WO2025085731A1 - TRANSFORMING GROWTH FACTOR ß (TGF-ß) OLIGONUCLEOTIDE COMBINATION THERAPY FOR TREATING MUSCULOSKELETAL DISEASES - Google Patents

Abstract

A method for lowering TGFß in injured musculoskeletal tissues through the use of TGFß inhibiting antisense oligonucleotides (AO) that enables improved regeneration of dystrophic skeletal muscles. TGFß inhibiting AO, when used in combination with a Dystrophin upregulating AO, enhances restoration of Dystrophin protein expression in the dystrophic muscles.

Description

TRANSFORMING GROWTH FACTOR p (TGF-β) OLIGONUCLEOTIDE COMBINATION THERAPY FOR TREATING MUSCULOSKELETAL DISEASES

GOVERNMENT SUPPORT

This invention was made with government support under W81XWH-21-1 -0680 awarded by Department of Defense. The government has certain rights in the invention.

REFERENCE TO A RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 63/591,431, filed October 18, 2023, which is incorporated by reference for all purposes.

REFERENCE TO A SEQUENCE LISTING

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 "550269WO_101724_ST26” This .xml file was generated on October 17, 2024 and 22,290 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.

BACKGROUND

Field of the Invention. The invention relates to the fields of musculoskeletal disease, specifically to treatment of Duchenne muscular dystrophy (DMD), or other chronic disease musculoskeletal or neuromuscular diseases characterized by upregulated Transforming Growth Factor β (TGF-β) activity and associated disease pathogenesis, and the therapeutic use of gene therapy and antisense oligonucleotide (AO) therapy to reduce expression of TGF-p. TGF-fi-related musculoskeletal disorders include but are not limited to such disorders as Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Limb-girdle muscular dystrophy

(LGMD), Congenital Muscular Dystrophies (CMD), Emery-Dreifuss Muscular Dystrophy

(EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Osteogenesis Imperfecta, Marfan

Syndrome (MFS), and conditions such as Cachexia and Sarcopenia.

Description of Related Art. Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder affecting approximately 1 in 5000 male births DMD is caused by mutations in the DMD gene encoding the muscle structural protein dystrophin, which is crucial for maintaining muscle fiber integrity ²’³. DMD patients present with muscle weakness that manifests by 3 to 4 years of age, followed by progressive muscle wasting and fibrosis. The pathophysiology of DMD is characterized by myofiber fragility, poor myogenesis, inflammation, and fibrotic replacement of muscle resulting from the absence of dystrophin 4. Ultimately, DMD leads to shortened lifespans in the third or fourth decade of life, driven by late-onset cardiomyopathy and cardio-respiratory failure ⁴’⁵. Therefore, dystrophin-restoring genetic therapies represent the fundamental clinical strategy for DMD with numerous antisense oligonucleotides (AO) and viral gene therapies in clinical development over the last decade.

Gene-based therapeutic strategies approved by the U.S. Food and Drug Administration

(FDA) include the exon skipping phosphorodiamidate morpholino AO (PMO), first approved in

2016, and more recently the adeno-associated virus (AAV) micro-dystrophin gene replacement vectors, the first of which was approved in 2023. Although AAV vectors target skeletal and cardiac muscles with high efficiency, their packaging capacity prohibits inclusion of the full length DMD gene; thus, a partially functional microdystrophin found in Becker muscular dystrophy was developed for use in DMD ⁶-⁷. Contrary to expectations, AAV-microdystrophin therapy was found to have weak net benefits due to insufficient motor function improvement in DMD patients, together with significant side effects such as serious liver injury, myocarditis, and immune- mediated myositis ⁸. On the other hand, therapeutic use of PMOs in DMD, which promote skipping of mutated dystrophin exons, allow correction of the reading frame and production of a minimally truncated dystrophin protein ^9-11. This protein performs the essential functions of the full-length dystrophin protein restoring myofiber stability. Emerging evidence suggests that PMO therapies have had a positive therapeutic effect on DMD patients by improving exercise capacity, slowing disease progression, and prolonging survival, with a favorable safety profile ¹²‘¹⁴. However, despite the growing promise of exon skipping PMO therapies, a paramount challenge remains towards achieving their full clinical impact, primarily enhancing their relatively poor delivery into skeletal muscle myofibers and cardiomyocytes ¹⁵. Currently, their result is limited dystrophin protein restoration in DMD patient muscles following chronic, systemic dosing ^15-18. Therefore, development of strategies to enhance PMO bioavailability and myofiber/cardiomyocyte uptake may help overcome these barriers and improve patient response and prognosis.

Our recent work revealed that the dystrophic muscle relies on inflammatory macrophages and satellite cell-mediated muscle regeneration for the effective uptake of PMOs into myofibers

¹⁹ Using the B10-mdx model of DMD, we found macrophages infiltrating the damaged and inflamed regions of the dystrophic muscle sequester the circulating PMO that reach the muscle interstitium and serve as a local drug reservoir within die tissue. The PMOs are retained there, in close proximity to activated myoblasts, for up to 1 week, well after PMOs in the blood circulation have disappeared. During this time, PMO is transited to proximal differentiating and fusing myoblasts for efficient delivery to the regenerating myofibers where it elicits local dystrophin expression ¹⁹-²⁰. However, in contrast to the robust regeneration observed in the BlO-mdx model,

DMD patients exhibit limited satellite cell myogenic capacity with disease ^21,22. Thus, we can infer that in DMD patients, PMO delivery is impaired due to this limited myogenic potential, while the development of strategies to enhance regenerative myogenesis in patients could dramatically enhance their clinical impact for DMD.

Transforming growth factor β (TGF-β) signaling plays a central role in promoting muscle atrophy and fibrosis in different skeletal muscle myopathies ²³. In the case of DMD, the expression of TGF-β 1 isoform was positively correlated with the degree of pathology and clinical severity ^24>25. Inversely, inhibition of TGF-β signaling enhanced myoblast fusion and regenerative myogenesis, while also reducing muscle atrophy in the mdx model ^26-29 Thus, improving myogenic potential through TGF-β targeted approaches in DMD patients may be an attractive therapeutic intervention to enhance both regenerative myogenesis and the effectiveness of exon skipping PMO therapies. However, as TGF-β signaling controls numerous physiological processes such as angiogenesis, neurogenesis, and innate and adaptive immune responses ³⁰, TGF-β targeted treatments for muscular dystrophy that would require system delivery must be designed to minimize any detrimental off-target effects.

SUMMARY OF THE INVENTION

1. A method for treating a subject having an elevated TGF-beta level which comprises administering to the subject at least one TGF-beta lowering antisense oligonucleotide for a time and under conditions that inhibit TGF-beta expression.

In one embodiment, this method comprises administering at least one TGF-beta lowering antisense oligonucleotide.

