, in some aspects, the biomarker and the28335 / 70816 / PC2023-045-02 change in the level of the biomarker is indicative of a dystrophinopathy in females, and in other aspects, the biomarker and the change in the level of the biomarker is indicative of a dystrophinopathy in males.

[0060] In various aspects, any of these methods is performed on protein or nucleic acid isolated from or present in a biological sample obtained from a human subject. In some aspects, the protein or nucleic acid may also be a fragment of the protein or nucleic acid. In some aspects, the nucleic acid is a DNA, an RNA, or an mRNA.

[0061] In some aspects, the biological sample is blood. In some aspects, the sample is serum. In exemplary aspects, the biological sample is plasma. In even more exemplary aspects, the biological sample is an extracellular vesicle (EV) isolated from plasma, or in some other aspects an EV isolated from some other tissue or bodily fluid.

[0062] Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are naturally released from almost all types of cells but, unlike a cell, cannot replicate. EVs are cell- derived membrane-surrounded vesicles that carry bioactive molecules and are released by a cell to deliver such bioactive molecules into the extracellular environment to recipient cells. Thus, EVs primarily act in cell-to-cell communication, delivering cargo from donor to recipient cells and modulating their physiological condition. Classical EVs are exosomes, microvesicles, and apoptotic bodies. EVs are different sizes (ranging from 50 to 500 nm) and have different contents based on the type and state of their producer cells. EVs carry tissuespecific molecules (i.e. lipids, RNA, DNA, peptides and proteins), and thus are exploited herein for the identification of biomarkers specific for the detection of a muscular dystrophy. Additionally, such biomarkers specific for a muscular dystrophy could be used to diagnose and / or predict disease progression, stratify patients for clinical trials, and assess efficiency or efficacy of potential muscular dystrophy therapies.

[0063] In some aspects, the sample or biological sample is taken prior to treatment to measure the level of the biomarker before treatment. In some aspects, the sample is taken before and after treatment with a therapeutic agent or therapeutic regimen to measure the level of the biomarker before or after treatment. In some aspects, the sample is taken over multiple time points to monitor the effect of a therapeutic treatment or therapeutic regimen over time. Thus, in some aspects, a first value of the biomarker, e.g., protein level, is compared to a second value or is compared to multiple values taken over a period of time. In some aspects, the sample is taken before, during, and after treatment with a therapeutic agent or therapeutic regimen to measure the level of the biomarker before, during, or after treatment to determine the effect of a biomarker during treatment. In some aspects, patients28335 / 70816 / PC2023-045-02 have a full clinical assessment at baseline (i.e., time 0) or prior to a first treatment with a therapeutic agent.

[0064] “Measuring” or “measurement” means assessing the presence, quantity or level of a biomarker, e.g. a protein or a nucleic acid, within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substance, or otherwise evaluating the values or categorization of a subject’s clinical parameters.

[0065] The terms “level” and “amount” are used herein interchangeably to mean the concentration of biomarker, e.g., protein or nucleic acid, present in a biological sample. In some aspects, protein and / or nucleic acid is prepared from the sample and the “level” and / or “amount” is the level or amount of a particular protein and / or nucleic acid of interest. In some aspects, it is the level or amount of the protein or nucleic acid biomarker. In some aspects, the level or amount is relative to a reference level or an initial level in the subject. In some aspects, the level or amount is relative to a control level. In some aspects, such control level is the biomarker level from a healthy subject known not to have muscular dystrophy. In some aspects, such control level is the mean level of the biomarker in a population of healthy subjects known not to have muscular dystrophy. In some aspects, the change in level is a change in level which is determined to be a statistically significant change in level relative to the initial level or to a previous level, or to a reference level.

