Method of treating muscular dystrophy and enhancing muscle regeneration

Abstract

A method for treating muscular dystrophy of a subject comprising administrating a N-formyl peptide receptor (FPR) agonist to the subject and compositions therefore. An FPR agonist may be used to target the FPR2-mediated pro-resolving pathway, thus facilitating the clearance of inflammation, reducing inflammatory response, enhancing repair processes, enhancing regenerative capacity in muscles, and otherwise improving prognosis of muscular dystrophy.

Description

[0001] METHOD OF TREATING MUSCULAR DYSTROPHY AND ENHANCING MUSCLE REGENERATION

[0002] REFERENCE TO RELATED APPLICATIONS

[0003] This international application claims priority to U.S Provisional Application No. 63 / 663,242, filed June 24, 2024 and to U.S. Provisional Application No. 63 / 769,828, filed March 11, 2025. Where permitted by law, the specification and drawings of both of these provisional applications are incorporated by reference for all purposes.

[0004] REFERENCE TO A SEQUENCE LISTING

[0005] In accordance with 37 CFR §1.831-1835 and 37 CFR§ 1.77(b)(5), the specification makes reference to a Sequence Listing submitted electronically as a .xml file named "553799WO_ST26.xml”. This .xml file was generated on June 24, 2025 and is 4,600 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.

[0006] FIELD OF THE DISCLOSURE

[0007] This disclosure relates to the fields of neuromuscular medicine, including treatment of muscular dystrophy or regenerative medicine such as methods for enhancing muscle regeneration.

[0008] BACKGROUND OF THE DISCLOSURE

[0009] Duchenne Muscular Dystrophy (DMD) is a severe and progressive muscle disease caused by the absence of dystrophin protein. Patients with DMD experience chronic inflammation and failure of muscle regeneration with recurring muscle injury leading to muscle degeneration and fibro-fatty replacement over time. Dystrophin plays a crucial role in maintaining the integrity of the sarcolemmal membrane by facilitating the assembly and function of the dystrophin- associated protein complex, thus its absence renders skeletal muscle cells more susceptible to mechanical damage, and increased muscle degeneration.

[0010] Dystrophin deficiency in cardiomyocytes also increases their vulnerability to sarcolemmal damage, and cell death, chronic inflammation and cardiac fibrosis. These pathologies also manifest in patients and lead to the thinning of the left ventricle wall, causing their progressive dilation and dilated cardiomyopathy that results in heart failure. Patients with DMD experience symptoms early in life, with cardiac deficits being a major contributor to premature mortality.

[0011] The foregoing Background description is for the purpose of generally presenting the context of the disclosure. Work of the inventors, to the extent it is described in this background section, as well as aspects of the description which may not otherwise qualify as prior art at the time of the filing, are neither expressly or impliedly admitted as prior art against the present disclosure.

[0012] SUMMARY OF THE INVENTION

[0013] The present application provides a method for treating muscular dystrophy, such as Duchenne muscular dystrophy (DMD), by targeting unresolved inflammation and immune cell dysregulation in affected muscle tissue. Whole muscle bulk RNA-sequencing analysis was performed in two established DMD mouse models — BlO-mdx, which exhibits consistent, gradual pathology progression after juvenile disease onset, and D2-mdx, which displays more aggressive muscle degeneration, pronounced inflammation, and regenerative failure that peaks during juvenile development. The D2-mdx model demonstrated significant upregulation of pro- inflammatory cytokines and chemokines compared to BlO-mdx, leading to heightened and prolonged recruitment of neutrophils and macrophages into muscle fibers. To address the severe pathology in D2-mdx, the invention promotes resolution of inflammation and immune cell clearance by utilizing FPR2 agonists, which enhance macrophage phagocytosis and neutrophil degranulation, thereby facilitating inflammation clearance and tissue repair. Administration of pro-resolving FPR2 agonists not only reduced inflammatory gene expression but also improved skeletal muscle regenerative capacity as well as reduced skeletal and cardiac muscle degeneration in juvenile D2-mdx mice, offering a novel therapeutic approach to attenuate unresolved inflammation and improve prognosis even in pediatric DMD patients.

[0014] A method for treating muscular dystrophy, especially DMD, by administering at least one agonist of a N-formyl Peptide Receptor (FPR), such as an agonist of FPRR2 to reduce chronic muscle loss. A method for facilitating clearance of inflammation in subjects with dystrophin-related disorders.

[0015] A method for enhancing muscle repair processes in subjects with dystrophin-related disorders.

[0016] Another aspect of the invention is a method of using a mdx mouse model on DBA / 2J genetic background (D2- / x) to model juvenile-onset cardiomyopathy and skeletal muscle loss as disclosed herein.

[0017] Another aspect of the invention is directed to therapeutic composition comprising one or more FPRR agonists, including FPR1, FPR2 or FPR3 agonists, peptides, or nucleic acids inducing or encoding such agonists.

[0018] More specific aspects of the invention include the following:

[0019] 1. A method for treating a chronic inflammatory muscle disease, such as muscular dystrophy of a subject, or by helping prevent degeneration or enhance muscle regeneration of a subject, comprising administering an N-formyl peptide receptor (FPR) agonist to the subject, wherein, optionally, the subject has or is at risk of developing Duchenne muscular dystrophy (DMD) and is administered an FPR2 agonist, such as BMS-986235 or Annexin Al N-terminal mimetic peptide AC2-26, and optionally, wherein said agonist is optionally selected based on its anti-inflammatory effects it exhibits in murine models of DMD.

[0020] 2. The method of embodiment 1, wherein the subject is treated for a chronic inflammatory muscle disease that is Duchenne muscular dystrophy (DMD)

[0021] 3. The method of any one of embodiments 1 or 2, wherein the subject is treated to reduce the severity of, or prevent, muscular degeneration or to enhance muscle regeneration.

[0022] 4. The method of any one of embodiments 1 to 3, wherein said agonist is selected based on its inflammation-regulatory effects it exhibits in at least one murine model of DMD comprising an mdx murine model.

[0023] 5. The method of any one of embodiments 1 to 4, wherein the subject has DMD and is in need of muscle regeneration.

[0024] 6. The method of any one of embodiments 1 to 5, wherein the subject is in need of treatment of muscle degeneration of cardiac, skeletal, or smooth muscle. 7. The method of any one of embodiments 1 to 6, wherein the subject is in need of preventing cardiac and skeletal muscle degeneration or supporting skeletal muscle repair caused by chronic muscle damage and inflammation including Becker muscular dystrophy. Limb Girdle Muscular Dystrophies (LGMD), Inclusion Body Myositis, Dermatomyositis, and / or Polymyositis.

[0025] 8. The method of any one of embodiments 1 to 7, wherein the subject is in need of regeneration of proximal or distal upper and / or lower body muscles.

[0026] 9. The method of any one of embodiments 1 to 8, wherein the subject is in need of regeneration of muscles of the pelvic girdle, thighs, or calves.

[0027] 10. The method of any one of embodiments 1 to 9, wherein the subject is in need of cardiac muscle repair.

[0028] 11. The method of any one of embodiments 1 to 10, wherein the subject is in need of respiratory muscle regeneration.

[0029] 12. The method of any one of embodiments 1-11, wherein the subject exhibits indicators of muscle degeneration including muscle fiber rupture, hematoma formation, release of damage-associated molecular patterns (DAMPs), abnormal immune cell recruitment or failure to resolve inflammatory signaling.

[0030] 13. The method of any one of embodiments 1-12 that facilitates clearance of inflammation, reduces an inflammatory response, enhances a repair process, enhances regenerative capacity in muscles, or otherwise improves prognosis compared to an untreated control subject.

[0031] 14. The method of any one of embodiments 1-13, wherein the FPR agonist is an endogenous FPR1, 2 or 3 agonist or a synthetic FPR1, 2 or 3 agonist.

[0032] 15. The method of any one of embodiments 1-14, wherein the FPR agonist is an FPR agonist other than an FPR2 agonist.

[0033] 16. The method of any one of embodiments 1-15, wherein the FPR agonist is an FPR2 agonist.

[0034] 17. The method of any one of embodiments 1-16, wherein the FPR agonist is selected from the group consisting of at least one of Ac2-26, BMS986235, lipoxin A4 (LXA4), MR-39, Compound 43, Compound 17b, LXA4, RvDl, a pyrrolidinone urea compound or WKYMV (SEQ ID NO: 1). 18. The method of any one of embodiments 1-17, wherein the FPR agonist is a FPR2 agonist which acts by enhancing the phagocytic capacity of macrophages and / or by driving the degranulation of neutrophils, to facilitate amelioration or resolution of inflammation and to enhance at least one repair process.

[0035] 19. The method of any one of embodiments 1-18, wherein the FPR agonists is an FPR2 agonist that is used to target the FPR2-mediated pro-resolving pathway.

[0036] 20. A pharmaceutical composition suitable for administration of a subject having muscular dystrophy comprising an amount of at least one FPR2 agonist sufficient to improve muscle function or to reduce muscle degeneration and a pharmaceutically acceptable carrier, buffer, or excipient.

[0037] 21. The pharmaceutical composition of embodiment 20 that has been chemically formulated to extend its biological half-life in vivo or that has been formulated to provide for extended release of the FPR2 agonist.

[0038] 22. The composition of embodiment 20, further comprising administration of at least one co-therapy that comprises micro-dystrophin gene therapy to introduce a truncated but functional dystrophin gene into a subject with DMD, administration of dystrophin exon-skipping antisense oligonucleotides or TGF-beta-lowering oligonucleotides, and stem-cell based therapies such as administration of autologous stem cells corrected ex vivo to express functional dystrophin or administration of allogeneic stem cells from a normal donor.

[0039] 23. The composition of embodiment 20, further comprising at least one, two, or three other FPR2 agonists or conventional drugs for DMD in a form suitable for administration to a subject having DMD.

[0040] 24. The composition of embodiment 23, wherein the FPR2 agonists agonize FPR2 at different binding sites or by different biological mechanisms from each other such as induction of different conformational changes in FPR2, induction of pro- or anti-inflammatory effects, or by activation of different signaling pathways.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figures 1A-1E. Bulk sequencing shows temporal and pathological grouping from mouse skeletal muscle samples. Figure 1A. Experimental design displaying the number of mice per age, strain, and disease state used for RNA sequencing and parallel histological experiments.

[0043] Figure IB. PCA plot indicating the variance of samples at 2 weeks 1.5 months and 8 months of age in the in skeletal muscle of BlO-zwt / xand B10-WT strains.

[0044] Figure 1C. Heatmap showing DEGs (p value <0.5) between 2 weeks and 6 weeks in in skeletal muscle of BIO mdx along with expression at 8 months.

[0045] Figure ID. PCA plot indicating the variance of samples at 2 weeks 1.5 months and 8 months of age in the in skeletal muscle of D2-WT and V)2-mdx.

[0046] Figure IE. Heatmap showing DEGs (p val <0.5) between 2 weeks and 6 weeks in in skeletal muscle of D2- / wt& along with expression at 8 months. Heatmaps are clustered hierarchically using spearmen correlation.

[0047] Figures 2A-2E. At disease onset, D2- / Wx exhibit higher expression of cytokines associated with inflammation in skeletal muscle.

[0048] Figure 2A. Heatmap of significant DEGs (pval <0.05) in skeletal muscle of BlO-mr&and D2- / ??<A samples at 1.5 months.

[0049] Figure 2B. Dotplot of top 25 GO terms calculated from significant DEGs in skeletal muscle of BlO-mt / x and D2-mt / x samples.

[0050] Figure 2C. Boxplot expression of top 25 DEGs associated with inflammatory GO terms in in skeletal muscle of B I 0- / Wx and D2- / Wx at disease onset (all genes have pval < 0.05).

[0051] Figure 2D. Heatmap displaying the expression of 15 chemokines in in skeletal muscle of BlO-mdx and D2-mdx and their respective WT at 1.5 months.

[0052] Figure 2E. Heatmap showcasing the expression of 21 cytokines in in skeletal muscle of B I 0- / Wx and D2-Wx and their respective WT at 1.5 months. All heatmaps were scaled by row and hierarchical clustering was performed by spearmen correlation.

[0053] Figures 3A-3F. Longitudinal analysis suggests inflammatory gene expression peaks in skeletal muscle at 1.5 months in D2- / wtfr but increases through disease progression Bl 0>-mdx.

[0054] Figure 3A. Dotplot of top 10 GO terms activated in in skeletal muscle of BlO-wcA at 1.5 months compared to 8 months

[0055] Figure 3B. Dotplot of top 10 GO terms suppressed in in skeletal muscle of B 10- / Wx at 1.5 months compared to 8months Figure 3C. Boxplot of the top 25 inflammatory DEGs from disease onset comparing expression in skeletal muscle of B I 0- / »c / x at 1.5 and 8 months

[0056] Figure 3D. Dotplot of top 10 GO terms activated in in skeletal muscle of D2- / Wx at 1.5 months compared to 8 months.

[0057] Figure 3E. Dotplot of top 10 GO terms suppressed in in skeletal muscle of D2-mdx at 1.5 months compared to 8 months.

[0058] Figure 3F shows a boxplot of showing decrease in expression in late-stage disease in skeletal muscle compared to their levels at disease onset.

[0059] Figures 4A-4D. Differences in inflammatory gene expression in skeletal muscle is less pronounced across D2- / Wx and BlO-mdx at late stage of disease.

[0060] Figure 4A. Heatmap of significant DEGs (pval <0.05) in BIO and D2 mdx skeletal muscle samples at 8 months of age, scaled by row.

[0061] Figure 4B. Dotplot of top and bottom 10 GO terms calculated from significant DEGs from D2-mdx to BlO-mdx in skeletal muscle samples at 8 months.

[0062] Figure 4C. Heatmap of the 204 inflammatory and immune genes from onset analysis with expression shown in in skeletal muscle at 8 months of age.

[0063] Figure 4D. Boxplot of gene expression in skeletal muscle at 8 months of age for the top 25 DEGs associated with inflammatory GO terms in B 10- / Wx and D2- / 7?6 / x at disease onset. Significance p < 0.05 (*), p < 0.01 (**), p< 0.001 (***).