In another embodiment, this method comprises administering at least one TGF-beta lowering antisense oligonucleotide and at least one dystrophin-restoring antisense oligonucleotide.

In another embodiment, the subject is human or non-human, such as a non-human mammal. The methods disclosed herein are primarily directed to modulation of TGF-beta 1. In some embodiments, TGF-beta 2 or 3 may be modulated. Unless otherwise indicated the methods and compositions described herein reference TGF-beta 1.

The methods disclosed here may use AO-mediated gene silencing or exon skipping for reducing TGF-beta expression or increasing dystrophin expression, respectively.

2. The method of claim 1, wherein the subject has an elevated TGF-beta driven myogenic deficit or tissue fibrosis, such as Duchenne Muscular Dystrophy (DMD), wherein the method comprises administering to a subject in need thereof at least one TGF-beta lowering antisense oligonucleotide, optionally in combination with at least one dystrophin-restoring antisense oligonucleotide for a time and under conditions that inhibit TGF-beta expression and, when the at least one dystrophin restoring antisense oligonucleotide is administered, under conditions that increase dystrophin expression; wherein, optionally, the antisense oligonucleotides comprise TPM02 (SEQ ID NO: 16) which targets human mRNA encoding TGF-beta- 1 and DPMO Eteplirsen which targets human mRNA encoding dystrophin.

3. The method of embodiments 1 or 2, wherein the subject has a single exon deletion or duplication of a dystrophin gene which may be a human or non-human gene; or wherein the subject has a multi-exon deletion or duplication of a dystrophin gene which may be a human or non-human gene.

4. The method of embodiments 1, 2 or 3, wherein the subject has a nonsense or missense mutation in the dystrophin gene, has a small deletion, duplication, or insertion in a dystrophin gene which may be a human or non-human gene. 5. The method of any one of embodiments 1-4, wherein the subject has a lower level of dystrophin compared to a healthy control.

6. The method of any one of embodiments 1-5, wherein the subject has elevated TGF-beta as compared to a healthy control. In some embodiments, a normal level of TGF-beta 1 in human plasma will range from 2.0 to 12.0 ng/ml or any intermediate value or subrange. See Wakefield, et al., Clin Cancer Res. 1995 Jan; 1(1): 129-36 which is incorporated by reference. Normal human subjects had 4.1 +/- 2.0 ng/ml TGF-betal (range, 2.0-12.0; n = 42), <0.2 ng/ml TGF-beta2, and

<0.1 ng/ml TGF-beta3 in their plasma. Also, TGFB plasma levels in controls and in patients with different neuromuscular diseases can be found in and are incorporated by reference to Ishitobi et al. 2000. Motor Neuron; . See page 2, results section for TGFB levels in DMD patients and other diseases.

7. The method of any one of embodiments 1-6, wherein the subject is no more than five years old.

8. The method of any one of embodiments 1-6, wherein the subject is more than five years old.

9. The method of any one of embodiments 1-8, wherein a dystrophin-restoring phosphorodiamidate morpholino oligomer (DPMO) and /or a TGF-beta 1 targeting phosphorodiamidate morpholino oligomer (TPMO) is locally administered to muscle or to damaged muscle lesions in the subject.

10. The method of embodiment 9, wherein the muscle is skeletal muscle.

11. The method of embodiment 9, wherein the muscle is cardiac muscle. 12. The method of any one of embodiments 1-11, wherein a dystrophin-restoring phosphorodiamidate morpholino oligomer (DPMO) and a TGF-beta 1 targeting phosphorodiamidate morpholino oligomer (TPMO) is systemically administered to the subject.

13. The method of any one of embodiments 1-12, wherein the TGF-beta 1 targeting phosphorodiamidate morpholino oligomer (TPMO) is TPMO2, or a corresponding orthologous human sequence.

14. The method of any one of embodiments 1-13, wherein the TGF-beta 1 targeting phosphorodiamidate morpholino oligomer (TPMO) is TPMO 1, 3, 4, 5 or 6, or the corresponding orthologous human sequences.

15. The method of any one of embodiments 1-14, wherein the dystrophin-restoring

PMO (DPMO) comprises etepliresn, goldirsen, viltolarsen or casimersen or a variant thereof and the TPMO is at least one of TPMO 1, 2, 3, 4, 5, or 6 (SEQ ID NOS: 15-20).

16. The method of any one of embodiments 1-15, further comprising administering an agent that increases macrophages infiltrating the damaged and inflamed regions of dystrophic muscle.

17. The method of any one of embodiments 1-16, further comprising a TCF-beta targeting antibody, small molecule or other agent other than a TPMO that reduces a level of, or expression of, phospho-SMAD3 (pSMAD3).

18. A method for treating a subject in need of muscle regeneration or repair or a muscular disease or condition other than Duchenne Muscular Dystrophy (DMD) comprising administering to a subject in need thereof TGF-beta lowering antisense oligonucleotides for a time and under conditions that inhibit TGF-beta expression. 19. A pharmaceutical composition comprising a combination of TGF-beta and dystrophin antisense oligonucleotides.

20. The pharmaceutical composition of embodiment 19, further comprising an agent that reduces SMAD3 expression other than the TGF-beta antisense oligonucleotides and/or an agent that promotes macrophage infiltration in and around damaged muscle tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures accompanying this application include micrographs that were originally captured in color. For the purposes of this PCT application, these micrographs have been converted to black and white images. This conversion has been performed to show the relevant details and structures visible in the original color images are adequately represented in the black and white versions. Where necessary, different shades of gray have been used to distinguish features that were originally differentiated by color. Applicant notes that while the black and white versions are suitable for examination purposes, the original color images may be made available upon request for a more detailed view of the micrographs. The conversion to black and white does not affect the substantive disclosure of the invention, and all essential features remain discernible in the figures as presented.

FIGS. 1A-1F. Decreased dystrophin restoration following systemic DPMO in D2-mdx compared to BlO-mdx. D2-mdx or B10-mdx mice were administrated systemic DPMO via the retroorbital sinus (400 mg/kg, exon 23 skipping PMO) at 20 weeks old. (A, B) Dystrophin expression shown in D2-mdx or B10-mdx triceps 2 weeks after IV injection of DPMO. Scale bar represents 100 μm. (C) Quantification of the number of dystrophin-positive fibers per triceps muscle in the total area. (D) Exon skipping was quantified from the triceps by RT-qPCR. The degree of exon skipping was calculated as the percentage of exon 22-24 mRNA expression of Dmd relative to exon 2-3 mRNA expression. (E) Quantification of the number of dystrophin positive clusters in the triceps muscle. (F) Quantification of the average area of dystrophin positive clusters within the triceps muscle. Data is represented as a scatter plot with SD; Saline n=4, DPMO n=6.

Statistical analysis was performed using a one-way ANOVA; **p<0.01.