[0066] In some aspects, the methods include measuring the level of at least one or more of the biomarkers. In some aspects, the methods include measuring the level of a combination of biomarkers. In some aspects, the level is a protein level in a biological sample. In some aspects, the level is a nucleic acid level in a biological sample. In some aspects, the level is measured in the EV, or in the EV after the EV is isolated from the sample. In some aspects, plasma EVs are isolated by SEC and then lysed to extract protein or mRNA. In some aspects, EVs are lysed with a detergent or a protein denaturant. In some aspects, such detergent is sodium dodecyl sulfate or other organic sodium salt. In some aspects, such detergent is 5% SDS. In some aspects, after the EVs are lysed, the EVs are sonicated to extract proteins or mRNA. In some aspects, the methods of the disclosure simply detect the presence or the absence of the biomarker as an indicator of disease. In some aspects, the methods of the disclosure detect a change in the level of the biomarker as an indicator of disease. In some aspects, the methods include

[0067] In some aspects, the methods of the disclosure comprise treating a subject. As used herein, the terms "treat", “treating”, and "treatment" refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or disorder.28335 / 70816 / PC2023-045-02Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or disorder prior to other treatments, such as surgery, stabilization of a disease or disorder (i.e., where the disease or disorder does not worsen), delay or slowing of the progression of a disease or disorder, amelioration or palliation of the disease or disorder, and remission (whether partial or total) of the disease or disorder, whether detectable or undetectable, and prevention of disease or disorder recurrence. "Treatment", in some aspects, also means prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder, those prone to or at risk of having the disease or disorder, or those in which the disease or disorder is to be prevented.

[0068] In some aspects, the disclosure includes treating a muscular dystrophy (MD). "Treating" includes ameliorating or inhibiting one or more symptoms of a muscular dystrophy including, but not limited to, muscle wasting, muscle weakness, myotonia, skeletal muscle problems, abnormalities of the retina, hip weakness, facial weakness, abdominal muscle weakness, joint and spinal abnormalities, lower leg weakness, shoulder weakness, hearing loss, muscle inflammation, and nonsymmetrical weakness. In some aspects, the treating includes ameliorating or inhibiting recurrent respiratory tract infections and cardiac complications resulting from the MD. In various aspects, such MD is DMD or BMD.

[0069] DMD is one of the severe forms of muscular dystrophy, affecting approximately one in every 3,600-5,200 newborn males and characterized by progressive muscle wasting and weakness, cardiovascular, and respiratory complications. These lead to loss of ambulation by age 12 and premature death by the 3rd or 4th decade of life. DMD is caused by mutations in the dystrophin gene leading to absence of dystrophin protein (427 kDa) in skeletal and cardiac muscles, as well as the gastrointestinal tract and retina. Dystrophin not only protects the sarcolemma from eccentric contractions, but also anchors a number of signaling proteins in close proximity to sarcolemma. Another form of MD is Becker Muscular Dystrophy (BMD). BMD, like DMD, is a genetic disorder that gradually makes the body's muscles weaker and smaller. BMD affects the muscles of the hips, pelvis, thighs, and shoulders, as well as the heart, but is known to cause less severe problems than DMD.

[0070] Many clinical cases of DMD are linked to deletion mutations in the DMD gene. In contrast to the deletion mutations, DMD exon duplications account for around 5% of disease-causing mutations in unbiased samples of dystrophinopathy patients [Dent et al., Am J Med Genet, 134(3): 295-298 (2005)], although in some catalogues of mutations the number of duplications is higher, including that published by the United Dystrophinopathy Project by Flanigan etal. [Hum Mutat, 30(12): 1657-1666 (2009)], in which it was 11%. BMD is also caused by a change in the dystrophin gene, which makes the protein too short. The28335 / 70816 / PC2023-045-02 flawed dystrophin puts muscle cells at risk for damage with normal use. See also, U.S. Patent Application Publication Nos. 2012 / 0077860, published March 29, 2012; 2013 / 0072541 , published March 21 , 2013; and 2013 / 0045538, published February 21 , 2013.