[0064] Figures 5A-5B. D2- / Wx samples express macrophage and neutrophil specific cytokines in in skeletal muscle peaking at 1.5 months but return to a level similar to age matched B I O-wc / x by late-stage disease. qPCR quantification of chemokines expressed primarily in macrophages (Fig. 5A) and monocytes (Fig. 5B) or neutrophils. Statistical significance is indicated as p < 0.05 (*), p < 0.01 (**), p< 0.001 (***).

[0065] Figures 6A-6O. FPR2 agonists lower expression of cytokine and inflammatory markers in in skeletal muscle while increasing regeneration in D2- / Wx.

[0066] Figure 6A. Schematic of inflammation in skeletal muscle after acute injury.

[0067] Figure 6B. Schematic explaining drug dosage of mice during the trial.

[0068] Figure 6C. immunofluorescent imaging of Quads DAPl=blue, eMHC=green,

[0069] Laminin=Pink in in skeletal muscle.

[0070] Figure 6D. Quantification of eMHC positive fibers. Figure 6E Centrally nucleated fibers (CNFs) per area of whole tissue in skeletal muscle.

[0071] Figure 6F. Damaged area in in skeletal muscle as a percent of whole tissue.

[0072] Figure 6G. Percent of whole skeletal muscle tissue section stained as F4 / 80 positive.

[0073] Figure 6H. iNos positive macrophages per mm2of damaged area in in skeletal muscle.

[0074] Figure 61. CD206 positive macrophages per mm2.

[0075] Figure 6J-6O. qPCR values from some of the top gene markers dysregulated in D2-mdx in skeletal muscle compared to WT at 1.5 months. Y axis is Fold Change, p < 0.05 (*), p < 0.01 (**), p< 0.001 (***).

[0076] Figure 7. Bulk sequencing of skeletal muscle shows WT across strains cluster together compared while D2-m<ir and B I 0- / ??t / x differences appears at disease onset.

[0077] Figures 8A-8C. Immune regulation and response pathways are the most prominent GO terms associated with upregulated genes at disease onset between B I 0- / x and D2-m7x in skeletal muscle.

[0078] Figures 9A-9B. Strain background does not account for the difference in expression of 25 inflammatory related DEGs in the mdx pairwise comparison at disease onset in skeletal muscle.

[0079] Figures 10A-10D. Show longitudinal differences in gene expression in skeletal muscle across models.

[0080] Figures 10A. Heatmap of significant DEGs (pval <0.05) in BlO-mdx in 1.5 month and 8- month-old samples.

[0081] Figures 10B. Heatmap showing expression of the 204 immune and inflammatory genes curated from the 1.5-month cross strain analysis.

[0082] Figures 10C. Heatmap of significant DEGs (pval <0.05) in D2-mdx in 1.5 month and 8- month-old samples.

[0083] Figures 10D. Heatmap showing expression of the 204 immune and inflammatory genes curated from the 1.5-month cross strain analysis.

[0084] Figure 11A-11B. Average expression of genes of interest plotted over time from single cell data with cells being collected 0, 1, 3, 5 and 7 days post injury in skeletal muscle.

[0085] Figure 11A. Average expression of macrophage-related genes of interest.

[0086] Figure 11A-11B. Average expression of neutrophil-related genes of interest. Figures 12A-12H. Histopathology associated with disease onset in juvenile D2-mdx hearts. Images show hearts harvested from juvenile (6 ± 0.5 wk) U2-mdx and B 10- / W.r mice at disease onset.

[0087] Figures 12A-12B. Whole tissue images with matched orientation of Vdl-mdx (Fig. 12A) and Bl Q-mdx (Fig. 12B) hearts showing ventricular and atrial fibro-calcified damage.

[0088] Figures 12C-12D. Cross-sectional images of juvenile D2-mt / x and B l O-wr / .r hearts through the ventricular lumen, stained for histological features by H&E (Fig. 12C), and for fibrosis by Sirius Red (Fig. 12D). Arrowheads mark areas of fibrosis.

[0089] Figures 12E-12F. Image (Fig. 12E) and quantification (Fig. 12F) showing a portion of heart cross-section from juvenile D2-mtfr and BlO-mtfc hearts, showing damaged tissue areas characterized by the presence of interstitial fibrosis, inflammatory cells and damaged cardiomyocytes.

[0090] Figures 12G-12H. Image (Fig. 12G) and quantification (Fig. 12H) showing a portion of heart cross-section from juvenile D2- / w<A and B I O- / W.r hearts labeled with Sirius Red to mark fibrotic tissue area in hearts from juvenile V)2-mdx and B I 0- / W.V mice. Data represent median ± IQR from n = 12 hearts per cohort. Statistical analyses performed using non-parametric Mann- Whitney test; **p < 0.01, ***p < 0.001. Refer to Figs. 17-18 for additional details.

[0091] Figures 13A-13E. RNAseq analysis at disease onset in D2-mdx and age-matched B10- mdx hearts. Juvenile D2-m<A and BlO-wt / x hearts at disease onset (6 wk ± 0.5 wk) were analyzed by bulk tissue RNAseq.

[0092] Figure 13A. Dimensionality reduction of whole transcriptomic data via PCA (n=3-4 hearts / genotype) to assess sample clustering and inter- / intra-sample variance.

[0093] Figure 13B. Differential gene expression analysis depicted via heatmap plot of 2,719 DEGs observed between D2-mt& (blue) and BlO-mdv (red), with 1,586 genes upregulated for D2-mdx and 1, 133 genes upregulated for B 1 Q-mdx. Expression is z-score values of variancestabilizing transformation (VST) normalized data. 13C. Gene Ontology (GO) analysis performed using Cytoscape and EnrichmentMap plugins to identify networks of related GO terms groups found upregulated (red dots) in juvenile D2- / W.r hearts relative to B l O-mZv. Pink clusters refer to inflammatory-related GO terms, while blue clusters 793 refer to extracellular matrix-related GO terms. Figures 13D-13E. Boxplots showing VST normalized gene expression levels for top 20 differentially expressed inflammation-related (Fig. 13D; GOBP: Inflammatory Response) and extracellular matrix-related (Fig. 13E; GOBP:External Encapsulating Structure) transcripts observed between juvenile D2- / w<7x and B10- / Wx hearts. Refer to Fig. 19 for additional details.

[0094] Figures 14A-14D. Targeted analysis of inflammatory response at disease onset in D2- mdx BlO-mdx hearts.

[0095] Figure 14A: qRT-PCR analysis of a distinct cohort of V)2-mdx and B l O-wt / x hearts to assess the expression of inflammatory genes identified by RNAseq analysis cohort to be differentially expressed. Transcripts include top dysregulated genes involved in leukocyte activation, migration and chemotaxis and regulation of inflammatory response and leukocyte- mediated immunity (Ccl3, Ccl8, Stab2, Adam8, Trem2, Il7r). Relative gene expression values normalized to internal Hprt transcript levels.

[0096] Figure 14B qRT-PCR analysis of a distinct cohort of D2-mdx and B l O-mt / .r hearts to assess inflammatory genes that show broad dysregulation of neutrophil and macrophage response in juvenile D2- / «<7x hearts (Illb, Lgals3, Argl, Fprl, Fpr2, Anxal). Relative gene expression values normalized to internal Hprt transcript levels.

[0097] Figures 14C-14D. Images showing immunostaining for pan-macrophage marker, F4 / 80 (green), and pro-inflammatory, pathogenic macrophage marker, GAL-3 (red), in juvenile D2- mdx (C) and B l O-mZr (Fig. 14D). Data represent median ± IQR from n = 7-9 hearts per cohort. Statistical analyses performed using non-parametric Mann-Whitney test; **p < 0.01, ***p < 0.001. For age-matched WT controls, refer to Fig. 20.

[0098] Figures 15A-15C. Targeted analysis of extracellular matrix remodeling response at disease onset in D2-mdx BlO-mdx hearts.

[0099] Figure 15A qRT-PCR analysis of a distinct cohort of D2-mdx and B l O-mt / x hearts to assess the expression of extracellular matrix-associated genes involved in matrix organization / re- organization (818 Fnl, Col lai, Itgax, Sppl, Mmpl2, Timpl) that are identified to be dysregulated by the RNAseq cohort. Relative gene expression values normalized to internal Hprt transcript levels.

[0100] Figures 15B-15C. Images showing extracellular matrix distribution visualized using wheat germ agglutinin (WGA, pink) within, and surrounding, areas of cardiac damage in juvenile D2- / Wr (Fig. 15B), and B I 0- / «tZr (Fig. 15C). Fig. 15B’-15C. Zoom of the dotted area from whole cross-sectional images showing immunostaining for COL1 A1 within the extracellular matrix shows increased COL1A1 expression in damaged D2-mtA hearts (Fig. 15B), relative to Bl O-W.r (Fig. 15C), indicative of early-onset endomysial fibrosis. Data represent median ± IQR from n = 7-9 hearts per cohort. Statistical analyses performed using nonparametric Mann-Whitney test; **p < 0.01, ***p < 0.001.

[0101] Figures 16A-16H. Pro-resolving therapy to mitigate cardiac disease onset in juvenile D2- mdx.

[0102] Figure 16A. Schematic describing the inflammatory response following cardiac injury in health (black trace) or dystrophic (red trace), showing acute versus chronic inflammation respectively. Use of anti-inflammatory drug (purple trace) lowers inflammatory response blunting inflammation, instead use of pro-resolving therapy (green trace) does not impact the onset of inflammation but helps clear inflammation preventing the inflammation to become chronic.

[0103] Figure 16B. Schematic detailing the pre-clinical testing of pro-resolving drug, BMS- 986235 (6.0 mg / kg, 3 wk daily administration) in D2- / w<7x mice (n=6) just prior to disease onset (18-19 days old).

[0104] Figure 16C. Whole tissue images of the matched orientation of hearts showing ventricular and atrial fibro-calcified damage in saline or BMS-986235-treated D2- / 77c / x mice.

[0105] Figure 16D. Image showing cross-section of D2-mt / x heart stained for histological features by H&E from mice treated with saline or BMS-986235.

[0106] Figure 16E. Images showing cross-section of D2- / 7?t& heart immunostained for panmacrophage marker, F4 / 80 (red) and counterstained with WGA (green) and DAPI (blue) to mark the ECM and nuclei, respectively.

[0107] Figure 16F. Image showing heart cross-section immunostained for COL1A1 and counterstained with DAPI (blue) to visualize nuclei.

[0108] Figures. 16G-16H. qRT-PCR analysis of inflammatory (Fig. 16G) and extracellular matrix genes (Fig. 16H) to assess the effect of drug treatment of D2-mdx mice (red triangles), as compared with saline-treated controls (black triangles). Relative gene expression values normalized to internal Hprt transcript levels. Data represent median ± IQR from n = 6 hearts per cohort. Statistical analyses performed using non-parametric Mann- Whitney test; *p < 0.05, **p < 0.01, ***p < 0.001. In Figs. 16-16H, red triangles in groups on right side of the figures. Figures 17A-17C. Cardiac histopathology in juvenile D2-mcZr model.

[0109] Figure 17A. illustrates Alizarin Red staining of juvenile D2-mdx and BlO-mdx hearts (whole cross-section), highlighting right ventricular (RV) heart damage and calcification in juvenile D2-mdx hearts; scale bars indicate 200 pm.

[0110] Figure 17B-17C. High magnification images from Fig. 17A, showing Alizarin Red staining of juvenile D2-mdx and BlO-mdx hearts, with corresponding quantification of calcified area per total tissue area, fibrosis, damage, and calcification highlighted by black arrowheads in Fig. 17C; scale bars indicate 100 pm. Statistical analyses were performed using Mann-Whitney U test: *p < 0.001.

[0111] Figures 18A-18F. GSEA enrichment plot analysis of top gene ontology (GO) hits from differential gene expression analysis in juvenile D2- Zr and BlO-mtA hearts.

[0112] Figure 18A. Inflammatory Response

[0113] Figure 18B. Extracellular Structure Organization

[0114] Figure 18C. Myeloid Leukocyte Migration

[0115] Figure 18D. Connective Tissue Metabolic Process

[0116] Figure 18E. Eosinophil Chemotaxis

[0117] Figure 18F. Granulocyte Chemotaxis

[0118] Figures 19A-19B Gene expression analysis of D2-WT and B10-WT hearts for selected immune and extracellular matrix targets.

[0119] Figure 19A relative gene expression analysis for dysregulated immune-related genes between D2-mdx versus BlO-mdx (as shown in Figure 19), assessed in juvenile D2-WT and B10-WT hearts.

[0120] Figure 19B relative gene expression analysis for dysregulated immune-related genes between D2-mdx versus BlO-mdx (as shown in Fig. 15), also assessed in juvenile D2-WT and B10-WT hearts.

[0121] DETAILED DESCRIPTION OF THE INVENTION

[0122] Throughout this description, the preferred embodiments and examples provided herein should be considered as exemplar, rather than as limitations of the present application.

[0123] Definitions Dystrophin is a 427 kDa rod-shaped protein encoded by the DMD gene (Xp21 locus), the largest human gene spanning 2.4 megabases. It contains four domains: N-terminal actin-binding domain (ABDI) linking to the cytoskeleton, a central rod domain with 24 spectrin repeats for flexibility, a cysteine-rich domain binding to sarcolemmal glycoproteins and a C-terminal domain interacting with syntrophins. Dystrophin stabilizes muscle membranes by connecting the cytoskeleton to the extracellular matrix via the dystrophin-associated protein complex (DAPC). This prevents damage during muscle contraction and regulates calcium homeostasis. Brain isoforms (e.g., Dp 140, Dp71) influence synaptic function and cognitive development.