FIGS. 2A-2J. Reduced DPMO delivery in D2-mdx, despite elevated levels of inflammation.

(A, B) D2-mdx or B10-mdx mice were administrated systemic FITC-DPMO (F-DPMO) via the retroorbital sinus (400 mg/kg) at 20 weeks old. IF images showing F-DPMO and F4/80 (marker for macrophage) localization in degenerating lesion and regenerating lesion in or BlO-mdx mice or D2-mdx triceps. (C) IF images from BlO-mdx and D2-mdx (20-weeks-old) triceps muscle sections stained to F4/80 and PDGFRa (marker for fibro-adipogenic progenitor (FAP) cells). (D,

E) Quantification of F4/80+ area (%) and PDGFRa+ area (%) per total cross-sectional area (n=10).

(F. G) Gene expression analysis of fibrosis markers, Tgfbl and Fnl from B10-mdx (n=10) and D2- mdx (n=8) triceps muscle. (H) IF images of muscle sections from B10-mdx and 2-mdx (20- weeks-old) triceps muscle sections stained to identify muscle fibers BrdU+ CNFs; sections co- stained with Laminin-a2 and DAPI. Mice were administrated BrdU for one week and sacrificed two weeks later. (I) Quantification of BrdU+ CNFs in total triceps muscle from BlO-mdx (n=10) and D2-mdx (n=8). Scale bar represents 50 μm. Statistical analysis was performed using Mann-

Whitney test; **p<0.01.

FIGS. 3A-3G. Design and development of Tgfbl -targeted PMOs (TPMOs). (A) Scheme representing PMO target sites within the 5’ UTR and exon boundaries to block translation or elicit out-of-frame premature termination. (B) Active levels of TGF-β in LPS-stimulated RAW 264.7 cells (macrophages) treated with TPMOs targeting Tgfhl. (C) Schematic showing intramuscular

(tibialis anterior: TA) TPMOs injection with multiple needle injury experimental design to identify the efficacy of TPMOs in D2-WT (12-week-old, n=4). (D) Heat map of Tgfbl and TGF-β target genes expression depicting fold change according to TPMOs in TA. (E) IF images showing control

PMO, TPMO2, and TPMO6 treated D2-WT TA cross-sections stained to pSMAD3, which is a mediator of TGF-β signaling, and eMHC; sections co-stained with Laminin-a2 (red) and DAPI

(blue). Scale bar represents 50 μm. (F, G) Quantifying the percentage of pSMAD3 positive area relative to DAPI area in the damaged muscle area (F) and the number of eMHC+ fibers in damaged area (G) from control PMO, TPMO2, and TPMO6 treated D2-WT TA. Statistical analysis was performed using a one-way ANOVA and Mann-Whitney test; *p<0.05, **p<0.01, ***p<0.001,

****p<0.0001.

FIGS. 4A-4G. Attenuation of downstream TGFfi activity and regenerative dysfunction following systemic TPMO administration inD2-mdx. (A) Experimental design schematic showing

IV TPMO administration (200 mg/kg) in juvenile D2-mdx mice (3 weeks old, n=6) to assess systemic drug impact. (B, C) IF images showing pSMAD3 expression and numbers of eMHC+ regenerating myofibers following systemic administration of control PMO or TPMO2 in D2-mdx.

(D, E) Corresponding quantification of pSMAD3 expression (D) and eMHC+ fibers (E) in damaged muscle areas following TPMO therapy. Data is represented as a scatter plot with SD from n=12 per group. (F) Relative frequency according to eMHC+ myofiber cross-sectional area

(CSA) following TPMO therapy. Statistical analysis was performed using the Mann-Whitney test;

*p<0.05.

FIGS. 5A-5F. Enhancement of dystrophin restoration following dual TPMO and DPMO therapy in D2-mdx. (A) Experimental design schematic showing IM TPMO2 (100 ug/TA) delivery prior to systemic DPMO treatment (200 mg/kg) in adult D2-mdx mice (n=6) to access combinational PMO therapy. TA muscles (n=12) were harvested three weeks after systemic

DPMO treatment, and IF was performed. (B) IF images showing dystrophin expression, BrdU+ regenerating myofibers, F4/80 expression, and PDGFRa expression following IM TPM02+IV

DPMO administration, relative IM control PMO+IV DPMO. Scale bar represents 50 μm. (C)

Quantification of dystrophin+ myofibers per TA muscle following therapy (n=12). (D)

Quantification of BrdU+ centrally-nucleated fibers (CNFs) per TA muscle (n=12). (E, F) The percentage of F4/80 (E) or PDGFRa expression (F) in damaged TA muscle areas after treatment was quantified and shown as a violin plot. Data is represented as a scatter plot with SD. Statistical analysis was performed using the Mann-Whitney test; **p<0.01, ****p<0.0001.

FIGS. 6A-6K. Figure 6. Improvement of muscle junction following long-term systemic dual TPM02 and DPMO therapy in D2-mdx. (A) Experimental design schematic showing long- term TPMO2+DPMO treatment in juvenile D2-mdx mice (3 weeks old, n=7-8). A cocktail of

TPM02 (200 mg/kg) and DPMO (200 mg/kg) was injected via the netroorbital sinus twice a week

(saline used as control). Functional analysis was performed after 11 dosing and harvesting skeletal muscles and the heart after 12 dosing. IF was performed on the left side of the gastrocnemius, which did not perform electric stimulation to measure isometric torque. (B, C) In vivo isometric force frequency plot and maximal isometric force in dual TPMO2+DPMO treated vs saline treated

D2-mdx gastrocnemius muscle. (D, E) Forelimb grip strength (D) and timed wire hang (E) functional outcome measure following treatment. (F-H) IF images showing dystrophin expression levels (F) and corresponding quantification of dystrophin+ fibers (G) and CNFs (H). Scale bar represents 100 μm. (I) Dystrophin protein levels were determined by capillary electrophoresis immunoassay. (J) Wes quantification in gastrocnemius of dual PMO-treated or saline-treated D2- mdx. The dystrophin expression of saline-treated or dual PMO-treated D2-mdx muscle is shown compared to the dystrophin expression of WT muscle. Data is represented as a scatter plot with

SD from n=7-8 per group. Statistical analysis was performed using the Mann-Whitney test;

*p<0.05, ***p<0.001, ****p<0.0001. (K) IF images showing expression and localization of b- dystroglycan, dystrobrevin, or a-sarcoglycan in dystrophin restored regions of dual PMO-treated

D2-mdx gastrocnemius muscle, compared to expression in saline-treated mice. Sections were co- stained with WGA. Asterisks mark the orientation of myofibers between adjacent serial sections.

Scale bar represents 50 μm.