[0071] Despite many lines of research following the identification of the dystrophin gene, treatment options are limited. The most advanced therapies include those that aim at restoration of the missing protein, dystrophin, using various means of gene therapy, including mutation-specific genetic approaches, such as antisense oligonucleotide (AON)- mediated exon skipping. U7 snRNA have been found to be important tools in exon skipping and splicing modulation [Goyenvalle et al., Mol Ther 17(7):1234-40 (2009)]. Moreover, splicing modulation using antisense oligonucleotides (AONs) has been developed for the past two decades as a potential treatment for many diseases, most notably Duchene muscular dystrophy (DMD). This includes pre-clinical and clinical trials [Mendell et al., Ann Neurol 74 Q37-47 (2013)]. However, such AONs were only shown to mediate weak exon skipping due to the fact that they penetrate the heart and diaphragm (i.e., the most affected muscles in DMD boys) only weakly and they are not stable, i.e., requiring reinjection of DMD patients. It is therefore described herein that AAV.micro-dystrophin and AAV-based U7 snRNA gene therapy approaches help circumvent the aforementioned potential delivery problems of AONs. There thus remains a need in the art for treatments for MDs, as well as better means for diagnosing MDs, monitoring the progression of MDs, and for monitoring the responsiveness of MDs to treatment.

[0072] In various aspects, the methods of the disclosure are carried out after diagnosis and, therefore, are methods of treating or ameliorating disease. In some aspects, efficacy of treatment is monitored by observing outcome measures. Such outcome measures include, but are not limited to, measuring to determine if there is improved muscle strength, improved muscle function, improved mobility, improved stamina, or a combination of two or more thereof in the subject after treatment. Such outcome measures are important in determining muscular dystrophy progression in the subject and are measured by various tests known in the art.

[0073] Currently, there is no effective treatment for muscular dystrophy (MD), but a variety of approved treatments can help to manage the condition and halt disease progression. As different types of MD can cause quite specific problems, the treatment administered is tailored to the subject’s needs. For example, as symptoms develop, healthcare professionals advise on various options. In some aspects, treatment of muscular dystrophy comprises administering to the subject an effective amount of any one or more of a) a steroid, b) eteplirsen (EXONDYS 51), c) golodirsen (VYONDYS 53), d) viltolarsen (VILTEPSO), e) casimersen (AMONDYS 45), f) micro-dystrophin gene therapy (ELEVIDYS),28335 / 70816 / PC2023-045-02 g) another not yet approved gene therapy or nucleic acid therapeutic, h) a creatine supplement, i) physical therapy, and / or j) mobility and / or breathing assistance, or a combination of any thereof.

[0074] Combination therapies are also contemplated by the disclosure. Combination as used herein includes simultaneous treatment or sequential treatments. Combinations of methods of the disclosure with standard medical treatments (e.g., corticosteroids and / or immunosuppressive drugs, micro-dystrophin) or with other inhibitory RNA constructs are specifically contemplated, as are combinations with other therapies known in the art, including, but not limited to, those disclosed in International Publication Nos.WO2016057975, WO2018170408, WO2019245973, WO2021108755, WO2021257595, which are incorporated by reference herein in their entireties.

[0075] A combination therapy, as used herein, includes both simultaneous treatment(s) and sequential treatment(s). Combinations of methods described herein with standard medical treatments and supportive care are specifically contemplated, as are combinations with therapies, such as glucocorticoids. All types of glucocorticoids are included for use in the combination therapies disclosed herein. Such glucocorticoids include, but are not limited to, prednisone, prednisolone, dexamethasone, deflazacort, beclomethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, and triamcinolone.

[0076] The disclosure also provides a kit comprising reagents to determine the presence of the biomarker in the sample from the subject. Thus, in some aspects, the kit comprises antibodies or nucleic acids that could bind to and detect the presence of the biomarker in the sample of the subject. Thus, in some aspects, such kit comprises an antibody or a combination of antibodies to any one or more of the biomarker(s) or a nucleic acid or a combination of nucleic acids that bind to any one or more of the biomarker(s), or other reagents to detect the presence or change in level of the biomarker protein or nucleic acid.