[0124] Dystrophin deficiency. Mutations such as deletions, duplications, substitutions or point mutation in DMD disrupt dystrophin production. Duchenne muscular dystrophy (DMD): Frameshift mutations cause near-complete dystrophin loss, leading to progressive muscle degeneration. Becker muscular dystrophy (BMD): In-frame mutations produce truncated but partially functional dystrophin, resulting in milder symptoms. Loss of DAPC components destabilizes the sarcolemma, causing membrane tears and calcium influx. Chronic calcium overload triggers necrosis, fibrosis, and impaired muscle regeneration. Cardiac and respiratory muscles deteriorate, leading to cardiomyopathy and respiratory failure. Muscle weakness with an onset by age 2-5, with gait abnormalities, calf hypertrophy, and loss of ambulation by adolescence. Intellectual disability, ADHD, or autism in 30-50% of cases, have been linked to mutations affecting brain isoforms (Dpl40 / Dp71) and dilated cardiomyopathy and arrhythmias are leading causes of mortality due to deficiency. The methods disclosed herein may be used to prevent, reduce the severity of, or treat diseases, disorders or conditions associated with, or characterized by dystrophin deficiencies.

[0125] Duchenne Muscular Dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness due to alterations in the production of the protein dystrophin, which is essential for maintaining the integrity of muscle cells.

[0126] DMD primarily affects boys, as it is inherited in an X-linked recessive pattern, though it can rarely affect girls who are carriers or have specific genetic circumstances. The disease typically manifests in early childhood, with symptoms such as muscle weakness and wasting (atrophy) appearing between ages 2 and 6, often starting in the pelvic and thigh muscles and later spreading to other muscle groups. As DMD progresses, affected individuals usually lose the ability to walk by their early teenage years and may develop serious complications such as cardiomyopathy (heart muscle disease) and respiratory difficulties due to weakening of the heart and breathing muscles. DMD is ultimately fatal, with most individuals succumbing to heart or respiratory failure in their late teens to early 30s. There is currently no cure for DMD, but treatments focus on managing symptoms, improving quality of life, and slowing disease progression. The methods disclosed herein may be used to prevent, reduce the severity of, or treat diseases, disorders or conditions associated with, or characterized by dystrophin deficiencies including DMD.

[0127] Duchenne Muscular Dystrophy (DMD) Disease Models include both the BlO-mdx (C57BL / 10ScSn) and D2-mdx (DBA / 2J_ murine models. The BlO-mdx model produces mild, minimal clinical symptoms and the D2-mdx model more severe symptoms that closely mimic human DMD including pediatric onset cardiac damage and severe fibrotic skeletal muscle loss starting in early childhood.

[0128] DMD phases. DMD muscular degeneration progresses through five distinct ultrastructural stages: (1) subcellular changes (Stage 1): Subtle volume alterations in muscle components despite normal appearance; (2) myofibril overcontraction (Stage 2): Localized sarcomere hypercontraction causes adjacent filament stretching; (3) Contraction clump formation (Stage 3): Homogeneous masses of contractile filaments replace normal myofibril architecture; (4) macrophage invasion (Stage 4): Immune cells infiltrate to clear cellular debris; and (5) complete structural collapse (Stage 5): Loss of all normal fiber features, replaced by fibrotic or adipose tissue. By adolescence, distal muscles (arms, lower legs) and cardiopulmonary systems become affected, leading to wheelchair dependence by age 12 and lifethreatening respiratory / cardiac complications in late teens. Intervention as disclosed herein may occur before or after one or more of these stages by administration of agents or therapies agonizing FPR, such as FPR1, FPR2 or FPR3.

[0129] DMD conventional therapy. One or more conventional DMD therapies may be performed before or after or concurrently with treatment with the methods disclosed herein using FPR agonists. These include pharmacological therapy with corticosteroids, such as prednisone, prednisolone, or deflazacort to delay disease progression.

[0130] Cardiac medications such as angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and beta blockers can be used to manage cardiac complications by slowing the progression of cardiomyopathy. Mineralocorticoid receptor antagonists (e.g., eplerenone) may also be considered.

[0131] Physical and Occupational Therapy including physical therapy focusing on maintaining joint mobility, preventing contractures or delaying scoliosis may be used. Occupational therapy may also be considered, such as that helps a patient maintain independence in daily activity and cope with progressive muscle weakness. Other therapies include respiratory therapy and orthopedic interventions, as well as diagnostic testing for muscle strength and lung and heart function. These too may be practiced before, concurrently, or after the therapeutic methods disclosed herein involving use of FPR agonists.

[0132] DMD therapies disclosed herein involve use of FPR agonists, such as agonists for FPR2 or other FPRs such as FPR1 or FPR3. Such therapies can involve administering an FPR agonist to subject under conditions that allow it to contact FPR receptors, including oral, nasally, parenterally, intraperitoneally, intravenously, intramuscularly, intrathecally, intracerebroventricular (ICV), intraci sternal magna (ICM, or other conventional modes for administering drugs or biologies.

[0133] FPR2 agonists are compounds that activate the formyl peptide receptor 2 (FPR2), a G- protein coupled receptor involved in regulating inflammation and immune responses. These agonists modulate FPR2 to promote anti-inflammatory or pro-resolving effects, making them potential therapeutics for conditions like chronic inflammation, neurodegenerative diseases, and cardiovascular disorders. Those skilled in the art may select a suitable FPR agonist. Some examples of FPR2 agonists include, but are not limited to, lipoxin A4 (LXA4), MR-39, BMS- 986235, C43, pyrrolidinone urea compounds, endogenous FPR2 agonists, and synthetic FPR2 agonists such as FPR2 Agonist 2. Annexin Al (ANXA1) an FPR agonis is a 37 kDa protein belonging to the annexin superfamily of calcium-dependent phospholipid-binding proteins is mentioned in the Examples herein. BMS-986235 is an FPR agonist appearing in the Examples.

[0134] Other FPR agonists, including FPR1, FPR2 and FPR3 agonists include endogenous agonists such as N-formyl peptides often derived from bacterial or mitochondrial proteins such as N-formyl-Met-Leu-Phe (fMLF), which is a high-affinity agonist for FPR1 and has lower affinity for FPR2; Lipoxins and pro-resolving mediators such a Lipoxin A4 (LXA4), aspirin- triggered 15-epi-lipoxin A4 (ATL), and resolvin DI are endogenous agonists with higher affinity for FPR2. These are involved in promoting the resolution of inflammation; annexin Al and its fragments which can act as agonists at FPR1 and FPR2. Exogenous and Synthetic Agonists such as peptide agonists: Synthetic peptides such as WKYMVm (a hexapeptide)(SEQ ID NO: 1 where m is D-met) are potent agonists for FPR1, FPR2, and FPR3, though with varying affinities. Small-molecule agonists: Recent medicinal chemistry efforts have identified various small synthetic molecules that selectively activate FPR1 or FPR2, including compounds like Quin Cl and TC-FPR 43 for FPR2, and others targeting FPR1. Lipidated peptidomimetics: These are synthetic lipidated peptides or peptidomimetics designed to mimic endogenous lipid mediators and can selectively activate FPR2, sometimes with biased signaling properties. Other or additional agonists are described by, and incorporated by reference to Tsai, et al., Formyl peptide receptor modulators: a patent review and potential applications for inflammatory diseases (2021-2024), EXPERT OPINION ON THERAPEUTIC PATENTS, DOI: 10.1080 / 13543776.2025.2515882 These include but are not limited to AnxAl peptide AMVSEFLKQAWFIENEEQEYVQTVKSSKGGPGSAVSPY PTFNPSSDVAALHKA (SEQ ID NO: 2), a polypeptide or polypeptide analog of

[0135] MVSEFLKQX1WFIENEEQEYX2QTX3KSSKGGPGSAX4SPYPTFNPSS (SEQ ID NO: 3), peptide comprising the peptide backbone AMVSEFLKQAWFIENEEQEYVQTVKS (SEQ ID NO:4, wherein XI or X2 are any conventional or non-standard amino acid, urea derivatives such as oxopyrrolidine urea (comprising formula 11), oxopyrrolidine urea (comprising formula 12), phenylurea (comprising formula 13), and cyclopropyl urea, (comprising formula 14). In some alternative embodiments an FPR2 modulator such as those described below may be used.

[0136] One skilled in the art may select a suitable aryl (AR) or R group, for example, where aryl is phenyl, halogenated (e.g., Cl, F, Br) phenyl, or C1-C6 alkyl- or alkoxyl-substituted phenyl; or where R is C1-C6 alkyl or alkoxy, phenyl or substituted phenyl, amide, azole, or a carboxylic acid or ester.

[0137] Inflammation of Heart (cardiac inflammation). The methods disclosed herein may be applied to ameliorating or blocking one or more types, stages or symptoms of cardiac inflammation. These include pericarditis, myocarditis and endocarditis. Modulation, including reduction of inflammation or acceleration of healing, of the acute phase or initial inflammatory responses, subacute phase or ongoing inflammation, chronic phase or persistent inflammation, and recovery / resolution phase of cardiac inflammation may be treated with the methods disclosed herein.

[0138] Modulation of cytokines and other biological agents. The present disclosure identifies specific cytokines associated with cardiac inflammation or muscular dystrophy such as DMD. In addition to the agonists described herein the proinflammatory effects of such cytokines and biological molecules may be modulated or reduced by methods known in the art including by use of monoclonal antibodies to the cytokines, such as antibodies to TNF, IL-6 IL-ip or other cytokines or to their receptors. Active immunization or gene therapy against proinflammatory agents such as those disclosed herein may be considered as well as small molecule drugs or natural products which reduce inflammation. Anti-inflammatory cytokines such as IL-10, TGF- P or other cytokine antagonists may be administered. Innate immune sensors may be targeted by ssRNA or drugs such as chloroquine. Such methods may be evaluated using the animal models and protocols described and exemplified herein.

[0139] Muscular dystrophy comprises a group of more than 30 inherited genetic diseases that cause progressive weakness and degeneration of skeletal muscles. These conditions result from mutations in genes that affect the production of proteins needed for healthy muscle function, leading to muscle fibers becoming damaged and eventually replaced by fibrous tissue or fat. Over time, this muscle wasting leads to decreased mobility, difficulty performing daily activities, and, in many cases, affects other organs such as the heart and lungs.

[0140] Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease resulting from the absence of dystrophin protein, which causes early-onset cardiac dysfunction in patients. As conventional mouse models of DMD do not manifest cardiac deficits until late adulthood, models with early-onset cardiac impairment are needed to study the mechanisms and potential therapies to target early-onset cardiac deficit in DMD. Unlike the commonly used DMD mouse model BlO-mdx (C57BL / 10ScSn-Dmdmdx / J), D2-mdx (DBA / 2J-mdx) model displays juvenileonset cardiac damage and impaired function. Molecular and histopathological examinations of juvenile D2-mdx hearts revealed cardiac fibrosis accompanied by abnormal extracellular matrix (ECM) organization and heightened leukocyte inflammatory response. This involved altered N-formyl peptide receptors (FPRs) pathway for resolving inflammation. A synthetic FPR2 agonist was used to target the FPR-2 mediated pro-resolving pathway to counter excessive cardiac inflammation in the D2-mdx model. This effectively mitigated the cardiac inflammation, prevented cardiac damage, and reversed ECM and inflammatory response. The present application provides a method associated with spontaneous early-onset cardiac damage caused by dystrophin deficit. A pro-resolution therapy is provided as a promising approach for treating cardiac damage in DMD patients. The present application provides a novel approach to treat cardiac pathology in DMD.

[0141] Glucocorticoids are the standard of clinical care for treating muscle inflammation associated with DMD but this is ineffective in treating cardiac damage and its chronic use by pediatric patients causes significant growth stunting and other side effects. The present application provides a mouse model for studying the spontaneous pediatric onset of cardiac damage and myogenic deficit in DMD. The present application provide a method for clearing excess inflammation (instead of blocking all inflammation) as a suitable therapeutic approach to treat cardiac damage and poor muscle regeneration.

[0142] Other therapeutic methods. The method disclosed herein that administers or induces FPR receptor agonists is beneficial for a range of therapeutic applications beyond muscular dystrophy, including but not limited to: promoting muscle regeneration or repair to accelerate recovery following physical injury or burns; enhancing metabolic efficiency, such as by augmenting aerobic glycolysis for rapid energy production during high-intensity exercise, or by reducing fat mass and promoting thermogenesis. Additionally, FPR agonists may be employed to reduce inflammation, mitigate post-exercise muscle damage, and shorten recovery time. These agents may also be utilized to address neuroinflammation, counteract age-related neurodegeneration, and diminish chronic inflammation as a driver of aging. Furthermore, administration of FPR receptor agonists may improve cardiovascular resilience by enhancing heart function and suppressing cardiac inflammation, thereby providing a multifaceted approach to improving health and longevity.

[0143] Abbreviations. Duchenne muscular dystrophy (DMD), Becker Muscular Dystrophy (BMD), extracellular matrix (ECM), principal component analysis (PCA), gene set enrichment analysis (GSEA), N-formyl peptide receptor (FPR), , Annexin Al (AnxAl), quantitative reverse transition polymerase chain reaction (qPCR), transforming growth factor beta (TGFa), glucocorticoids (GCs), angiotensin-converting enzyme inhibitors (ACEi), differentially expressed genes (DEGs), gene ontology (GO), biological processes (BP), normalized enrichment scores (NES), Angiotensin receptor blocker [ARB], Left Ventricle (LV), Right Ventricle (RV), Induced Pluripotent Stem Cells (iPSCs).

[0144] The features and properties of the present invention are shown in the following examples which illustrate the benefits and advantages of the present invention.