FIG. 7A-7C. Restoration of dystrophin in D2-mdx hearts following long-term systemic dual TPM02 and DPMO therapy. (A) IF images showing dystrophin expression levels within

D2-mdx cardiomyocytes following long-term systemic dual PMO therapy. Scale bar represents

50μm. (B, C) Corresponding quantification of dystrophin+ cardiomyocytes per heart (B) and percentage of dystrophin+ cardiomyocytes to total cardiomyocytes (C). (D) Quantification of dystrophin+ cardiomyocyte clusters per heart cross-section following long-term systemic dual

PMO therapy. Data is represented as a scatter plot with SD from n=7-8 per group. Statistical analysis was performed using the Mann-Whitney test; ***p<0.001.

FIGS. 8A-8E. Decreased dystrophin restoration in quadriceps ofD2-mdx mice compared to B10-mdx mice after systemic DPMO treatment. D2-mdx or BlO-mdx mice (20 weeks old) were administrated systemic DPMO (400 mg/kg) via the retroorbital sinus (IV) to assess efficacy between models. (A-B) Dystrophin expression shown in B10-mdx (A) and D2-mdx (B) quadriceps

2 weeks after IV injection of DPMO. Scale bar represents 100 μm. (C) Quantification of dystrophin-positive fibers per area (mm2) in quadriceps. (D) Quantification of dystrophin positive myofiber clusters per cross-section in quadriceps. (E) Quantification of the average area (mm2) of dystrophin-positive clusters within quadriceps. Data is represented as a scatter plot with SD; Saline and DPMO n=4-5/cohort. Statistical analysis was performed using a one way ANOVA; *p<0.05,

**p<0.01.

FIGS. 9 A and 9B. Reduced DPMO efficiency in D2-mdx mice despite robust DPMO localization throughout inflammatory lesions. (A-B) Whole cross-sectional IF images of BlO-mdx

(A) and D2-mdx (B) triceps, corresponding to Figures 2A-B, showing localization of FITC-DPMO

(FDPMO) and F4/80 (marker for macrophage); sections co-stained with WGA and DAPI. Scale bar represents 200 μm.

FIGS. 10A-10D. Assessment of TPMO effect on TGF-β signaling and regenerative capacity. (A-D) TPMOs were injected into the TA (100 ug/TA) with concurrent needle injury and harvested four days later. (A-B) IF images showing control PMO- and TPMO-treated D2-WT TA cross sections stained for pSMAD3, a downstream mediator of TGF-β signaling (A), and eMHC

(B); sections co-stained with Laminin-α2 (red) and DAPI (blue). Scale bar represents 50 μm. (C-

D) Quantification of the percentage of pSMAD3 positive area relative to DAPI area in damaged muscle regions (C) and the number of eMHC+ fibers localized within damaged muscle regions

(D). Data is represented as a scatter plot with SD; n=4 TA muscles per group. Statistical analysis was performed using a one-way ANOVA. Data corresponds to Figure 3.

FIGS. 11A and 11B. TPMO provides acute therapeutic response following concurrent intramuscular injury and treatment. (A) IF images showing pSMAD3 expression following IM

TPMO2+FV DPMO or IM control PMO+TV DPMO administration. Scale bar represents 50 μm.

(B) Quantification of the percentage of pSMAD3 expression per damaged TA muscle area after

PMO treatment. Data is represented as a scatter plot with SD from n=6 per group. Statistical analysis was performed using the Mann-Whitney test. FIGS. 12A-12E Assessment of dystrophin restoration in gastrocnemius following chronic, dual TPM02 and DPMO therapy. (A-B) Whole cross-sectional IF images of gastrocnemius muscles (corresponding to Figure 6F). IF staining for dystrophin (green), laminin-α2 (gray) and

DAPI (blue) shown. Scale bar represents 500 μm. (C) Longitudinal assessment of body weight for the duration of treatment regimen (described in FIG. 6A). (D) Terminal kidney and spleen weights measured at the conclusion of the chronic trial. (E) Terminal heart, quadriceps (quad), triceps, TA and gastrocnemius (GC) weights measured at the conclusion of the chronic trial.

FIGS. 13A-13C. . Dystrophin restoration in the heart following chronic, dual TPM02 and DPMO therapy. (A-B) Whole cross-sectional IF images of the heart, corresponding to

Figure 7 A. IF staining for dystrophin (green), laminin-α2 (gray) and DAPI (blue) shown. Scale bar represents 500 μm. (C) IF images showing expression and localization of b-dystroglycan, dystrobrevin, or a-sarcoglycan in dual PMO-treated D2-mdx gastrocnemius muscle, compared to saline-treated controls. Sections were co-stained with WGA. Scale bar represents 50 μm.

DETAILED DESCRIPTION OF THE INVENTION

Poor dystrophin restoration is a major challenge towards recognizing the full potential of the phosphorodiamidate morpholino oligomers (PMO)-based antisense oligonucleotide (AO) gene therapy for Duchenne Muscular Dystrophy (DMD). Poor myogenesis and excessive fibrosis induced by high TGFβ activity in DMD muscle contribute to poor efficacy of dystrophin PMO therapy. Using the severe D2-md&r model of DMD that exhibits greater TGFβ activity and poor myogenesis as compared to the milder B10-ma!r model, we find poor PMO-mediated dystrophin restoration in D2-mdx muscle. Here, we describe use of a combinatorial PMO therapy that simultaneously inhibits macrophage TGFβ production and enhances myofiber dystrophin production. We developed a TGFβ inhibiting AO that reduced expression of TGFβ-responsive genes and TGF-β-mediated SMAD3 signaling to enhance muscle regeneration following muscle injury. Acute combinatorial use of this TGF-β-targeting PMO with dystrophin exon skipping PMO led to significant increases in dystrophin restoration. Chronic use of this dual PMO therapy recapitulated these benefits and enhanced muscle functional capacity. Further, combinational

PMO therapy promoted dystrophin restoration in cardiomyocytes in vivo, another major challenge for PMO therapies in DMD. Our findings offer the first combinatorial PMO gene therapy for DMD to tackle challenges associated with poor efficacy of PMO-mediated gene therapy, while simultaneously improving muscle restoration and function.

Here, we report that dystrophin-restoring PMO (DPMO) efficacy was reduced in muscles of the more severe D2-mdx (DBA/2J genetic strain) model of DMD, that share similar pathophysiologic features with DMD patients (i.e. heighted degeneration and limited regenerative capacity), in contrast to the (C57BL/6 genetic strain) model that is regeneration competent and only displays mild muscle degeneration and fibrosis. T overcome this, we developed TGF- 0 1 -targeting PMO (TPMO) and a dual TPMO and DPMO strategy to simultaneously enhance regenerative myogenesis and dystrophin exon skipping and protein restoration. We found locally- and systemically-delivery TPMO effectively rescued myogenic capacity and regeneration in the D2-mdx muscle. Further, administration of combinational TPMO and DPMO therapy in D2-mdx mice recapitulated these findings and led to dramatic enhancements in Dmd exon skipping and dystrophin restoration as well as muscle functional improvements following chronic administration. Interestingly, this combinational TPMO and DPMO approach also led to dystrophin restoration in cardiomyocytes, which has been a major challenge for unconjugated PMOs to date. Thus, this work demonstrates the therapeutic potential of the first

PMO-based dual target, combinatorial gene therapy for DMD to effectively overcome the major challenges associated with poor DPMO delivery and efficacy in DMD patients, while simultaneously improving muscle niche dynamics and muscle functional capacity.