[0077] In the context of the disclosure, the term "kit" means two or more components, one of which corresponds to a reagent to detect the presence of the biomarker in a sample, and the other which corresponds to a container, recipient, instructions, or otherwise. A kit, therefore, in various aspects, is a set of products that are sufficient to achieve a certain goal, which can be marketed as a single unit.

[0078] The kit may additionally contain directions or instructions for use (e.g. in the form of a leaflet or instruction manual), means for detecting the presence or the level of the biomarker protein or nucleic acid in the sample, and / or means for diluting the protein and / or28335 / 70816 / PC2023-045-02 nucleic acid. In some aspects, the kit comprises a label and / or instructions that describes use of the reagents provided in the kit.

[0079] This 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 combination of features are not found together in the same sentence, or paragraph, or section of this document. The disclosure also includes, for instance, all embodiments of the disclosure narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the disclosure described as a genus, all individual species are considered separate aspects of the disclosure. With respect to aspects of the disclosure described or claimed with "a" or "an," it should be understood that these terms mean "one or more" unless context unambiguously requires a more restricted meaning.

[0080] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the disclosure.

[0081] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term."

[0082] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having."

[0083] When used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0084] In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[0085] It should be understood that this disclosure is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular28335 / 70816 / PC2023-045-02 embodiments only and is not intended to limit the scope of the subject matter of the disclosure, which is defined solely by the claims.

[0086] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0087] A better understanding of the disclosure and of its advantages will be obtained from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.EXAMPLES

[0088] Additional aspects and details of the disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting.Example 1Materials and Methods

[0089] Patient selection:

[0090] Plasma samples from the dystrophinopathy cohort were obtained from patients and healthy controls recruited by Dr Kevin Flanigan’s lab at the Muscular Dystrophy Clinic (Nationwide Children’s Hospital, OH, USA) (Table 1). For all samples, plasma was collected using anti-coagulant (EDTA) treated collection tubes, and each sample is assigned a blinding code so that all batches of samples were tested by the lab of Dr. Nizar Saad (Nationwide Children’s Hospital, OH, USA) in a blinded fashion.28335 / 70816 / PC2023-045-02

[0091] Table 1 . Patient clinical data of patients.CSS: Clinical Severity Score. CM: Cardiomyopathy.

[0092] Extracellular vesicle (EV) isolation:

[0093] For proteomic analyses, plasma EVs were isolated by size-exclusion chromatography (SEC). This method allows for efficient EV separation from soluble components and most lipoproteins. While there are various other ways to isolate EVs (e.g., filtration, differential centrifugation, density gradient and immunoprecipitation), most of these methods are exclusively targeted for either EV yield or specificity. In contrast, SEC is efficient in both parameters, and therefore ideal for biomarker discovery. 500uL of each precleared plasma sample was loaded onto Izon qEV35 chromatography columns, and using an automated fraction collector (Izon), the first five fractions were collected and combined. EV size and concentration were monitored by Nanoparticle Tracking Analysis (NTA, Nanosight). The EV structure was characterized by transmission electron microscopy (TEM). EV quality was assessed by western blot to evaluate the presence of serum EV markers (e.g., CD9), serum proteins (e.g., ApoB, C3). To eliminate bias in subsequent analyses, EV isolation was performed blinded. Samples were then batched to ensure dystrophinopathy patient samples and healthy controls were analyzed together.

[0094] Protein isolation and Mass spectrometry:

[0095] Plasma EVs isolated by SEC were lysed with 5% SDS and sonicated to extract proteins. A typical sample preparation yielded at least 200 pg of protein from 500 pL of plasma. A total of 100 pg of protein was used for mass spectrometry (MS), while the rest was stored for Western Blot analysis. For MS, the proteins were digested via Strap or28335 / 70816 / PC2023-045-02Rapigest depending on the sample quantity and following the Core’s protocol. Liquid Chromatography was then carried out with tandem MS (LC-MS / MS). Data were searched using Mascot Daemon by Matrix Science version 2.7.0 (Boston, MA) via Proteome Discoverer (version 2.4 Thermo Scientific) and searched the database against the most recent Uniprot databases. Label free quantitation was performed using the spectral count approach, in which the relative protein quantitation was measured by comparing the number of MS / MS spectra identified from the same protein in each of the multiple LC-MS / MS datasets.