[0145] EXAMPLE 1

[0146] Targeting resolution of inflammation to attenuate muscle regenerative dysfunction in Duchenne Muscular Dystrophy

[0147] Summary. Patients with Duchenne Muscular Dystrophy (DMD) experience chronic inflammation and a failure of muscle regeneration, with recurring muscle injury leading to progressive muscle degeneration and fibrofatty replacement over time. To better understand the factors influencing disease progression, the inventors performed whole muscle bulk RNA- sequencing analysis in two disease models used to study DMD: the 810-mdx and D2-mdx strains. The 810-mdx model is characterized by a consistent, gradual worsening of pathology following juvenile onset, while the D2-mdx model exhibits more aggressive degeneration and inflammation with regenerative failure that peaks early in juvenile development. Given the differences in disease progression between these models, the inventors aimed to uncover transcriptional differences at disease onset and throughout progression, while ruling out inherent strain differences by including wild type controls. The analysis revealed that pro-inflammatory cytokines and chemokines were differentially upregulated in the juvenile D2-mdx model compared to the milder 810-mdx model; notably, these genes are involved in immune cell communication and chemoattraction and are associated with increased and prolonged neutrophil and macrophage extravasation in D2-mdx muscles. While being unbound by any particular theory or explanation, these findings suggest that promoting resolution of inflammation and immune cell clearance can mitigate severe pathology in the D2-mdx model. To test this, the inventors employed FPR2 agonists, which act by enhancing the phagocytic capacity of macrophages and driving neutrophil degranulation, thereby facilitating inflammation clearance and enhancing repair processes. The inventors found that use of pro-resolving FPR2 agonists not only led to downregulation of inflammatory genes but also enhanced regenerative capacity in juvenile D2-mdx muscles, supporting the therapeutic potential of FPR2-targeting pro-resolving drugs to attenuate the effects of unresolved inflammation and improve prognosis in DMD.

[0148] Background. Duchenne muscular dystrophy (DMD) is a degenerative muscle disease affecting approximately 1 in 5,000 male births worldwidel. The disease arises from a mutation in the dystrophin (DMD) gene, leading to a lack of the dystrophin protein. This deficiency results in chronic muscle damage, persistent inflammation, and impaired muscle regeneration23. Over time, these processes drive muscle degeneration and fibro-fatty replacement as the disease progresses4. DMD typically manifests in the skeletal muscles during early childhood, with cardiac and respiratory involvement emerging in late childhood and adolescence. Ultimately, patients often succumb to the disease in their third or fourth decade of life.

[0149] While the primary cause of DMD is the absence of dystrophin, inflammation plays a critical role in both the pathology and progression of the disease. In healthy muscle, injury triggers a coordinated immune response: neutrophils are rapidly recruited to the site of damage, where they release cytokines such as interferon-gamma and TNF. These cytokines further attract and activate macrophages to a pro-inflammatory state within two days post-injury. Subsequently, the expression of interleukin- 10 (IL- 10) increases, leading to a shift toward pro-regenerative macrophages that dominate the injury site by day five. These pro-regenerative macrophages facilitate extracellular matrix remodeling, myoblast fusion, and muscle regeneration.

[0150] In dystrophic muscle, however, the injury response becomes disorganized. Areas of damage, inflammation, and regeneration overlap spatially, disrupting the normally well- coordinated waves of inflammation and repair. As a result, inflammation fails to resolve properly, leading to an aggravated inflammatory response that further impairs muscle regeneration.

[0151] To investigate the role of inflammation in DMD, researchers have employed mdx mouse models. The standard model, C57BL / 10-mdx (BlO-mdx), carries a genetic mutation causing dystrophin deficiency. BlO-mdx mice experience chronic cycles of muscle damage and regeneration, leading to increased immune cell infiltration and progressive degenerative pathology, such as fibro-fatty replacement. Notably, muscle fibrosis in this model is only observed in the late stages of disease, making the phenotype milder than that seen in human DMD, although many disease mechanisms are recapitulated.

[0152] The DBA2 / J-mdx (D2-mdx) mouse model shares the same genetic mutation as BlO-mdx but exhibits more aggressive myofiber damage and inflammation at disease onset (around 6 weeks of age). This heightened inflammation closely mimics the pathogenesis observed in young DMD patients. The early onset of fibrosis in D2-mdx mice impairs myogenic cell fate and accelerates pathological progression compared to the more gradual course seen in BlO-mdx mice.

[0153] To better understand the mechanisms underlying these differences in disease onset and progression, the inventors performed large-scale, longitudinal bulk RNA sequencing of skeletal muscles (quadriceps) from BlO-mdx and D2-mdx mice at presymptomatic (2 weeks), disease onset (1.5 months), and late-stage (8 months) time points, alongside age-matched wild-type controls. The inventors’ data revealed elevated cytokine and chemokine expression in juvenile D2-mdx muscles, which plateaued to levels comparable to those in BlO-mdx muscles by 8 months of age. These cytokines and chemokines are predominantly expressed by macrophages and neutrophils and are involved in immune cell signaling that regulates their timely accumulation and clearance after injury.

[0154] Based on their findings, and while not being bound by any particular theory or explanation, these findings show that promoting inflammation resolution through FPR2 agonist therapies which enhance phagocytosis and neutrophil degranulation — can restore the resolution of inflammation, thereby improving repair processes and mitigating downstream pathologies of DMD.

[0155] This Example demonstrates that treatment with FPR2 -targeted drugs not only reduced the expression of disease-specific inflammatory genes but also improved the regenerative capacity of juvenile D2-mdx muscles. These data further characterize the transcriptional factors involved in the heightened immune response observed in dystrophic pathology and support the therapeutic development of pro-resolving drugs to effectively clear chronic inflammation associated with recurring injuries, ultimately improving outcomes for individuals with DMD.

[0156] METHODS— EXAMPLE 1.

[0157] Animals. All animal procedures were conducted in accordance with guidelines for the care and use of laboratory animals as approved by the Institutional Animal Care and Use Committee (IACUC) of Children’s National Research Institute (CNRI). The C57BL / 10ScSn- DMDmdx / J (810-mdx) and D2.B10-DMDrndx / J mouse models of Duchenne muscular dystrophy (DMD) were utilized for all experiments; both models harbor the same nonsense point mutation in exon 23 of the dystrophin (Dmd) gene, abolishing dystrophin protein expression. The C57BL / 10ScSn / J (810-WT) and DBA2 / J (D2-WT) mouse models served as age- matched, model-specific controls. Mice were obtained from the Jackson Laboratory and were housed at the CNRI Comparative Medicine Unit, where they received daily monitoring, food, water, and enrichment ad libitum, while being maintained under a 12-hour light / dark cycle.

[0158] Tissue Harvesting and Sample Collection. At designated ages corresponding to specific stages of disease progression, mice were euthanized via CO2 inhalation and cervical dislocation. The inventors surgically removed muscles, mounted them on cork using tragacanth gum, flash-froze the tissues in liquid nitrogen-chilled isopentane, and stored the samples at - 80°C8. For all assays, samples were collected from matched regions of the same muscles. Cryosections were prepared using a Leica CM 1950 cryostat for RNA analyses, histology, and immunostaining assays.

[0159] RNA extraction, RNA library preparation, RNA sequencing and bioinformatic analyses. Total RNA was extracted using TRlzol RNA isolation (Life Technologies) from frozen muscle samples. RNA was purified using RNeasy mini elute columns (Qiagen) and DNAse treated using Turbo DNA-free kit (Invitrogen™). Purified, DNAse-treated RNA was quantified by NanoDrop and quality was assessed using Qbit RNA Assays (Thermofisher™) and BioAnalyzer nano chips (Agilent) (RIN> 7.3, Average 8.4±0.33). RNAseq library preparation and sequencing was performed using the TruSeq mRNA stranded kit (Illumina™) and the Illumina NovaSeq

[0160] SP100 Flow Cell with an average coverage of 39.66 million read pairs per sample at 2x150 base pair read length. The quality of the raw fastq reads from sequencer were evaluated using FastQC version 0.11.9. followed by adapter and quality trimming using Trimgalore. Overall analysis summary reports were analyzed using MultiQC vl .11. STAR 2.7.6a and was used to map the reads to the reference mouse genome (GRCm39-mmlO) and RSEM 1.3.1 was used to estimate counts. Differential gene expression analysis was performed using default parameters with Deseq2 version 1.26 (R 3.6). Visualization was performed by PCA, heatmap, Enhanced Volcano and ggplot2 R packages. We set a threshold for log2 fold change (Log2FC)change of greater than an absolute value of 1 to select for the genes with significant differential expression. Gene lists were sorted by Log2FC (highest to lowest) to obtain the ranked list of differentially expressed genes and a padj value cutoff of 0.05 was used to assess statistical significance.

[0161] Gene Set Enrichment Analysis (GSEA) was performed using unthresholded results from DESeq2 pairwise comparisons, which were ranked by Log2 fold change and exported as tab- delimited rank files (.rnk) for upload into the standalone desktop version of GSEA (v4.E0). The GSEA pre-ranked analysis was conducted with most default parameters except that the Ranked list was set to the pairwise comparison .mk file, the gene sets database was specified as "c5. bp. v7.4. symbols" (incorporating Hallmark gene sets, Gene Ontology biological processes, and gene symbols), and the Chip platform was set to "Mouse_Gene_Symbol_Remapping_to_Human_Orthologs_MSigDB v? .4. chip" .

[0162] Gene ontology analysis with Cytoscape and EnrichmentMap. For subsequent gene ontology analysis, the GSEA pairwise comparison results were imported into Cytoscape (v3.9.0) and analyzed using the EnrichmentMap plugin collection (vl.1.0); comparisons were loaded into EnrichmentMap, and the AutoAnnotate function was used with the MCL Cluster Annotation algorithm configured to generate three-word cluster labels, resulting in networks of related GO terms grouped and named according to the most common words within each cluster. Autogenerated names of networks were renamed to fix grammar and nodes arranged to improve legibility. The leading-edge genes for each cluster (node) from the immune and extracellular matrix networks were exported for further analysis. The clusterProfiler and enrichplot packages were used to run additional GO analysis in R under the gseGO function, using a minimum gene set size of 3 and a maximum of 100.

[0163] Deconvolution. Single cell data from Mckellar, Walter et al. was used to determine cell type specific expression of the genes validated by qPCR. Cell type expression was normalized and plotted over 7 days post notexin injury in 20-month-old B10-WT mice to show both cell profile expression and changes to expression temporally through the repair process.

[0164] Histology and histological analyses. Frozen heart tissues were removed from -80°C cryostorage and sectioned at an 8 pm thickness using a Leica CM1950 cryostat chilled to -20°C, where tissues were then mounted on slides and stained using Hematoxylin and Eosin (H&E), Alizarin Red, Picosirius red, and Masson’s Trichrome according to TREAT-NMD Standard Operating Procedures (SOPs) as described previously. Thresholding parameters were applied uniformly to whole cross-section tiled images acquired on the Olympus VS120-S5 Virtual Slide Scanning System using CellSens Version 1.13 and ImageJ FIJI Version 2.1. / 1.53c. For H&E- stained sections, areas of damage were selected using CellSens and quantified as percent damaged tissue area per total cross-sectional muscle area. For Alizarin Red stained sections, calcified areas were selected and quantified using CellSens as percent calcified tissue area per total cross-sectional muscle area. For Masson’s Tri chrome stained sections, areas of fibrosis were calculated using FIJI (Image 174 J) and reported as percent fibrosis per total crossectional muscle area.

[0165] Immunofluorescence. Frozen muscles were removed from -80°C cryostorage and sectioned at an 8 pm thickness and mounted on slides for immunostaining procedures. Muscle sections were stained with anti-eMHC (1 :25, Fl .652, DSHB) and anti-Laminin-a2 (1 :250, L0663, Sigma).

[0166] First, muscle sections were fixed in ice-cold PFA for 10 min, washed in PBS (0.1 % Tween-20), and blocked for 1 h in PBS supplemented with 10% goat serum (Gene Tex), 0.1 % Tween-20 (Sigma- Aldrich), and 10 mg / ml BSA (Sigma-Aldrich). Then sections were incubated with primary antibodies overnight at 4 °C and subsequently probed with Alexa Fluor secondary antibodies, including goat anti-mouse IgGl AlexaFluor 488 (1 :500, A-21121, Thermo Fisher) and goat antiO-rat IgG (H+l) AlexaFluor 647 (1 :500, A-21247, Thermo Fisher). Sections were also counterstained Prolong Gold Antifade with DAPI (P36935, Thermo Fisher) for nuclear staining.

[0167] Gene expression analysis. RNA extracted from Quadriceps from 6week and 8 month old WT and dystrophic mice were used to perform gene expression analysis. Gene expression analysis was performed on D2-mdx samples treated with BMS, Ac-226 or a Saline control. In brief, total RNA was extracted from muscle samples by standard TRlzol (Life Technologies) isolation. Purified RNA (l,000ng) was reverse-transcribed using Random Hexamers and High- Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The mRNAs were then quantified using individual TaqMan assays on an ABI QuantStudio 7 Real-Time PCR machine (Applied Biosystems) using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). Specific mRNA transcript levels were quantified using individual TaqMan assays (Thermo Fisher)specific for each mRNA target, including Itgax (Mm00498701_ml ), Mmpl2 (Mm00500554_ml ), Trem2 (Mm04209424_gl ), Argl (Mm00475988_ml ), Sppl (Mm00436767 ml ), Ccr2 (Mm99999051_gH), Cxcrl (Mm00731329_sl), Ccl3 (Mm00441259 gl), Cxcr2 (Mm99999117 si ), Cxcr4 (MmO 1996749 si ), Tnf (Mm00443258_ml ), Cxcl2 (Mm00436450_ml ), Nos2 (Mm00440502_ml ), 111 b (Mm00434228_ml ), ltgb2 (Mm00434513_ml ), Fprl (Mm00442803), lgals3

[0168] (Mm00802901_ml ). Gene expression for all mRNA targets was normalized to internal Hprt mRNA transcript levels using Hrpt Taqman assay (Mm03024075 m 1 > VIC-MGB).

[0169] Pro-resolution preclinical drug trial. D2-mdx mice (18-19 days-old, n=6, males and females) were treated daily with pro-resolving drugs, FPR2 agonist BMS-986235 (6mg / kg, oral gavage; HY-131180, MedChemExpress) or Ac2-26 (5mg / kg, I.P.; 34429, Cayman Chemical), for 37 weeks. Control D2-mice were administered saline (18-19-day-old, n=6, males and females) for 3 weeks. BMS-986235 was initially resuspended in 10% DMSO (Sigma) and 90% corn oil (Sigma) and further diluted in cherry syrup (NDC-0395-2662-16, Humco) for oral gavage. Ac2-26 was resuspended in PBS for I.P. administration. After the conclusion of the treatment regimen, mice were euthanized and the gastrocnemius, quadriceps, tibialis anterior, and triceps were harvested, flash-frozen in liquid nitrogen-chilled isopentane and stored at -80°C for molecular and histopathological analyses.