Results

D2-mdx as a model to evaluate poor DPMO efficacy with systemic treatment.

Our previous work demonstrated the value of the D2-mdx model for investigating the drivers of myogenic failure in DMD ^31>32. To assess the relevance of the D2-mdx model to investigate how poor myogenic capacity dictates the delivery and efficacy of PMO therapies in

DMD, we first treated D2-mdx and BlO-mdx mice with equivalent doses of DPMO (400 mg/kg,

IV) and compared the resulting dystrophin expression in skeletal muscles between these models.

As we hypothesized based on our prior work demonstrating the requirement of regeneration for

PMO delivery in skeletal muscles ^19,20, we found the number of dystrophin positive fibers and corresponding level of Dmd exon skipping were markedly reduced in both triceps and quadriceps of D2-mdx muscle after systemic DPMO treatment, in comparison to B 10-mdx muscles; see FIGS.

1A-1D, 9A-9C, 8A-8C).

Additionally, we observed limited numbers of dystrophin-expressing clusters in treated

D2-mdx muscles, relative to B10-mdx, while the size of each cluster, which corresponds to the extent of actively regenerating myofibers per lesion at the time of PMO administration ¹⁹’²⁰, was also significantly reduced in treated D2-mdx muscles

Figures 1E-1F, 8D-8E S1D-E. To investigate whether reduced DPMO efficacy in D2- mdx muscles was the result of poor drug retention within the muscle, or the impaired myogenic capacity of this model, we administered fluorescein (FITC)-conjugated DPMO (F-DPMO) and assessed PMO localization, retention, and myofiber uptake in both D2-mdx and B10-mdx muscles 4 days following systemic F-DPMO (400 mg/kg) administration. Interestingly, we found similar

F-DPMO accumulation and retention within degenerating inflammatory lesions that were inundated with macrophages regardless of mdx strain; see FIGS. 2A-2B, 9A-9B .

Furthermore, as the extent of macrophage infiltration per damaged lesion was considerably higher in the D2-mdx muscles (FIGS. 2A-2D), consistent with our previous report ³², we also found heightened F-DPMO signal within these degenerating sites in D2-mdx as compared to B10- mdx muscles (FIGS. 2A-2B). However, while in BlO-mdx muscles we observed PMO localized within the myonuclei of regenerating myofibers, this was a rare observation in D2-mdx muscles

(FIGS. 2A-2B). Overall, this result aligns well with our earlier work demonstrating the importance of inflammatory cells in providing a sustained reservoir of PMO within degenerating/regenerating muscle lesions ^19,2°, and further, suggests the lack of regenerative myogenesis in D2-mdx as the major culprit driving such as stark difference in PMO efficacy between mdx models.

Fibro-adipogenic progenitors (FAPs), marked by platelet-derived growth factor receptora

(PDGFRa), are skeletal muscle stromal cells that play critical roles in healthy muscle homeostasis and muscle repair following acute injury ^33-36. However, we previously showed that chronic damage in D2-mdx muscle induces high TGF-β production, resulting in abnormal and excessive accumulation of FAPs that can directly compromise SC function, myoblast fusion, and regenerative capacity following injuries ^31>32. As the interactions of macrophages and SCs required for myofiber PMO delivery occur within the dystrophic muscle interstitium together with this aberrant accumulation of FAPs, FAPs could play a direct (barrier) or indirect (suppression of fusing myoblasts) role on PMO delivery in this system. Thus, we evaluated FAP dynamics and related downstream effectors in D2-mdx and BlO-mdx muscles. Consistent with previous results,

FAPs, stained with PDGFRa, were significantly increased in the D2-mdx muscle interstitium compared to B10-mdx, while expression of fibronectin (Fnl) and Tgfbl, were also significantly elevated in D2-mdx muscle (FIGS. 2E-2G). Further, this was associated with reduced levels of

BrdU-positive centrally nucleated fibers (CNFs) in D2-mdx muscles compared to B 10-mdx (FIGS.

2H-2I). These results suggest that poor PMO delivery to the myofibers in D2mdx muscle is potentially driven by aberrant FAP expansion that hinders resolution of inflammation and SC- mediated repair.

Development of PMO-mediated Tgfbl knockdown (TPMO) strategy. Based on our previous reports ^31,32, we hypothesize that targeted inhibition of TGF-β could be an attractive therapeutic avenue for enhancing myofiber DPMO delivery as it promotes muscle regeneration through enhanced myoblast fusion ^26-29 and through clearance of excessive FAP accumulation in dystrophic muscles ³⁶. However, with the need for systemic dosing for body wide inhibition of TGF-β in dystrophic muscles, we were conscious of the need to specifically target the drug to the site of action (damaged muscle lesions) in order to minimize off-target effects that might otherwise hinder its suitability for translation in DMD. The above and previous results ^19,20 demonstrates that macrophages serve as local reservoirs for PMO within dystrophic muscles. Additionally, as the main TGF-β producing cells in dystrophic muscle are macrophages ^37,38, we hypothesized that systemic delivery anti-TGF-β PMOs designed to block mRNA translation or promote premature termination of mRNA translation by out-of-frame skipping would promote tissue- and cell-type specificity to enhance drug effects within the muscle. To test this hypothesis, we first designed and synthesized a series of anti-TGF-β PMOs (TPMOs) targeting various regions of the Tgfbl gene, including the 5’-UTR (translation blocking PMOs) and various exon-intron junctions (premature termination PMOs) (FIG. 3A). PMO target sites were selected based on a combination of optimal

AO G/C content (<60%), self-complementarity scores, length, and melting temperature (Tm, 90- 100°C). To examine each TPMO’s inhibitory effect of TGF-β expression, we first treated mouse macrophages (RAW 264.7) in vitro with lipopolysaccharide (LPS) to induce TGF-β expression and then with each individual TPMO for 48 hours. This resulted in a ~3.5-fold knockdown of active TGF-β compared to control (scrambled) PMO (FIG. 3B), while TPM04 and TPM05 were slightly less effective reducing active TGF-β by ~2-fold. These results validated our AO design strategy and identified potential lead candidates for further evaluation. Next, we assessed the in vivo efficacy of all TPMOs by eliciting a mild muscle injury in the tibialis anterior (TA) muscle

(multiple needle injuries) to trigger TGF-β expression in D2-WT TA muscles, together with concurrent intramuscular TPMO (100 pg/oligo/TA) injections (FIG.3C). Evaluation of Tgfbl and TGF-β downstream targets ^39,40 4 days post-injection revealed TPM02 and TPM06 as having greatest impact on Tgfbl and TGF-β downstream target transcript expression (FIG. 3D).