[0096] Statistical Analysis'.

[0097] The differential expression of both EV-associated protein levels was assessed using the Wald test with a significance cut-off of p <0.05 (R package: DESeq2). To control for testing of many proteins, false discovery rates (FDR) were computed from raw p values, with FDR <0.1 and log2FC >1 used as a cutoff for significance. Pearson correlation coefficients with p values are computed on scatter plots with the “stat_cor” function in R.

[0098] Immunoblotting:

[0099] Potential biomarkers were identified by mass spectrometry with western blot analysis. The location of the biomarkers (either inside or outside EVs) was then determined by treating the EVs with Proteinase K (20 pg / ml) which would only retain the molecules packed within the EVs because the protein would be protected in the EV by their lipid bilayer membrane. PMSF (20 pM) was added to the EVs one hour post treatment to inhibit Proteinase K and ensure it would not be accessible to EV proteins prior to western blot. As an additional control, EV membranes were treated simultaneously with Proteinase K and 1% Triton X-100, which ruptures EV membranes, allowing Proteinase K to digest intraluminal EV proteins.Example 2EV isolation from DMD and BMD patient plasma samples and mass spectrometry analysis

[0100] Extracellular vesicles (EVs) were isolated from patient plasma samples from a cohort containing BMD and DMD patients (Table 1) using size exclusion chromatography (SEC) following the most recent guidelines from the International Society for Extracellular Vesicles for isolation, storage, and characterization. The morphology of the isolated EVs were microscopically characterized using transmission electron microscopy (TEM), which displayed a nanoparticle circular shape delimited by a bilipid membrane (Fig. 1 A). Further characterization of the size and concentration of EVs, using Nanoparticle Tracking Analysis (NTA), revealed disease-specific differences (Fig. 1 B-E).28335 / 70816 / PC2023-045-02

[0101] Following EV characterization, protein content analysis was carried out by performing mass spectrometry (LC-MS / MS) analysis. 542 proteins (mean spectral count per condition > 3) (Fig. 1 D) were identified. The proteins indexed in ExoCarta EV plasma database (http_colon_forward slash forward slash_exocarta.org, accessed on July 23, 2024) were used as reference. Gene Ontology (GO) enrichment analysis was then carried out using the online DAVID tool with Cellular Compartment (CC) sub-analysis. The GO enrichment analysis illustrated that the analyzed EVs are enriched in the ‘extracellular region’, extracellular exosome’, and ‘extracellular space’ proteins (Fig. 1 E). To unveil any potential condition-specific clustering of overall protein content, a principal component analyses (PCA) also was carried out. As a result, no specific clustering of samples in BMD or DMD patients was detected (Fig. 1 F).Example 3 Disease specific differences in plasma EV proteomes

[0102] To reveal any potential plasma EV biomarkers, differential protein expression analyses was run to detect / analyze differences between patient samples and their corresponding healthy controls. Using stringent cut-off criteria (FDR<0.1 , pval<0.05 and log2FC >1 | log2FC <-1) proteins dysregulated in each disease were identified (Fig. 2A-F). In BMD and DMD cohorts formed of 7 BMD patient samples, 9 DMD patient samples, and 6 healthy control patient samples, all proteins that met the cut-off criteria were found to be exclusively expressed in patient EVs. These dystrophinopathy-exclusive proteins were identified as myomesin 3 (MYOM3), glycogen phosphorylase, muscle associated (PYGM), titin (TTN), filamin C (FLNC) (Fig. 2A-B). Additionally, DMD plasma EVs were also enriched for myosin heavy chain 2 (MYH2) (Fig. 2B). Notably, these dystrophinopathy-specific EV proteins are involved in key muscle functions such as muscle contraction and sarcomere organization, as illustrated by GO enrichment analyses (Fig. 2E). The major changes observed by mass spectrometry were validated with immunoblotting for MYOM3 and PYGM (Fig. 2F). In the DMD cohort, the specific PYGM band at 105 kDa was only observed in DMD patient samples (not in healthy controls), while MYOM3 staining yielded two distinct fragments in DMD patient samples (not in healthy controls), which is in line with previous reports on MYOM3 fragmentation (Fig. 2F).Example 4 Correlations of plasma EV proteins with confounding factors