[0170] Statistics. Sample size 226 estimations were based on similar studies previously performed by our team using juvenile mdx models43, 44. Data were analyzed using Prism GraphPad software (9.2.0). Data distribution was assessed by Shapiro-Wilks normality test. Statistical analyses were performed using non-parametric Mann-Whitney test based on outcome of Shapiro-Wilks normality test. All ?-values less than 0.05 were considered statistically significant; *p<0.05, **p<0.01, ***p<0.001. Data plots reported as scatter plots with median and Interquartile Range (IQR).

[0171] Microscopy. Images were acquired with an Olympus VS120-S5 Virtual Slide Scanning System with UplanSApo 20x / 0.75 objective and Olympus XM10 monochrome camera or Olympus VS200 Virtual Slide Scanning System with UPlanSApo 20x / 0.80 objective and Olympus VS-264C CMOS color camera. Analysis was performed using Olympus CellSens vl .13 and ImageJ FIJI Version 2.1. / 1.53c (National Institutes of Health) software packages. Statistics. Sample size 226 estimations were based on similar studies previously performed by our team using juvenile mdx models43, 44. Data were analyzed using Prism GraphPad software (9.2.0). Data distribution was assessed by Shapiro-Wilks normality test. Statistical analyses were performed using non-parametric Mann-Whitney test based on outcome of Shapiro-Wilks normality test. All / ?-values less than 0.05 were considered statistically significant; *p<0.05, **p<0.01, ***p<0.001. Data plots reported as scatter plots with median and Interquartile Range (IQR).

[0172] RESULTS— EXAMPLE 1

[0173] Trajectory of longitudinal immune-related gene expression for I) 2- mdx and Bl 0-mdx muscle align with model-specific disease severity and progression.

[0174] To better characterize the mechanisms driving disease onset and long-term progression in mdx models of DMD, we performed bulk RNA sequencing (RNAseq) of quadricep muscles harvested from Bl 0-mdx and D2-mdx at 2 weeks, 1.5 months and 8 months of age, representing pre-symptomatic, disease onset and late-stage disease timepoints, respectively. The inventors additionally included age-matched, strain-specific wild type (WT) controls to account for any inherent differences between the BIO and D2 genetic backgrounds. These data were then bioinformatically analyzed including gene ontology (GO) analysis and further validated through additional qPCR testing (Fig. 1A).

[0175] The PC A of the BIO and D2 samples from RNAseq showed that samples from the pre- symptomatic time point clustered closely together, with mdx and WT samples overlapping. This would indicate that at 2 weeks of age, disease and WT transcriptomic signatures exhibit very low variance (Fig. IB, ID).

[0176] Additionally, when all samples were plotted together on a correlation matrix, all 2-week samples, for both mdx and WT of the BIO and D2 backgrounds, clustered closely together, indicating this trend is consistent within and across strains (Fig. 7).

[0177] Interestingly though, at disease onset, we observed the most variance between D2-mdx and D2-WT samples, while B 10-mdx is most differentiated from B10-WT at late-stage disease. This is further demonstrated by the heatmaps of differentially expressed genes (DEGs) in mdx samples between 2 weeks and 1.5 months of age across all time points.

[0178] For the BlO-mdx strain, a large portion of genes were highly expressed at 1.5 months and only become more upregulated by 8 months (Fig. 1C).

[0179] In the D2- / 7?r / .v, however, the majority of DEGs were most highly expressed at disease onset (1.5 months) as opposed to late state disease (8 months) (Fig. IE). These results mirror the gradual progression of pathology in ^10-mdx compared to the sudden severity of disease pathology at disease onset in D2-mdx, that while progressive over time in terms of muscle wasting and fibrotic replacement, does not necessarily lead to increased expression of DEGs between 1.5 and 8 months of age.

[0180] Heightened cytokine and chemokine expression accompanies the sudden and severe pathologies at disease onset in D2-mdx mice. The inventors sought to further investigate the specific genes involved in the differential pathogenesis at disease onset between BlO-mdx and D2-mdx (1.5 months of age). It was found that although many genes peaked in D2-mdx at onset (Fig. IE), not all were differentially expressed genes in D2-mdx were upregulated compared to BlO-mdx at this time (Fig. 2A).

[0181] All significant DEGs between the two strains at disease onset were therefore used to perform gene ontology (GO) analysis. Many of the top activated GO terms were related to inflammatory response, leukocyte chemotaxis, and activation of macrophage- and neutrophilspecific inflammatory pathways (Fig. 8).

[0182] From this analysis we compiled a list of 204 significant DEGs (FC >± 1, pval < 0.05) at disease onset that were involved in any immune or inflammatory pathways as identified by GO analysis (Fig. 9A).

[0183] Of these immune related genes, the inventors plotted the expression of the top 25 most dysregulated highlighting their heightened expression in D2-mdx compared to H10-mdx (Fig. 2C). When the expression of the same 25 genes was plotted comparing 1.5-month-old WT from both strains, only 1 gene was significantly altered in expression between WT strains, indicating that the differential expression in mdx models is not attributed to genetic background (Fig. 9B).

[0184] The inventors also identified several chemokines (Cxcrl, Cc / 3, Ccr5) and cytokines Z / s B and 1 / lr) were among the top 25 dysregulated immune and inflammatory genes between mdx strains at disease onset. Cytokines and chemokines play a key role in leukocyte activation and clearance, processes known to be dysregulated in dystrophy23- 25. Based on the genes that appeared in the top 25 inflammatory DEGs, we plotted additional cytokine and chemokine expression at 1.5 weeks, which illustrates the trend of D2-mdx muscles having the highest expression as compared to BlO-mdx or either WT strain (Fig. 2D, 2E). These data point to inflammatory pathways and cytokine and chemokine signaling as key contributors in the differential pathologies displayed at disease onset between BlO-mdx and D2- mdx.

[0185] Longitudinal gene expression analysis indicates aberrant inflammatory signaling peaks at disease onset in D2-mdx while it progressively increases over time in Bl 0-nidx muscles. Given the prevalence of heightened expression of inflammatory genes at disease onset in D2-mdx compared to BlO-mdx we were curious how transcript levels of these genes changed along the course of disease progression. It was clear that many genes changed in expression in both disease strains from onset to late-stage disease (Fig. 10A, 10C).

[0186] To further investigate the dynamic of transcriptional changes over time, we performed extensive GO analysis with the most significant DEGs in both strains from onset to late-stage disease. In BIO- mdx the GO terms activated at 1.5 months of age were related to cell cycle and mineralization, while inflammatory response and leukocyte related terms appear suppressed in 1.5 months compared to 8 months (Fig. 3A, 3B). Interestingly, no inflammatory GO terms were in the top 10 activated at disease onset compared to late stage in D2-mdx, and all suppressed terms at 1.5 months were associated with mitochondrial respiration or lipid metabolism, which has been shown to be closely related to immune cell phenotype and function (Fig. 3D, 3E).

[0187] When the inventors plotted the list of the top 25 inflammatory related DEGs from the disease onset cross strain analysis longitudinally, we can see a trend that around half of these genes progressively increased in expression over the course of disease in BlO-mdx, mirroring the progression of pathology over time (Fig. 3C). However, in the D2-mdx model, the expression of the majority of these 25 inflammatory genes actually decrease in expression in late-stage disease compared to their levels at disease onset (Fig. 3F). These trends can also be seen throughout all 204 inflammatory genes identified by the cross strain 1.5-month analysis, where expression is highest in D2-mdx at onset, while many of these genes have their highest expression at 8 months of age in BlO-mdx (Fig. 10B, 10D). Overall, this indicates that the genes driving exacerbated pathology in D2-mdx at disease onset may plateau or even diminish over time, while in BlO-mdx their expression appear to continuously increase, illustrating a potential mechanism to explain why the two models diverge early on, but appear pathologically and functionally similar at later stages of disease progression. Differences in expression of key inflammatory genes is less notable between D2-mdx and BlO-mdx at 8 months compared to disease onset. To further investigate the changes in immune and inflammatory pathways in late-stage disease, the inventors ran a parallel pipeline to the 1.5-month-old cross strain analysis in our 8-month-old BlO-mdx and D2-mdx cohorts. There were still hundreds of genes that differed significantly in expression at this time point (Fig. 4A), prompting us to investigate what pathways these genes were associated with. The top 10 activated pathways in D2-mdx included ATP synthesis and respiratory chain complex, while activated pathways in BlO-mdx at 8 months were related to lipid storage and macrophage and foam cell differentiation (Fig. 4B).

[0188] Unlike the 1.5-month comparison, we did not observe an overabundance of inflammatory pathways upregulated in D2-mdx, which provides evidence that by late-stage disease inflammation levels and associated immune cell signaling become more comparable across these mdx strains. This is further demonstrated by the expression of our compiled 204 immune-related genes identified at disease onset, where although there is still visibly higher expression in D2- mdx as compared to BlO-mdx at 8 months, the difference is far less dramatic than what we observed at disease onset (Fig. 4C, 4D; Fig. 2C; Fig. 9A).

[0189] Chemokines and cytokine expression specific to macrophage and neutrophil activity peak in expression at disease onset in D2-mdx compared to late-stage disease or either disease stage in BlO-mdx. To confirm the peak of cytokines and chemokines in D2-mdx at disease onset, the inventors performed qPCR of several probes from our bulk RNAseq dataset. Along with several key cytokines and chemokines, they also included a few non-cytokine / chemokine immune related genes from the 1.5-month cross strain analysis in our experiments to further validate the immune gene signature across different age points. By utilizing publicly available single cell sequencing data of muscle and stromal cells collected at different time points post injury, they were able to confirm that our transcriptional targets were expressed primarily by macrophages, monocytes or neutrophils during the normal repair process (Fig. 10A and 10B). In all 12 immune genes evaluated, including macrophage-specific targets Itgax, Mmpl2, Trem2, ArgI, Sppl, Ccr2, and neutrophil-specific targets Cxcrl, Cc / 3, Cxcr2, Cxcr4, Tnf Cxc / 2, the highest expression levels were consistently found in 1.5-month D2-mdx muscles, relative to all other cohorts (Fig. 5A and 5B). However, for several genes, including Cc / 3, Sppl, asx& Argl, the inventors found no significant differences between the BlO-mdx and D2-mdx at the 8-month time point (Fig. 5A and 5B). This data points to the heightened inflammatory signals in D2-mdx contributing to the sudden and severe pathology at disease onset, which is far less pronounced by late-stage disease. This prompted us to investigate if we could promote resolution of aberrant inflammation in the D2-mdx model to lessen the severity of pathology associated by this asynchronous and unresolved pro-inflammatory response.

[0190] Use of FPR2 agonists promote resolution of inflammation and attenuate aberrant cytokine and chemokine responses to enhance repair in D2-mdx. As muscle fiber damage and death results in chronic inflammation and progressive muscle fibrosis, anti-inflammatory corticosteroids have been the mainstay of clinical management of DMD. However, these drugs still work by blocking inflammation instead of clearing inflammation (Fig. 6A).

[0191] Inflammation following skeletal or cardiac muscle injury is required for repair and supports greater regenerative ability. Despite the efficacy of corticosteroids to treat skeletal muscle symptoms and prolong ambulation, they have been shown to impair the regenerative capacity of skeletal muscle with chronic use from childhood, and further, there are varying reports on their cardio-respiratoiy benefits and side effects in DMD patients and DMD mouse models. Activating AnxAl-FPR2 signaling via FPR2 agonists offers an alternate to corticosteroids to address aberrant muscle inflammation in DMD by activating resolution of inflammation, instead of blocking inflammation (Fig. 6A).

[0192] Such an approach, termed as 'Resolution Pharmacology', has been enabled by harnessing the physiological mechanism that tapers and resolves inflammation activated due to tissue damage to return the inflamed tissue to its uninflamed functional state. Unlike anti-inflammatory therapy that work to prevent inflammation (Fig. 6A), these pro-resolving therapies work to resolve chronic inflammation, without the trade-off of blocking the acute inflammatory cues required for efficient tissue repair (Fig. 6A).

[0193] Recent findings by our laboratory revealed use of an FPR2 agonist helped physiologically resolve cardiac muscle inflammation and mitigate its downstream influence on fibrotic degeneration of cardiomyocytes, preventing juvenile onset cardiac muscle loss39. Thus, the inventors were interested in evaluating whether activation of pro-resolution FPR signaling using an FPR2 agonist would also prove beneficial to attenuate the pathologies associated with unresolved inflammation in the severe D2-mdx model. To test the effectiveness of this approach, they utilized synthetic FPR2 agonist BMS-986235 and ANXA 1 mimetic peptide Ac2-26 and initiated a 3 week long daily dosing regimen in D2-mdx mice starting at 2.5 weeks of age (n=6 / cohort), prior to disease onset, after which point muscles were harvested for subsequent analysis (Fig. 6B).

[0194] Analysis of quadriceps cross-sections indicated increased regenerative capacity in both BMS-986235 and Ac2-26 drug-treated groups compared to saline controls, based on quantification of embryonic myosin heavy chain positive fibers (eMHC+), centrally nucleated fibers (CNFs), and decreased areas of damage (Figs. 6C-6F). Considering the enhanced regeneration with treatment, we next wanted to confirm that treatments were promoting resolution of inflammation as anticipated. Treatment with FPR2 agonists lowered the overall fluorescent area of pan-macrophage marker F4 / 80 throughout cross-sections (Fig. 6G). More specifically, pro-inflammatory macrophage marker INOS was reduced in damaged areas in response to both treatments compared to saline controls, while pro-regenerative macrophages did not alter significantly in numbers across the cohorts within areas of damage (Fig. 6H and 61).

[0195] This indicated that the inflammatory process was able to effectively transition from proinflammatory to pro-regenerative stages with treatment. Additionally, qPCR was performed on BMS-986235-, Ac2-26- and saline-treated groups, which indicated that many of the key inflammatory genes identified as aberrant in D2-mdx muscles from our bulk seq data, were in fact now lower in expression following acute treatment when compared to the control group (Fig. 6J-6O).