Immunofluorescence for phospho-SMAD3 (pSMAD3), a direct downstream mediator of TGF-β signaling, and embryonic myosin heavy chain (eMHC), a marker of regenerating myofibers, revealed that TPM02 had the greatest net benefit in damaged muscle lesions. In comparison to control PMO or TPM06, TPMO2-treated muscles showed reduced pSMAD3 expression and increased numbers of eMHC+ myofibers (FIGS. 3E-3G, 11A-11D), while TPMO6-treated muscles failed to significantly deviate from those levels observed in control muscles. Interestingly, we found an association where damaged lesions with high local pSMAD3 expression also had the fewest number of eMHC+ myofibers (FIG. 3E). Together, these results identified TPM02 as our most efficacious AO both in vitro and in vivo for further develoμment and testing in dystrophic muscle.

Systemic TPMO 2 administration curbs TGF-P signaling and enhances regeneration following spontaneous injury in D2-mdx muscle. To further assess in vivo effects of our lead

TPM02 candidate, we next tested whether systemic delivery of TPM02 elicited muscle-specific therapeutic effects in vivo in D2-mdx mice, which mimic the limited myogenic capacity observed in DMD patients. We systemically injected TPM02 (200 mg/kg) via retro-orbital sinus into juvenile D2-mdx mice and examined the degree of spontaneous muscle regeneration eight days post-administration (FIG.4A). We found the effect of systemic TPMO2 treatment was maintained throughout the duration of the study, as evidenced by decreased pSMAD3 expression in the spontaneous muscle-damaged areas at end point (FIGS 4B, 4D). Consistent with our previous results following intramuscular injection of TPMO2 in D2-WT, we found the number of eMHC+ myofibers was significantly increased relative to our control group, confirming that effective TGF- β inhibition improves muscle regeneration in juvenile D2-mdx mice (FIGS.4C, 4E). Additionally, in control D2-mdx muscles, eMHC+ myofibers were noticeably smaller and failed to fuse with one another when present within the same basement membrane, as compared to TPMO2-treated muscles (FIGS. 4C, 4F). These results indicate that systemic TGF-β inhibition by way of PMO- mediated Tgfbl knockdown improves myoblast fusion and muscle regeneration in vivo.

Dual TPMO and DPMO therapy enhances regeneration and dystrophin restoration in D2- mdx muscle. We previously demonstrated the requirement of SC fusion and regeneration for efficient PMO delivery to dystrophic myofibers ¹⁹. As we found compromised regenerative capacity in the D2-mdx model impedes DPMO-mediated dystrophin restoration (Figure 1), we thus hypothesized that TPMO-mediated inhibition of TGF-β and resulting enhancement of regenerative potential would yield improved DPMO delivery and efficacy in D2-mdx muscle. To test this hypothesis, we evaluated the in vivo benefit of a dual PMO therapeutic strategy, where we first administered TPMO2 (100 μg) by intramuscular injection to the TA muscle of 20-week- old D2-mdx mice, followed 2 days later by systemic administration of DPMO (200 mg/kg), to assess therapeutic impact on dystrophin restoration 3 weeks thereafter (FIG. 5A). To monitor impact on regenerative myogenesis, cohorts also received BrdU for seven days starting two days after needle injury and TPMO administration to label subsequently regenerating myofibers (FIG.

5A). The benefit of dual TPMO+DPMO therapy was benchmarked against a cohort receiving scrambled PMO (IM; control for TPMO) and systemic DPMO. Assessment of dystrophin restoration by immunostaining revealed dual TPMO+DPMO therapy yielded increased numbers of dystrophin-expressing fibers compared to DPMO-treated D2-mdx muscles (FIGS. 5B-5C). As expected, this was associated with a ~2-fold increase in the numbers of BrdU+ CNFs per TA muscle in the dual TPMO+DPMO cohort, relative to controls. Examination of macrophage infiltration and FAP expansion within damage lesions, showed local macrophage response by

F4/80 immunostaining, was not impacted by TPM02. However, although the inhibitory effect on TGF-β signaling did not persist for three weeks following single intramuscular TPMO administration (FIGS. 12A, 12B), the expansion of FAPs as observed by PDFGRa immunostaining, a process regulated by TGF-β ^28.36 and which directly affects myogenesis, ⁴¹, were significantly reduced in damaged sites in dual PMO-treated muscles compared to control

DPMO-treated muscles (FIG. 5B, 5E, 5F). These results indicate that TGF-β inhibition via TPMO suppresses FAP accumulation and promotes muscle regeneration in the damaged lesions, which directly improves DPMO delivery to these regenerating myofibers and the resulting levels of dystrophin restoration in skeletal muscles of D2-mdx mice.

Long-term systemic dual TPMO and DPMO therapy improves skeletal muscle junction through enhanced dystrophin restoration in severe D2-mdx model. To validate our dual PMO strategy and investigate the long-term therapeutic benefits on muscle function in the D2-mdx model, we systemically treated D2-mdx mice with a TPMO+DPMO cocktail therapy twice a week for six weeks, starting from the onset of disease pathology at 3 weeks of age, and benchmarked outcomes measures against saline-treated control mice (FIG. 6A). After 5 weeks of dual

TPMO+DPMO therapy, we investigated the impact on muscle strength through a series of in vivo functional assays. Here, we found dual TPMO+DPMO-treated D2-mdx mice showed improvements in grip strength and inverted suspension hang time, relative to saline-treated D2- mdx mice (FIG. 6B-6C). Additionally, assessment of in vivo isometric force (torque) for the gastrocnemius muscle generated in response to tetanic stimulation of the tibial nerve, revealed improved functional capacity with TPMO+DPMO therapy at tetanic stimulations of 140-200 Hz, as well as, significantly increased maximal peak isometric force, as compared to the saline-treated control muscles (FIG. 6D-E). Pertinent to our functional assessments, we found no differences in longitudinal body weight that might impact functional testing outcomes such as hang time and grip strength assessments (FIG. 12C), however, we did observe slight increases in the weights of the gastrocnemius and TA muscles in our dual PMO cohort relative to saline-treated controls that might indicate therapeutic drug effects on muscle health (FIG. 12E). Based on functional benefits provided by our dual PMO therapy, we next sought to examine the extent of dystrophin restoration with chronic TPMO+DPMO treatment following conclusion of the dosing regimen (FIG. 6A).