[0103] Considering the dynamic and variable nature of dystrophinopathies, experiments were carried out to correlate the differentially expressed protein levels with confounding factors. For dystrophinopathy patients, the assessed confounding factors included age, ambulation status, steroid use, and presence of cardiomyopathy. Among these, patient age28335 / 70816 / PC2023-045-02 was a major factor determining PYGM and MYOM3 levels in dystrophinopathy patients (Fig. 3A-B). In both cases, significant negative correlations were observed with age. Furthermore, both PYGM and MYOM3 levels were significantly higher in patients with no reports of cardiomyopathy (PYGM FC: 3.7, pval: 0.03, MYOM3 FC: 4.3, pval: 0.02) (Fig. 3C-D).PYGM levels were also significantly lower (FC: 0.18, pval: 0.02) in patients who are dependent on full-time wheelchair use (Fig. 3E). Altogether, data suggests that plasma EV- associated PYGM, although relevant as a biomarker of DMD, appears to peak early in DMD pathogenesis, and appears to readily decline as the disease progresses.Example 5Proteinase K treatment separates plasma proteins from EV proteins

[0104] The plasma proteome mostly comprises albumins, globulins, and fibrinogens. Moreover, certain plasma proteins, such as complement pathway components and lipoproteins, naturally form a corona layer around circulating EVs. While SEC efficiently separates small nanoparticles from protein fractions, it is important to rule out any carryover plasma proteins in the EV analysis. Therefore, EVs were treated with 20 pg / ml of Proteinase K for 1 hour. Such treatment with Proteinase K selectively degrades proteins external to EVs. Because Proteinase K cannot penetrate through the lipid bilayer of the EVs, EV proteins are resistant to Proteinase K treatment. This treatment is then followed by Proteinase K inhibition using PMSF for 10 minutes. This is essential to prevent Proteinase K from degrading EV intraluminal proteins upon their extraction from EV in preparation for immunoblotting (Fig. 4A). This experimental set up / procedure allowed for the elimination of the lipoprotein ApoB, while retaining the canonical EV marker CD9.

[0105] Among the potential dystrophinopathy biomarkers, the smaller MYOM3 fragment was completely retained in EVs upon the full Proteinase K treatment, suggesting that it, i.e., the smaller MYOM3 fragment, is packaged within EVs (Fig. 4B). Similarly, PYGM was also partially retained, suggesting that it is associated with EVs (Fig. 4B).

[0106] Accordingly, both MYOM3 and PYGM are biomarkers present in EVs in plasma samples of DMD patients.

[0107] The foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the invention may be apparent to those having ordinary skill in the art.

[0108] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.28335 / 70816 / PC2023-045-02

[0109] Throughout the specification, where compositions are described as including components or materials, it is contemplated that the compositions can also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Likewise, where methods are described as including particular steps, it is contemplated that the methods can also consist essentially of, or consist of, any combination of the recited steps, unless described otherwise. The invention illustratively disclosed herein suitably may be practiced in the absence of any element or step which is not specifically disclosed herein.

[0110] The practice of a method disclosed herein, and individual steps thereof, can be performed manually and / or with the aid of automation provided by electronic equipment. Although processes have been described with reference to particular embodiments, a person of ordinary skill in the art will readily appreciate that other ways of performing the acts associated with the methods may be used. For example, the order of various of the steps may be changed without departing from the scope or spirit of the method, unless described otherwise. In addition, some of the individual steps can be combined, omitted, or further subdivided into additional steps.