[0196] While additional studies are required to evaluate the full promise of pro-resolving therapy in terms of attenuating long-term disease progression in dystrophic muscle, our data from this acute preclinical trial indicated this therapeutic approach served to effectively clear proinflammation and promote progression towards regeneration to enhance muscle repair while dampening pathogenic inflammatory signaling in vivo.

[0197] These results in conjunction with the inventors’ recent observations showing use FPR agonists to attenuate onset and severity of pathology in dystrophic hearts, demonstrates dual benefit of FPR agonists to treat both skeletal and cardiac muscle pathology in dystrophic mdx models and its potential application as an alternative to chronic corticosteroids use for DMD patients. EXAMPLE 2

[0198] Failure to resolve inflammation contributes to juvenile onset cardiomyopathy in a mouse model of Duchenne Muscular Dystrophy

[0199] Summary — Example 2. Absence of dystrophin protein causes cardiac dysfunction in patients with Duchenne muscular dystrophy (DMD). Unlike boys with DMS, the common mouse model of DMD (B l O-u / cZr) does not manifest cardiac deficits until later adulthood. This has limited our understanding of the mechanism and therapeutic approaches to target the onset of cardiac pathology in DMS. Here the inventors show that the mdx mouse model on the DBA / DJ genetic background (D2-mdx) displays juvenile onset cardiomyopathy. Molecular and histopathological analysis revealed to the inventors heightened leukocyte chemotactic signaling and failure to resolve inflammation leading to chromic inflammation and fibrotic organization of extracellular matrix (ECM) leading to cardiac pathology in juvenile D2-mdx mice. To treat this pathology, the inventors employed an N-formyl peptide receptor (FPR) agonist that helped physiologically resolve acute inflammation and mitigated inflammation and fibrosis, preventing juvenile onset cardiomyopathy. This work identifies events associated with pediatric-onset cardiac damage in a mdx model and demonstrates a new therapeutic avenue for treating cardiomyopathy in boys with DMD.

[0200] Background — Example 2

[0201] Duchenne Muscular Dystrophy (DMD) is a severe progressive muscle disease caused by the absence of the dystrophin protein. Dystrophin plays a crucial role in maintaining the integrity of the sarcolemmal membrane by facilitating the assembly and function of the dystrophin- associated protein complex; thus, its absence renders skeletal muscle cells more susceptible to mechanical damage and increased muscle degeneration.

[0202] Dystrophin deficiency in cardiomyocytes also increases their vulnerability to sarcolemmal damage and cell death, chronic inflammation, and cardiac fibrosis. These pathologies also manifest in patients and lead to thinning of the left ventricle (LV) wall, causing progressive dilation and dilated cardiomyopathy that results in heart failure.

[0203] Patients with DMD experience symptoms early in life, with cardiac deficits being a major contributor to premature mortality not only in DMD but also in DMD carriers and in Becker Muscular Dystrophy (BMD). While the commonly used B I 0- / x model exhibits skeletal myopathy at an early age, cardiac deficit is not evident until late adulthood. The advent of the D2-mdx model identified greater disease severity and fibrosis as compared to BlO-mdx, even in younger mice. Excessive skeletal muscle fibrosis in the D2-mdx model results from an increase in transforming growth factor beta (TGF-P) signaling, and cardiac deficit is reported as early as adulthood (16 weeks of age).

[0204] While therapeutic approaches to address cardiac deficits in patients with dystrophin deficiency are a topic of active investigation, anti-inflammatory glucocorticoids (GCs), angiotensin-converting enzyme inhibitors (ACEi), and angiotensin receptor blockers (ARBs) are commonly used for these patients. Anti-inflammatory therapy by GC has the longest history of use in DMD patients and has mixed reports of cardiac effect and side effects in both DMD patients and mouse models. An anti-inflammatory protein activated by GCs that mediates GC efficacy is Annexin Al (AnxAl). AnxAl works similarly to the endogenous pro-resolving lipid mediator Lipoxin A4 and Resolvin DI by helping to resolve acute inflammation by binding Formyl Peptide Receptors (FPRs. This feature of AnxAl has led to the advent of natural and synthetic agonists of FPR2, which, unlike GCs, reduce inflammation by promoting resolution of chronic inflammation instead of suppressing the tissue’s inflammatory response.

[0205] Use of FPR2 agonists reduces acute cardiac damage in various tissue injury models, including myocardial infarction, where it restricts premature heart failure and restores tissue function. Just as endogenous FPR2 agonists, nanomolar doses of a synthetic agonist (BMS- 986235) also resolve chronic inflammation in preclinical models, which has led to its progress to clinical studies (Clinical Trial NCT03335553). This drug activates macrophage transition to a pro-resolving (M2-like) state by enhancing phagocytosis and neutrophil apoptosis, which regulates their chemotaxis. All of which help improve mouse survival, reduce scarring, and preserve tissue degeneration. These are desirable features of therapies to target cardiac inflammation and fibrosis associated with the cardiac pathology observed in DMD patients.

[0206] To understand the early onset of cardiac dysfunction in the D2-mdx model, we investigated the factors that distinguish pediatric initiation of cardiac dysfunction as compared with the late adult onset in the BlO-mdx model. This revealed onset of cardiac pathology in juvenile (<6 weeks old) D2- / .r mice and showed that this is associated with excessive immune infiltration, fibrotic ECM replacement, and degenerative remodeling of cardiac ventricular walls. It identified increased leukocyte chemotactic signaling and failure to resolve inflammation as major contributors to the initiation of cardiac pathology. To address this deficit, we test a drugbased pro-resolution therapy for preclinical evaluation as a therapeutic approach to mitigate early-onset cardiac damage in DMD mouse models and patients.

[0207] Methods — Example 2

[0208] Animals. All animal procedures were conducted following guidelines for the care and use of laboratory animals as approved by the Institutional Animal Care and Use Committee (IACUC) of Children’s National Research Institute (CNRI). The C57BL / 10ScSn-DMDmdx / J (BlO-mdx) and D2.B10-DMD / J mouse models for DMD were utilized for experiments, and both harbor the same nonsense point mutation in exon 23 of the dystrophin (Dmd) gene, thereby abolishing dystrophin protein expression. The C57BL / 10ScSn / J (B10-WT) and DBA2 / J (D2- WT) mouse models were used as age-matched, model-specific controls. Mice were obtained from the Jackson Laboratory and housed at the CNRI Comparative Medicine Unit where they were provided daily monitoring, food, water, and enrichment ad libitum, while being maintained under 12 h light / dark cycles.

[0209] Tissue Harvesting and Sample Collection. Mice were euthanized via CO2 inhalation and cervical dislocation at designated ages corresponding to specific stages of disease progression. Muscles were surgically removed, mounted on cork with Tissue-Tek OCT compound, flash- frozen in liquid nitrogen-chilled isopentane, and stored at -80°C. For all assays, samples were collected from targeted regions of the same muscles by collecting cryosections (Leica CM1950 cryostat) for analyses or histology and immunostaining assays.

[0210] RNA Extraction, RNA Library Preparation, RNA Sequencing, and Bioinformatic Analyses. Total RNA was extracted using TRIzol RNA Isolation (Life Technologies) from frozen muscle samples. RNA was purified using RNeasy MiniElute columns (Qiagen) and DNase treated using the Turbo DNA-free kit (Invitrogen). Purified, DNase-treated RNA was qualified by NanoDrop and quality assessed using Qubit RNA Assays (Thermo Fisher) and BioAnalyzer Nano chips (Agilent) (RIN >7.8, average 8.3±0.32). RNA library preparation and sequencing were performed using the TruSeq mRNA stranded kit (Illumina) and the Illumina HiSeq4000 Flow Cell with an average coverage of 63.05 million read pairs per sample at 2><75 base pair read length. Quality of the raw FASTQ reads from the sequencer was evaluated using FastQC version 0.11.5, followed by adapter and quality trimming using Trim Galore. STAR 2.5.3a was used to map the reads to the reference genome (GRCm38-mmlO). The mapped reads were counted using RSEM (version 1.3.2) with a reference genomic feature fde (Gene Transfer Format, GTF). Overall analysis summary reports were analyzed using MultiQC vl.6. Differential gene expression analysis was performed using R Bioconductor packages. Briefly, tximport™ version 1.18.0, was used to extract the raw counts, estimated by RSEM. Deseq2 version 1.26 (R 3.6), was used for normalization and differential expression evaluation. Principal Component Analysis (PCA) was evaluated using the plotPCA function of DESeq2. Visualization of the first 2 Principal components was performed using the ggplot2 (version 3.3.5) (<https: / / www.bibguru.eom / r / how-to-cite-r-package-ggplot2 / >) package. We set a threshold for log2 fold change (Log2FC) change of greater than an absolute value of 0.6 to select for the genes with significant differential expression. Gene lists were sorted by Log2FC (highest to lowest) to obtain the ranked list of differentially expressed genes and a padj value cutoff of 0.05 was used to assess statistical significance. Heatmaps were created using pheatmap (version 1.0.12) (http: / / github.com / raivokolde / pheatmap), and volcano plots using EnhancedVolccmo (<https: / / bioconductor.org / packages / devel / bioc / vignettes / EnhancedVolcano / inst / doc / EnhancedV olcano.html>).

[0211] Gene Set Enrichment Analysis (GSEA). DESeq2 pairwise comparison results were filtered for 0.6 log2FC and 0.05 adjusted p value and exported as tab-delimited rank files (.rnk files) for upload into the stand-alone desktop version of GSEA (v4.1.0). The GSEA pre-ranked analysis was used with most default parameters except the following: the Ranked list = pairwise comparison ".rnk" file, Gene sets database = "c5.bp.v7.4. symbols" (Hallmark gene sets, GO biological processes), and the Chip platform = "Mou se_Gene_Sy mb ol_Rem apping_M SigDB . v7.4. chi p " .

[0212] Gene ontology analysis with Cytoscope and EnrichmentMap. The GSEA pairwise comparison results were uploaded into Cytoscape (v3.9.0) and analyzed using the EnrichmentMap pipeline collection plugins (vl.1.0). Comparisons were loaded into EnrichmentMap and the AutoAnnotate function was used with the MCL Cluster Annotation algorithm set to 5 words. The results are networks of related GO terms that are grouped together into a named network based on the most common words in each GO term within. Auto-generated names of networks were renamed to fix grammar, and nodes arranged to improve legibility. The leading-edge genes for each cluster (node) from the immune and extracellular matrix networks were exported for further analysis.

[0213] Histology and Histological Analyses. Frozen muscles were removed from -80°C cryostorage and sectioned at an 8 pm thickness using a Leica CM 1950 cryostat chilled to -20°C, where tissues were then mounted on slides and stained using Hematoxylin and Eosin (H&E), Alizarin Red, Picrosirius Red, and according to CNMC standard operating procedures (SOPs). Thresholding parameters were applied uniformly to whole cross-section tiled images acquired on the Olympus VS120-S5 Virtual Slide Scanning System using CellSens version 1.13 and Imaged FIJI version 2.1.0 / 1.53c. For H&E-stained images, areas of damage were selected using CellSens and quantified as percent damaged tissue area per total cross-sectional muscle area. For Alizarin Red-stained images, calcified areas were selected and quantified using CellSens as percent calcified tissue area per total cross-sectional muscle area. For Masson’s Trichrome-stained images, areas of fibrosis were calculated using FIJI (ImageJ) and reported as percent fibrosis per total cross-sectional muscle areal2.

[0214] Immunofluorescence. Frozen muscles were removed from -80°C cryostorage and sectioned at an 8 pm thickness and mounted on slides for immunostaining procedures. Muscle sections were incubated with anti-F4 / 80 (1 : 100, MCA497R, Bio-Rad), anti-COLlAl (1 : 100, ab21286, Abeam), and anti-GAL-3 (1: 100, ab76245, Abeam). First, muscle sections were fixed in ice-cold PFA for 10 min, washed in PBS (0.1% Tween-20), and blocked for 1 h in PBS supplemented with 10% goat serum (GeneTex), 0.1% Tween-20 (Sigma-Aldrich), and 10 mg / ml BSA (Sigma-Aldrich). Then sections were incubated with primary antibodies overnight at 4°C and subsequently probed with Alexa Fluor secondary antibodies, including goat anti-rat (H+L) Alexa Fluor 647 (1 :500, A-21247, Thermo Fisher), goat anti-rabbit (H+L) Alexa Fluor 488 (1:500, A-11008, Thermo Fisher), and goat anti-rabbit (H+L) Alexa Fluor 568 (1 :500, A-11011, Thermo Fisher). Sections were counterstained with wheat germ agglutinin (WGA) Alexa Fluor 647 (1 :500, W32466, Thermo Fisher) to delineate cardiomyocytes and ProLong Gold Antifade with DAPI (P36935, Thermo Fisher) for nuclear staining.

[0215] Gene Expression Analysis. Hearts from juvenile and adult dystrophic mice were used for gene expression analysis. In brief, total RNA was extracted from muscle samples by standard TRIzol (Life Technologies) isolation. Purified RNA (1000 ng) was reverse transcribed using random hexamers and the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The RNAs were then quantified using individual TaqMan assays on an ABT QuantStudio 7 Real Time PCR machine (Applied Biosystems) using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). Specific mRNA transcript levels were quantified using individual TaqMan assays (Thermo Fisher) specific for each mRNA target, including Ccl8 (Mm01297183_ml), Cd3 (Mm00441259_ml), Ccl2 (Mm00441242_ml), Illb

[0216] (Mm00434228_ml), Anxal (Mm00440225_ml), Lgals3 (Mm00802901_ml), Stab2

[0217] (Mm00454684_ml), Argl (Mm00475988_ml), I117r (Mm00434295_ml), Adam8

[0218] (MmOl 163449_ml), Trem2 (Mm04209421_ml), Fpr2 (Mm00484464_sl), Sppl

[0219] (Mm00436767_ml), Fnl (Mm01256744_ml), Collal (Mm00801666_gl), Itgax

[0220] (Mm00498701_ml), Mmpl2 (Mm00500554_ml), and Timpl (Mm01341361_ml)12.