Surprisingly, we found dual therapy yielded unexpectedly high levels of dystrophin expression in treated gastrocnemius muscles, achieving -25% dystrophin+ fibers on average and ranging from

-20-40% overall (FIGS. 6F-6G, 12A-12B). Similarly, the number of dystrophin-expressing myofibers with internal nuclei (CNF) was markedly increased, again in accordance with our mechanism of PMO delivery whereby efficient SC fusion enhances PMO uptake in regenerating dystrophic myofibers (FIG. 6H). Furthermore, quantification of dystrophin protein expression in protein lysates generated from these same gastrocnemius muscles using capillary Western immunoassays (Wes), showed our dual PMO therapy achieved restoration of ~7% of WT dystrophin levels, while no dystrophin was detected in saline-treated control muscles (FIG. 6I-6J).

As our dual PMO therapy yielded a significant functional benefit with chronic dosing, we next investigated its impact on restoration of the dystrophin-associated protein complex (DAPC), which is largely compromised in terms of localization and structure in the absence of dystrophin expression. Immunostaining serial muscle sections for dystrophin together with either dystroglycan, dystrobrevin, or a-sarcoglycan revealed their heightened expression properly localized along the sarcolemma in TPMO+DPMO-treated muscles in all fibers where dystrophin had been restored (FIG. 6K). Meanwhile as expected, saline-treated muscles were characterized as having low, diffuse expression of dystroglycan, dystrobrevin, or a-sarcoglycan, in addition to accumulation of protein aggregates along the sarcolemma, indicative of their poor localization and the compromised stability of the DAPC in the absence of dystrophin (FIG. 6K). Importantly, treatment with our dual PMO therapy resolved these aberrant protein aggregates and restored high level DAPC expression specifically at the sarcolemma without any apparent off-target localization

(FIG. 6K).

Dual TPMO and DPMO therapy promotes dystrophin restoration in cardiomyocytes in severe D2-mdx model. In both preclinical models and DMD patients, PMO therapy has failed to effectively target cardiomyocytes, potentially due to the lack of regeneration in cardiac muscle ¹⁹ or cardiomyocyte-specific endosomal PMO entrapment ⁴², which has been a major challenge of this therapeutic approach in DMD. To investigate whether dual TPMO and DPMO therapy could elicit dystrophin restoration in D2-mdx hearts through its stromal effects, we immunostained hearts harvested following chronic dual therapy (FIG. 6A). Quite surprisingly, we found dual PMO therapy was able to restore dystrophin in small clusters of cardiomyocytes scattered throughout the D2-mdx heart (FIG. 7 A), an observation which others have noted ⁴² and which we have not observed previously in response to DPMO therapy alone in the BlO-mdx model ⁴³. Quantification of dystrophin+ cardiomyocytes in response to dual therapy revealed dystrophin restoration in

150-300 cardiomyocytes per heart cross-section, equating to ~2% dystrophin+cardiomyocytes and a roughly 10-fold increase compared to revertant cardiomyocyte levels observed in saline- treated hearts (FIGS. 7B-7C, 13A-13B). Additionally, assessment of dystrophin+ cardiomyocyte clusters per heart, showed a >4-fold increase relative to those observed as revertant cardiomyocytes in saline-treated hearts. Although the sporadic nature of dystrophin restoration in dual PMO-treated hearts failed to exert any apparent benefit on the expression of dystroglycan, dystrobrevin, or a-sarcoglycan (FIG. 13C), as was shown in skeletal muscles harvested from this cohort (FIG. 6K), this result still represent a striking finding in light of the historical challenges for PMO delivery to cardiac muscle, and may indicate targeting of TGF-β in conjunction with

DPMO therapy as a potential therapeutic avenue to improve PMO-mediated dystrophin restoration in the hearts of DMD patients.

Discussion

FDA-approved, PMO-based exon skipping AO therapies represent one of the most promising therapeutic techniques for DMD and have shown positive clinical effects in DMD patients, attenuating ambulatory decline compared to natural history cohorts and placebo control groups ^12-14 However, their clinical impact has been more limited than initially projected based on preclinical data ^9.11.44 due to the sporadic and limited uptake in myofibers ^19.43. of penetrance in cardiomyocytes ⁴² Similar to DMD patients, we find that restoration of dystrophin protein using PMO AOs in the more severe D2-mdx model is strictly limited compared to that observed in BlO-mdx mice, which have far milder dystrophic pathologies. In accordance with our

‘inflammation-mediated PMO delivery’ mechanism ^19.20, while we find the PMO deposited and retained within inflammatory lesions throughout the dystrophic muscle, the reduced myogenic capacity exhibited by the D2-mdx model (a critical pathological feature of DMD) strictly limits that transit of PMO from inflammatory cells to fusing SCs and regenerating myofibers which dramatically curbs the extent of dystrophin restoration (Figure 1-2). Our prior work demonstrated aberrant TGF-β signaling in D2-mdx muscles, driven by a polymorphism in the Ltbp4 locus, aggravates immune cell and FAP dynamics which compromise SC function and regenerative potential in the D2-mdx model ³¹’³². These findings are clinically relevant for DMD patients, as

LTBP4 polymorphisms are also associated with the progression of fibrosis in DMD patients ⁴⁵.

Further, we found direct modulation of TGF-β using a potent small molecule inhibitor of TGF-β attenuated degenerative pathology and enhanced regenerative potential in vivo in this model ³²

Thus, direct targeting of TGF-β in severely dystrophic muscle represents a promising strategy to alter disease severity in DMD, mitigate progressive myogenic deficits, and potentially enhance the efficacy of current exon skipping AO therapies for DMD based on the requirement of proficient regeneration for PMO uptake in dystrophic muscles ^{19, 20. 15}

Based on the propensity of macrophages to phagocytize and retain PMO within degenerating lesions ^{19, 20}, in addition to these cells being the major producers of TGF-β in dystrophic muscles ³⁷, we thus hypothesized that development of a novel PMO-based approach that effectively targets TGF-β (TPMO) signaling would not only enhance myogenic potential of dystrophic muscle, but also promote timely delivery of dystrophin-restoring PMOs to regenerating dystrophic myofibers to overcome the major barrier limiting its efficacy in DMD patients. Importantly, development of TGF-β focused therapeutics would serve to benefit the DMD patient population as a whole, regardless of dystrophin mutation, as excessive TGF-β production is a common feature of DMD 23,24,46,47, even from infancy ⁴⁶, and positively correlates with disease severity and prognosis 23,25,45,46 Thus, we next developed and screened a series of novel TPMO

AO candidates to downregulate TGF-β that either work by blocking mRNA translation via targeting of the 5’-UTR and translation start site, or by initiating premature termination of mRNA translation through targeting of downstream exon-intron boundaries to promote out-of-frame‘exon skipping ’ (FIG. 3). Critically, initial in vitro screening in macrophage cultures showed downregulation of active TGF-β for both types of AO strategies and provided a list of candidates for further evaluation in vivo. Excitingly, we found a single intramuscular dose of our lead TPMO candidates (TPM02, TPM06) effectively reduced TGF- β -mediated downstream pSMAD3 expression and increased regenerative capacity following acute needle-induced injury in D2-WT muscles (FIG. 3).