[0111] All patents, publications and references cited herein are hereby fully incorporated by reference. In case of conflict between the present disclosure and incorporated patents, publications and references, the present disclosure should control.

Claims

28335 / 70816 / PC2023-045-02CLAIMSWe claim:1 . A method of diagnosing a muscular dystrophy in a subject, the method comprising detecting the presence of a biomarker or a combination of biomarkers or detecting a change in level of a biomarker or a combination of biomarkers in a biological sample from the subject, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), or filamin-C (FLNC), and the combination is a combination of any two or more of MYOM3, PYGM, TTN, or FLNC.

2. The method of claim 1 , wherein the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy.

3. The method claim 1 , the method comprising detecting the presence of a biomarker or a combination of biomarkers or detecting a change in level of a biomarker or a combination of biomarkers in a biological sample from the subject, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), filamin- C (FLNC), or myosin-2 (MYH2), and the combination is a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2.

4. The method of claim 1 or 3, wherein the muscular dystrophy is Duchenne muscular dystrophy.

5. The method of any one of claims 1-4, wherein when the method comprises detecting the presence of the biomarker or the combination of biomarkers in the sample, the subject is diagnosed as having a muscular dystrophy.

6. The method of any one of claims 1-4, wherein the method comprises detecting a change in the level of the biomarker or the combination of biomarkers in the sample.

7. The method of any one of claims 1-6, wherein when the change in the level of the biomarker or the combination of biomarkers is an increase in the level of the biomarker or the combination of biomarkers compared to a control or to a reference level, the subject is diagnosed as having a muscular dystrophy.

8. The method of any one of claims 1-7, wherein when the change in the level of the biomarker or the combination of biomarkers is an increase in the level of MYOM3, PYGM, TTN, or FLNC, or an increase in level of the biomarker in a combination of any two28335 / 70816 / PC 2023-045-02 or more of MYOM3, PYGM, TTN, or FLNC compared to a control or reference level, the subject is diagnosed as having a muscular dystrophy.

9. The method of claim 8, wherein the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy.

10. The method of any one of claims 1 -7, wherein when the change in the level of the biomarker or the combination of biomarkers is an increase in the level of MYOM3, PYGM, TTN, FLNC, or MYH2 compared to a reference level or an increase in level of the biomarker in a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2 compared to a reference level, the subject is diagnosed as having a muscular dystrophy.11 . The method of claim 10, wherein the muscular dystrophy is Duchenne muscular dystrophy.

12. The method of any one of claims 1-11 , wherein the change in the level of the biomarker is measured as being statistically significant compared to the reference level or to the control level.

13. The method of any one of claims 1-12, wherein the sample is tissue or fluid isolated from the subject.

14. The method of claim 13, wherein the fluid is plasma or serum.

15. The method of claim 13 or 14, wherein the fluid is plasma.

16. The method of any one of claims 1-15, wherein the sample is an extracellular vesicle (EV) isolated from plasma of the subject.

17. The method of any one of claims 1-16, wherein the biomarker is a protein.

18. The method of any one of claims 1-16, wherein the biomarker is a nucleic acid.

19. The method of any one of claims 1-18 further comprising treating the subject after diagnosing the subject with the muscular dystrophy, wherein treating comprises administering to the subject an effective amount of any one or more of a) a steroid, b) eteplirsen (EXONDYS 51),28335 / 70816 / PC2023-045-02 c) golodirsen (VYONDYS 53), d) viltolarsen (VILTEPSO), e) casimersen (AMONDYS 45), f) micro-dystrophin gene therapy (ELEVIDYS) g) another not yet approved gene therapy or nucleic acid therapeutic, h) a creatine supplement, i) physical therapy, and / or j) mobility and / or breathing assistance, or a combination of any thereof.