[0221] Pro-Resolution Preclinical Drug Trial. D2-mdx mice (18-19 days old, n=6, males and females) were treated daily with pro-resolving drug BMS-986235 (6 pg / g, oral gavage; HY- 131180, MedChemExpress) for 3 weeks. Control D2-mdx mice were administered saline (18-19 days old, n=6, males and females) for 3 weeks. BMS-986235 was initially resuspended in 10% DMSO (Sigma) and 90% corn oil (Sigma) and further diluted in cherry syrup (NDC-0395-2662- 16, Humco) for oral gavage. After treatment, hearts were harvested, imaged, flash-frozen in liquid nitrogen-chilled isopentane, and stored at -80°C for molecular and histopathological analyses.

[0222] Microscopy. The inventors used the Olympus VS120-S5 Virtual Slide Scanning System with a UPlanSApo 40* / 0.95 objective, Olympus XM10 monochrome camera, and Olympus VS ASW FL 2.7 imaging software. Analysis was performed using Olympus CellSens 1.13 and ImageJ FIJI version 2.1.0 / 1.53c software (National Institutes of Health). Brightfield whole tissue imaging was performed using a Leica MZ6Z Digital HD Stereo Microscope with a 10* / 22 mm objective and camera system.

[0223] Statistics. Sample size 226 estimations were based on similar studies previously performed by our team using juvenile mdx models43, 44. Data were analyzed using Prism GraphPad software (9.2.0). Data distribution was assessed by Shapiro-Wilks normality test. Statistical analyses were performed using non-parametric Mann-Whitney test based on outcome of Shapiro-Wilks normality test. All / 2-values less than 0.05 were considered statistically significant; *p<0.05,**p<0.01, ***p<0.001. Data plots reported as scatter plots with median and Interquartile Range (IQR). RESULTS— EXAMPLE 2

[0224] I)2-mdx model Exhibits Pediatric-Onset Cardiac Damage.. The inventors have previously described the use of D2- / MtZr model of DMD to define mechanisms contributing to pediatric-onset severe skeletal muscle degeneration. Here we observed extensive pericardial damage of the ventricular wall in juvenile (<6-week-old) T)2-mdx hearts (Fig. 12A). In contrast, the age-matched milder DMD model, B I 0- / Wx, did not demonstrate any conspicuous histopathology (Fig. 12B). In D2-mdx, the histopathological damage extended to both the right and the left ventricular walls (Fig. 12C). Use of Sirius Red staining revealed extensive fibrosis, which manifests at notable levels in both the endomysium and perimysium of the ventricular walls in D2- / ?itZr (Fig. 12D). Whole tissue cross-sectional analysis of H&E and Sirius Red stained hearts revealed increased cardiac wall damage and cardiomyocyte degeneration (p<0.001) (Fig. 12C, 12E, 12F), and a concomitant increase in fibrosis (p<0.05) in the juvenile D2-mdx hearts compared to age-matched B 10- / ??<7x hearts (Fig. 12D, 12G, 12H). The extent of damage in D2-mdv mice varied between the mice, reaching up to nearly 20% of total cardiac muscle cross-sectional area in the most affected case (Fig. 12E, 12F). Reminiscent of the skeletal muscle damage in D2-mtfr, the cardiac muscle also exhibited increased calcification in damaged areas, which was exclusive to juvenile D2- / 7?cZr hearts (Fig. 17). With mixed reports of cardiac pathology in older DBA2 / J wildtype (D2-WT) hearts we assessed if the juvenile-onset cardiac damage was also a feature of 252 the juvenile D2-WT mice. Hearts from juvenile D2- WT showed no signs of gross pathology and were comparable to the hearts from the age- matched BIO wildtype (B10-WT) mice (Fig. 18A, 18B). This was further confirmed by the microscopic evaluation that showed absence of any endomysial or perimysial fibrosis or calcification in the D2-WT hearts throughout the left and right ventricular walls (Fig. 18C, 18D). Overall, these histopathological analyses reveal early onset spontaneous cardiac damage with significant fibro-calcification in the V)2-mdx, offering a model to investigate the mechanisms of pediatric-onset cardiac damage and accompanying endomysial fibrosis observed in DMD.

[0225] Dysregulated inflammatory response characterizes cardiac damage in juvenile D2- mdx. To identify the molecular alterations associated with the pediatric- versus adult-onset cardiac damage caused by dystrophin deficit, we performed a comparative transcriptomic analysis of hearts from D2- / ??<7x and Bl O-zwt / x mice. Using bulk RNA sequencing we examined genes that show significant differential expression (Log2FC 0.6 and padj value 0.05) between the 6-week-old male B I 0- / Wx and D2-m / r hearts. This identified a total of 5,344 differentially expressed genes (DEGs) representing 22.3% of all protein-coding murine genes. Principal component analysis (PCA) identified that gene expression profiles of D2- / Wx hearts were distinctly segregated from B l O-W.r along both PCI (72% variance) and PC2 (15% variance) axes (Fig. 13A). Intra- and inter sample variability of DEGs by heatmap analysis of the top 2,719 DEGs revealed consistent trends for both up- and down-regulated genes based on the genotype at disease onset, with 1,586 genes upregulated in D2-mdx and 1,133 genes upregulated in B l O-m / .v (Fig. 13B).

[0226] To identify the functions of the DEGs and assess how they may contribute to the observed histopathology and functional deficit in juvenile D2- / «tZr hearts, we performed Gene Set Enrichment Analysis (GSEA) using our DESeq2 pairwise comparison results (0.6 log2FC and 0.05 adjusted p-value), followed by gene ontology (GO) analysis using Cytoscape’s EnrichmentMap pipeline (Fig. 13C). GO EnrichmentMap analysis predominantly indicated that upregulated DEGs in the juvenile D2-mdv fell within biological processes (BP) specific to immune response and extracellular matrix organization and remodeling. These included GOBP terms implicated in regulation of the inflammatory response, immune cell activation, leukocyte chemotaxis and migration, and cytokine signaling (Fig. 13C, red clusters, left four ovals), as well as extracellular matrix organization and disassembly, osteoblast differentiation, substrate adhesion and bone remodeling, and overall tissue homeostasis (Fig. 13C, blue clusters (right four ovals). Outputs from GSEA provided a comprehensive GO analysis of all upregulated and downregulated GOBP terms, their respective normalized enrichment scores (ES), p-values, and enrichment plots identifying alterations in inflammatory and extracellular matrix GOBPs have strongest positive enrichment scores (ES) (Fig. 19). The top 20 upregulated GOBP terms identified dysregulation of the inflammatory response or extracellular matrix architecture that included a total of 331 upregulated DEGs common between them .

[0227] This was validated by the quantification of the normalized expression for the top 20 most upregulated DEGs specific to unique GOBPs such as inflammatory response (GO:0006954)(Fig. 13D) and external encapsulating structure (extracellular matrix) organization (G0:0045229) (Fig. 13E). The leading-edge genes identified increased expression of pro- inflammatory chemokines, and components of fibrotic extracellular matrix remodeling as potential drivers of overt pediatric-onset cardiac damage in the D2- / wt& (Fig. 13, Fig. 19).

[0228] To independently validate the role of aberrant acute inflammatory response of granulocyte and leukocyte chemotaxis by way of chemokine / cytokine signaling pathways, we examined these genes by qPCR in an expanded (n = 9 D2-mt / x and n = 7 B10- / »c / .r) cohort of juvenile mdx mice. This validated our findings from RNAseq analysis and confirmed significant upregulation of macrophage-secreted pro-inflammatory C-C family chemokines, Cc / 3 (macrophage inflammatory protein- la) and Ccl8 (monocyte chemoattractant protein-2), that regulate neutrophil, monocyte and 304 lymphocyte chemotaxis following acute tissue damage (Fig. 13A; p<0.001}.

[0229] Similarly, expression of Il7r (interleukin-7 receptor), that promotes neutrophil and monocyte recruitment, was also found upregulated in D2- / Wv hearts (Fig. 14A; p<0.001), while, expression of Stab2 (stabilin-2), a macrophage-expressed phosphatidylserine (PS) surface receptor that mediates phagocytosis and extracellular matrix remodeling during inflammation was also upregulated in D2- / 7?<A hearts (Fig. 14A; p O.OOl). Expression ofAdam8 (a disintegrin and metalloproteinase 8), that promotes release of pro-inflammatory cytokines and cell adhesion molecules and degradation of extracellular matrix 48, was also highly overexpressed in D2-mdx hearts in accordance with bulk RNAseq results (Fig. 14A; p<0.001}. Trem2 (triggering receptor expressed on myeloid cells 2), which drives NF-kB signaling and production of pro inflammatory cytokines including IL-6 and TNFa, was also upregulated in juvenile D2-wc& hearts (Fig. 14A; p<0.01}.

[0230] With the abundant increase in inflammatory cell chemokines, we examined the abundance of macrophages using the pan-macrophage marker F4 / 80. This confirmed extensive presence of macrophages in the damaged regions in V)2-mdx hearts, which is distinct from what is observed in the B l O-zi / t / x hearts (Fig. 14C, 14D; top panels}. To assess the attributes of infiltrating macrophages in the D2- / W.r hearts, we next profiled transcript levels of Il-lb (interleukin- lb), a pro-inflammatory macrophage marker, Argl (arginase-1), a pro-regenerative macrophage marker, and Lgals3 (galectin-3), a macrophage marker associated with chronic (pathogenic) muscle inflammation. While the inflammatory and regenerative marker genes were increased by 3-, and 9-folds respectively in D2- / wt&, compared to Bl O-wc / r hearts, the level of pathogenic marker (Lgals3) was elevated by ~45-fold (p<0.00I) (Fig. 14B). To determine if the different genetic background may contribute to this dysregulation we examined these transcripts in age-matched juvenile D2-WT and B10-WT mice. This showed no influence of genetic background for Adam8, Illb, Lgals3, and Argl between strains, and minimal impact on the expression of Ccl3 (p<0.01\ Ccl8 (p<0.01), Trem2 (p<0.01\ and Il7r (p ().O5) (Supplemental Fig. 4A). As our previous investigations 330 have revealed galectin-3 enriched macrophages as a driver of fibrotic degeneration of D2-mdx skeletal muscles51, we assessed the tissue localization and abundance galectin-3 protein (GAL-3) in D2-mtfr. This identified that GAL-3+ macrophages were highly abundant and nearly exclusively localized within the damaged regions of D2- / ; / CZY hearts (Fig. 14C), while in accordance with qPCR results, these pathogenic macrophages were absent in B l O-wt / .r hearts (Fig. 14B, 14D)

[0231] Increased expression of chemokines observed in these tissues explains the excess o macrophages in D2-w<A. However, during acute injury, the inflammatory response is controlled by a pro-resolving response that follows cytokine-mediated activation of inflammation and involves activation of the G protein coupled receptors including Formyl peptide receptors (FPRs). FPRs are activated by the endogenous ligands produced in response to tissue damage, including lipids (Resolvin DI, Lipoxin A4) and protein (Annexin Al; AnxAl) that bind FPR1 / 252, and serve as the master switch at the site of damage that helps resolve the inflammation. They do so by promoting macrophage skewing from pro- to anti-inflammatory fates and regulating signaling pathways that help clear immune cell infiltration by activating their apoptosis and non-phlogistic clearance as well. To assess if the excessive inflammatory responses in D2- / W.V hearts is due to reduced FPR signaling, we examined the expression of FPRs (Fprl, Fpr2)~ and its ligand Anxal. This revealed over 4-fold upregulation of FPRs (both Fprl and Fpr2) expression in D2- / wt / x relative to B \Q-mdx heart, but no change in the expression of Anxal between D2-mdx and B l O-mt / x (Fig. 14B). These results validated the findings from the previous cohort used for bulk RNAseq analysis (Fig. 13). Together, they suggest reduced activation of FPR signaling hinders resolution of inflammation and may be a driver of the excessive inflammation seen in juvenile D2-mt& hearts, which in turn is linked to their fibro- degenerative state.

[0232] Fibrotic ECM remodeling drives early onset cardiac fibrosis in juvenile D2-mdx. To evaluate the prominent upregulation of ECM remodeling pathways implicated by the RNAseq analysis, we performed qPCR for multiple extracellular matrix-associated components and remodeling enzymes identified by the analysis of our bulk RNAseq cohort. This validated the observed upregulation of Fnl (fibronectin; p '0.01), Collal ("collagen 1A; p=0.0712), and Itgax (integrin alpha X; p<0.001) in T)2-mdx hearts, relative to (Fig. 15A).

[0233] Assessment of Sppl (osteopontin), a DMD genetic modifier that links inflammation to extracellular matrix assembly and fibrosis and macrophage-expressed matrix remodeling enzyme Mmpl2 (matrix metalloproteinase 12), both showed over 200-fold upregulation in D2-mdx hearts, compared to Bl O- / W.r (Fig. 15A). While other ECM regulator and structural components including Timpl (TIMP metallopeptidase inhibitor 1), Itgax, Fnl, and Collal showed between 4-40-fold upregulation in D2-mt& hearts (Fig. 15A). These validate the findings from the bulk RNAseq cohort and implicate a nexus of inflammatory-ECM dysregulation in pediatric-onset cardiac pathogenesis in the D2-mt&. To monitor the sites of fibrosis in juvenile D2-mdx hearts, we immunostained heart cross-sections for C0L1A1 and co-stained with wheat germ agglutinin (WGA), which showed dense C0L1A1 expression in VF2-mdx in the damaged regions along the RV wall and throughout the endomysium (Fig. 15B, 15B’), while endomysial C0L1A1 staining in B I O-wc / x counterparts was minimal in comparison (Fig. 15C, 15C’). To address the influence of genetic background on the above findings, we assessed expression in D2-WT and B10-WT hearts, which indicated no genotype-related differences in the expression of Fnl, Collal, Itgax, ox Timpl, and a comparatively modest increases in the expression of Sppl (p<0.0T) and Mmpl2 (p<0.0T) in D2-WT hearts, relative to B10-WT (Fig. 15B), when compared to differences between mdx strains (Fig. 15A). This identified the site and composition of the fibrotic ECM in the T)2-mdx heart. It also highlighted the potential of targeting the aberrant proinflammatory response to attenuate fibrotic cardiac degeneration of the D2- / m& dystrophic heart.