20. A method for determining efficacy of a therapeutic treatment for a muscular dystrophy in a subject, the method comprising measuring the level of a biomarker or the levels of biomarkers in a combination of biomarkers in a biological sample from the subject before and after the treatment, and determining that the treatment is effective in treating the muscular dystrophy if the level of the biomarker or the levels of the biomarkers in the combination of biomarkers is decreased relative to a reference level or to the level of the biomarker or the level of the biomarker in the combination of biomarkers in the sample from the subject before treatment.21 . The method of claim 20, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), or filamin-C (FLNC), and the combination of biomarkers is a combination of any two or more of MYOM3, PYGM, TTN, or FLNC.

22. The method of claim 20 or 21 , wherein the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy.

23. The method of claim 20, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), filamin-C (FLNC), or myosin-2 (MYH2), and the combination of biomarkers is a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2.

24. The method of claim 21 or 23, wherein the muscular dystrophy is Duchenne muscular dystrophy.

25. The method of any one of claims 20-24, wherein the sample is tissue or fluid isolated from the subject.28335 / 70816 / PC2023-045-0226. The method of claim 25, wherein the fluid is plasma or serum.

27. The method of claim 25 or 26, wherein the fluid is plasma.

28. The method of any one of claims 20-27, wherein the sample is an extracellular vesicle (EV) isolated from plasma of the subject.

29. The method of any one of claims 20-28, wherein the biomarker is a protein.

30. The method of any one of claims 20-28, wherein the biomarker is a nucleic acid.31 . The method of any one of claims 20-30, wherein the treatment comprises administering to the subject an effective amount of any one or more of a) a steroid, b) eteplirsen (EXONDYS 51), c) golodirsen (VYONDYS 53), d) viltolarsen (VILTEPSO), e) casimersen (AMONDYS 45), f) micro-dystrophin gene therapy (ELEVIDYS) g) another not yet approved gene therapy or nucleic acid therapeutic, h) a creatine supplement, i) physical therapy, and / or j) mobility and / or breathing assistance, or a combination of any thereof.

32. A method for monitoring disease progression of a muscular dystrophy in a subject, the method comprising: measuring the level of a biomarker or the levels of the biomarkers in a combination of biomarkers in a biological sample from the subject at a first time point and at one or more subsequent time points to obtain biomarker level data across time points; comparing the level of the biomarker or the levels of the biomarkers in the combination of biomarkers across time points to determine if there is an increase or28335 / 70816 / PC2023-045-02 decrease in the level of the biomarker or the levels of the biomarkers in the combination of biomarkers; and determining that the muscular dystrophy is not progressing if the level of the biomarker or the levels of the biomarkers in the combination of biomarkers is decreased relative to a reference level or to the level of the biomarker or the level of the biomarker in the combination of biomarkers in the sample from the subject at the first time point or at an earlier time point.

33. The method of claim 32, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), or filamin-C (FLNC), and the combination of biomarkers is a combination of any two or more of MYOM3, PYGM, TTN, or FLNC.

34. The method of claim 32 or 33, wherein the muscular dystrophy is Becker muscular dystrophy or Duchenne muscular dystrophy.

35. The method of claim 32, wherein the biomarker is myomesin-3 (MYOM3), glycogen phosphorylase, muscle form (PYGM), titin (TTN), filamin-C (FLNC), or myosin-2 (MYH2), and the combination of biomarkers is a combination of any two or more of MYOM3, PYGM, TTN, FLNC, or MYH2.

36. The method of claim 33 or 35, wherein the muscular dystrophy is Duchenne muscular dystrophy.

37. The method of any one of claims 32-36, wherein the sample is tissue or fluid isolated from the subject.

38. The method of claim 37, wherein the fluid is plasma or serum.

39. The method of claim 37 or 38, wherein the fluid is plasma.

40. The method of any one of claims 32-39, wherein the sample is an extracellular vesicle (EV) isolated from plasma of the subject.41 . The method of any one of claims 32-40, wherein the biomarker is a protein.

42. The method of any one of claims 32-40, wherein the biomarker is a nucleic acid.

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