[0234] Activation of pro-resolving 382 FPR signaling prevents cardiac damage in D2-mdx hearts. The above findings link inflammation and cardiac fibrosis in T)2-mdx. With mixed success of corticosteroid use in treating this in heart by dampening inflammation, coupled with our findings of poor activation of pro-resolving FPR signaling, we hypothesized use of FPR- targeting therapy may resolve the chronic inflammation, without blocking acute inflammation, which is required for the reparative ability of the cardiac injury (Fig. 16A).

[0235] To assess the benefit of a pro-resolving FPR-agonist therapy for pediatric-onset cardiac pathology in V)2-mdx, we tested use of the synthetic FPR agonist BMS-986235 versus saline (n = 6 animals / cohort). Mice were orally dosed with drug beginning at 2.5-weeks of age (just prior to the onset of cardiac pathology) and were maintained on drug (or saline) till 6-weeks of age when the tissues were harvested for further analysis (Fig. 16B). Analysis of histopathology of the cardiac tissue cross-section revealed a clear lack of fibro-calcified damaged areas along the RV and LV walls in drug-treated hearts, compared to saline controls (Fig. 16C). Histological analyses performed by H&E staining confirmed this observed therapeutic effect with some of the drug treated hearts showing only small and discrete areas of damage within the RV wall, and the rest lacked any signs of damage or inflammation altogether, while the saline-treated mice showed extensive cardiac damage (Fig. 16D). To further assess this impact of pro-resolving therapy on inflammation and extracellular matrix remodeling in D2- / .r hearts, we immunostained tissue cross-sections for macrophages, which confirmed the reduction in macrophage infiltration through the heart and within and surrounding any small sites of damage that existed in our treated cohort (Fig. 16E). This was in stark contrast to the heightened macrophage infiltration both within and surrounding sites of damage in saline control hearts (Fig. 5E). Next, to more directly assess fibrotic gene expression we immunostained these hearts for C0L1A1 and found the drug treatment also significantly reduced the C0L1A1 expression throughout the heart, when compared to the expression in the saline controls (Fig. 16F).

[0236] Next, to assess the effect of acute BMS-986235 treatment on inflammation and extracellular matrix remodeling pathways, we performed targeted qPCR for both inflammatory and extracellular matrix remodeling-associated transcripts previously shown to be dysregulated in D2- / 7?t / x hearts (Figs. 13-15). We found acute BMS-986235 treatment of juvenile D2- / 7?c / x mice resulted in a significant reduction in the levels of inflammatory transcripts Trem2, Lgals3, and Anxal (Fig. 16G), confirming the efficacy of this pro-resolving therapy to attenuate aberrant inflammatory signaling via the FPR2-ANXA1 axis. Similarly, quantification of extracellular matrix remodeling targets, Sppl, Timpl, and Colla showed significant depletion of Sppl and 77 / 7? / ) / transcripts (Fig. 16H), and a trending reduction in Collal transcript levels (Fig. 16H) which aligns with C0L1A1 immunostaining results (Fig. 16F). Future chronic studies will be required to assess the full benefit of chronic pro-resolving therapy to delay the onset and lessen the severity of fibrotic cardiac degeneration with disease progression in older D2- / ??t / x mice.

[0237] While there has been a long-standing recognition of early-onset cardiac deficit and its lethal consequences for boys with DMD, our experimental understanding of the molecular deficits and preclinical interventions have been based on the use of adult mouse models. This is in part due to significant differences between the cardiac pathology between the patients and mdx model. The mdx model shows late onset cardiomyopathy with mild to moderate inflammation and fibrosis, which is rarely lethal. Our findings support the pediatric-onset of cardiac damage in the T)2-mdx model, with early onset spontaneous cardiac damage and significant fibro- calcification early in life. This offers an opportunity to investigate the mechanisms of pediatric- onset cardiac damage and accompanying endomysial fibrosis observed in DMD.

[0238] The inventors here identify a dysregulation of inflammatory and ECM remodeling as two such pathological pathways. Next, they focused on harnessing underlying molecular pathways that distinguish the cardiac deficit in D2- / 7?r / x model as compared to the adult-onset cardiac deficitB 10- / 7?cZr model. This analysis allowed distinguishing the differences driving the onset of mild and severe cardiac muscle degeneration and cardiomyopathy independent of the presence / absence of dystrophin protein. Such differences are reminiscent of the DMD patients, who manifest varying level and severity of cardiomyopathy despite the lacking dystrophin expression.

[0239] They also identify excessive an inflammatory response that fails to resolve through FPR2- mediated pro-resolving pathway prevents restoring the injured heart tissues to their uninflamed state. While the infiltrating leukocytes are needed in damaged heart to clear the dead cells, FPR2 and other mediators that repress inflammation are released leading to predominance of anti-inflammatory cells - a response associated with activation of cardiac repair. We find that this latter repressive response is poor, and that the excessive inflammatory signaling proceeds via Sppl and TGFa pathway to activate downstream cardiac fibrosis and other degenerative response. Activation of these profibrotic degenerative response have long been recognized as a feature of the D2-m<7r model 17 which we previously showed contributes to excessive skeletal muscle pathology in the juvenile D2- / .r mice by suppression of skeletal muscle regeneration. Unlike skeletal muscle, cardiac muscle does not undergo regeneration and our comparative analysis of T)2-mdx and B10-mc7x identify cardiomyocyte degeneration due to chronic inflammation and loss by way of fibro-calcified ECM. We observe the ventricular pericardium as the region most affected by this damage, but this can progress to the atria as well (Fig. 12). This variability in the affected region is in addition to the variability we observe in the severity of cardiac damage between individual mice. This hints at a level of stochasticity in the degeneration. We believe this may plausibly reflect the level of initial damage to the affected heart, which is then amplified as the ensuing inflammation becoming chronic.

[0240] We find chronic cardiac inflammation in D2- / ??<A is marked by higher level of activation of inflammatory and immune response in part by higher expression of chemokines that attract these immune cells (Figs. 13, 14). This inflammatory response involves accumulation of ,S / ; / 9 / (OPN) / 459 A c7 / .s3(GAL-3) expressing pathogenic macrophages that we recently identified by single cell RNAseq analysis of the skeletal muscles from mdx mice51. Osteopontin secreted by these macrophages promote skeletal muscle fibrosis by activating the stromal progenitors and we suggest a similar mechanism may be in place following accumulation of these macrophage in the damaged heart (Fig. 14). In support of this mechanism, we find these GAL-3+macrophages enriched in the same pericardial region that are enriched in C0LA1 indicative of active fibrosis (Fig. 14). The indication that this is an active phenomenon comes from the concomitant enrichment of other ECM building (fibronectin) and degrading (TIMP / MMP) components along with leukocyte attracting (CCCL3 / 8) and resolution (FPR1 / 2) signaling (Figs. 13-15). This dynamic indicated that there is a likely shift in the equilibrium of these two opposing inflammation building and resolving signals and hence rebalancing this could provide a likely beneficial effect for the affected region. This is in line with the fact that while acute inflammation following tissue damage is essential for cardiac repair, chronic inflammation can be disruptive. To address this ensuing imbalance of these two opposing inflammatory signaling we made use of the pro-resolving therapy that unlike GCs, precisely targets the resolution of inflammation by activating FPR2 signaling but not blocking activation of inflammation by NFkb or related proinflammatory pathways. This approach showed excellent promise such that a short (3 week) treatment of juvenile D2- / .r mice allowed full resolution of inflammation and prevented any subsequent fibrotic cardiac damage detected histologically as well as by way of aberrant molecular signature including Gal3ISppl macrophages and CollAl and Fnl expressing stromal cells (Fig. 16A-16H).

[0241] In summary, our studies introduce the D2-m<A as a model that manifests pediatric-onset cardiac damage, which provides opportunities to investigate the drivers of early-onset cardiomyopathy in DMD patients. We identified dysregulation of inflammatory and ECM remodeling pathways as key contributors. Specifically, our findings highlight excessive an unresolved inflammatory response involving pathogenic macrophages and neutrophils, contributes to chronic inflammation 485 and progressive cardiomyocyte degeneration and fibrotic replacement in the D2- / .r model. Finally, our use of FPR2 -targeting therapy provides a potential therapeutic avenue to prevent or mitigate pediatric-onset cardiac pathologies in DMD.

[0242] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ”. A and / or B includes A, B, and (A + B).

[0243] Any numerical range recited herein is intended to include all sub-ranges and values subsumed therein. Where a range of values is provided, it is to be understood that each intervening value between an upper and lower limit of the range and any other stated or intervening value in that stated range is encompassed within the disclosure. Where the stated range includes upper and lower limits, ranges excluding either of those limits are also included.

[0244] Disclosure of values and ranges of values for specific parameters (such as temperatures, molecular weights, weight percentages, etc.) are not exclusive of other values and ranges of values useful herein. As used herein in the specification, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “substantially”, “about” or “approximately,” even if the term does not expressly appear. As used herein, the words “we”, “our” or “us” typically refer to the inventors.

[0245] Color designations such as “red” or “blue” correspond to different shadings in the black- and-white or grayscale figures herein or may be discerned from the contest of their use.

[0246] All publications and patent applications mentioned in this specification or in the two provisional application priority documents are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, especially referenced is disclosure appearing in the same sentence, paragraph, page or section of the specification in which the incorporation by reference appears.

[0247] The citation of references herein does not constitute an admission that those references are prior art or have any relevance to the patentability of the technology disclosed herein. Any discussion of the content of references cited is intended merely to provide a general summary of assertions made by the authors of the references and does not constitute an admission as to the accuracy of the content of such references.

Claims

1. CLAIMSWhat is claimed is:

1. A method for treating a chronic inflammatory muscle disease, such as muscular dystrophy of a subject, or by helping prevent degeneration or enhance muscle regeneration of a subject, comprising administering an N-formyl peptide receptor (FPR) agonist to the subject, wherein, optionally, the subject has or is at risk of developing Duchenne muscular dystrophy (DMD) and is administered an FPR2 agonist, such as BMS-986235 or Annexin Al N-terminal mimetic peptide AC2-26, and optionally, wherein said agonist is optionally selected based on its anti-inflammatory effects it exhibits in murine models of DMD.

2. The method of claim 1, wherein the subject is treated for a chronic inflammatory muscle disease that is Duchenne muscular dystrophy (DMD).

3. The method of claim 1, wherein the subject is treated to reduce the severity of, or prevent, muscular degeneration or to enhance muscle regeneration.

4. The method of claim 1, wherein said agonist is selected based on its antiinflammatory effects it exhibits in at least one murine model of DMD comprising an mdx murine model5. The method of claim 1, wherein the subject has DMD and is in need of muscle regeneration.

6. The method of claim 1, wherein the subject is in need of treatment of muscle degeneration of cardiac, skeletal, or smooth muscle.

7. The method of claim 1, wherein the subject is in need of preventing cardiac and skeletal muscle degeneration or supporting skeletal muscle repair caused by chronic muscle damage and inflammation including Becker muscular dystrophy. Limb Girdle Muscular Dystrophies (LGMD), Inclusion Body Myositis, Dermatomyositis, and / or Polymyositis.

8. The method of claim 1, wherein the subject exhibits indicators of muscle degeneration including muscle fiber rupture, hematoma formation, release of damage-associated molecular patterns (DAMPs), abnormal immune cell recruitment or failure to resolve inflammatory signaling.

9. The method of claim 1 that facilitates clearance of inflammation, reduces an inflammatory response, enhances a repair process, enhances regenerative capacity in muscles, or otherwise improves prognosis compared to an untreated control subject.

10. The method of claim 1, wherein the FPR agonist is an endogenous FPR1, 2 or 3 agonist or a synthetic FPR 1, 2 or 3 agonist.

11. The method of claim 1, wherein the FPR agonist is an FPR agonist other than an FPR2 agonist.

12. The method of claim 1, wherein the FPR agonist is an FPR2 agonist.

13. The method of claim 1, wherein the FPR agonist is selected from the group consisting of at least one of Ac2-26, BMS986235, lipoxin A4 (LXA4), MR-39, Compound 43, Compound 17b, LXA4, RvDl, a pyrrolidinone urea compound, or WKYMV (SEQ ID NO: 1).

14. The method of claim 1, wherein the FPR agonist is a FPR2 agonist which acts by enhancing the phagocytic capacity of macrophages and / or by driving the degranulation of neutrophils, to facilitate amelioration or resolution of inflammation and to enhance at least one repair process.

15. The method of claim 1, wherein the FPR agonists is an FPR2 agonist that is used to target the FPR2-mediated pro-resolving pathway.

16. A pharmaceutical composition suitable for administration of a subject having muscular dystrophy comprising an amount of at least one FPR2 agonist sufficient to improve muscle function or to reduce muscle degeneration and a pharmaceutically acceptable carrier, buffer, or excipient.

17. The pharmaceutical composition of claim 16 that has been chemically formulated to extend its biological half-life in vivo or that has been formulated to provide for extended release of the FPR2 agonist.

18. The composition of claim 16, further comprising administration of at least one cotherapy that comprises micro-dystrophin gene therapy to introduce a truncated but functional dystrophin gene into a subject with DMD, administration of dystrophin exon-skipping antisense oligonucleotides or TGF-beta-lowering oligonucleotides, and stem-cell based therapies such as administration of autologous stem cells corrected ex vivo to express functional dystrophin or administration of allogeneic stem cells from a normal donor.

19. The composition of claim 16, further comprising at least one, two, or three other FPR2 agonists or conventional drugs for DMD in a form suitable for administration to a subject having DMD.

20. The composition of claim 19, wherein the FPR2 agonists agonize FPR2 at different binding sites or by different biological mechanisms from each other such as induction of different conformational changes in FPR2, induction of pro- or anti-inflammatory effects, or by activation of different signaling pathways.

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