Meanwhile, a single systemic dose of TPM02, which we identified as our most promising

TPMO candidate, recapitulated these findings even in the context of the highly aggravated degenerative pathologies specific to juvenile D2-mdx mice (FIG. 4). Interestingly, newly regenerated myofibers were observed specifically in damaged areas with low expression of pSMAD3, validating our therapeutic strategy of targeting TGF-β in dystrophic muscle tissue to attenuate myogenic deficit in severe DMD (FIG. 3-4). With our initial testing demonstrating the efficacy of our TPMO strategy to rescue regenerative deficits in the D2-mdx model, we next sought to test our hypothesis that TPMOs would work synergistically with dystrophin-restoring exon skipping PMOs (DPMO) to enhance their uptake in dystrophic myofibers, based on our earlier work ¹⁹’²⁰. Use of either acute intramuscular TPMO delivery followed by systemic DPMO delivery, or a chronic combinational TPMO and DPMO regimen in D2-mdx mice, proved combinational use of these drags were in fact synergistic as evidenced by the significant increase in numbers of dystrophin-expressing myofibers following dual PMO therapy (FIGS. 5-6). Considering the major pharmacological challenges that have limited the clinical impact of exon skipping therapies since their inception, our results suggest co-administration of TGF-β-modulating PMOs may improve outcomes for DMD patients receiving dystrophin-restoring PMO therapies, irrespective of their mutation specific exon skipping therapy. Critically, as exon skipping therapies in DMD are each tailored to specific mutation locus and thus are only applicable to a subset of DMD patient pool, use of TGF-β-modulating PMO therapy could also serve to benefit the broader DMD population irrespective of dystrophin mutation (or available exon skipping therapy) to attenuate myogenic deficits and accompanying pathologies.

Pharmacokinetically, PMO is rapidly cleared from the blood within just hours following systemic administration ^48,49. However, PMO actively sequestered by macrophages within damaged muscle is retained for at least a week in close proximity SC undergoing fusion for myofiber repair ¹⁹. As TGF-β is a multifunctional cytokine that regulates embryonic development, cell proliferation and differentiation, immune response and wound healing, as well as, extracellular matrix deposition and fibrosis, persistent systemic inhibition of TGF-β may have undesirable effects ³⁰. In a previous study ³¹, intramuscular treatment of D2-mdx mice with ITD-1, which is a specific inhibitor of the TGF-β pathway that targets selective proteasomal degradation of type n TGF-β receptor, reduced the degeneration of D2-mdx muscle without significantly altering the extent of regenerative myogenesis. However, in the prior study we were limited to using intramuscular delivery of ITD-1 in order to avoid potential systemic insults. Here, our use of PMO approach to effectively downregulate TGF-β signaling, serves to more precisely target the drag to macrophages where it exerts its effects. As macrophages responding to injury promote the proliferation of SC through direct contact ⁵⁰, our results also indicate that PMO mediated inhibition of macrophage-secreted TGF-β effectively alters the fate of both FAPs and SCs within the stromal to attenuate disease pathologies, as evidence by reduced FAP-specific PDGFRa expression and increased levels of myoblast fusion and nascent fiber formation (FIG. 5). Thus, use of PMO-based

AOs to downregulate TGF-β expression and activity offers the unique advantage of enhancing macrophage-specific delivery within dystrophic muscle to mitigate potential off-target systemic effects, and in fact, chronic systemic administration of our TGF-β -inhibiting TPMO, in combination with DPMO, over 6 weeks did not cause any apparent behavioral side effects or changes in body or organ (kidney, spleen) weights (FIG. 12C-12D), or signs of any gross hepatic or renal toxicity. Further, while we observed no difference in heart weight at conclusion of the trial, we did find a slight but non-significant increases in the weights of the quadriceps and triceps and significant increases in the weights of the gastrocnemius and TA muscles with dual PMO therapy, relative to saline-treated controls (FIG. 12E), which we attribute to the high levels of dystrophin restoration and regeneration achieved with our dual PMO therapy.

Rather surprisingly, we show for the first time that co-administration of TPMO together with DPMO promotes DPMO delivery to cardiomyocytes resulting in dystrophin restoration (FIG.

7). The lack of effective PMO delivery into dystrophic cardiomyocytes is well documented ^9,43 and has become the major limitation of this therapeutic approach for DMD. It has been speculated that

PMO-based approaches have been unsuccessful in restoring heart dystrophin expression due to limited cardiomyocyte regeneration ⁵¹, in light of our mechanism of efficient PMO delivery in dystrophic skeletal muscle ¹⁹. However, curiously pharmacological inhibition of TGF-β has been reported to enhance the differentiation of uncommitted mesoderm ¹⁸ into cardiomyocytes ⁵² and improve cardiac regeneration and function in a myocardial infarction model ⁵³. Furthermore, in a y-sarcoglycan deficient mouse model (Sgcg null), increased TGF-β signaling was observed in the heart, while inhibition of this signaling improved cardiac function ⁵⁴’⁵⁵. Based on this evidence, we propose that TGF-β inhibition through our PMO-based strategy may promote cardiac regeneration to encourage PMO uptake within cardiomyocytes and elicit dystrophin expression.

In summary, we have developed a novel therapeutic approach using FDA-approved PMO- based AO to simultaneously inhibit TGF-β to mitigate its detrimental effects on myogenesis and stromal cell fate and enhance dystrophin restoration in dystrophin muscles. We found use of a dual TGF-β-targeting and dystrophin-restoring PMO therapy, not only enhanced myogenic capacity of dystrophic skeletal muscle, but also promoted the efficient delivery of dystrophin-restoring PMO therapy by way of this enhanced regenerative myogenesis, in accordance with our published mechanism of PMO delivery ^19,20. Notably, this approach restored stromal cell dynamics and improved muscle health and function in the severe D2-mdx model.

Further, we revealed a novel therapeutic approach to promote the update of dystrophin restoring PMOs in cardiac muscle and elicit dystrophin expression through dual targeting of TGF-β.

Further, while our dual PMO approach would be anticipated to work synergistically with exon skipping therapies, regardless of exon mutation, our TGF-β-targeting PMO therapy could stand alone as a monotherapy for broad application in DMD. Importantly, our findings have revealed a promising and novel therapeutic approach to overcome the major barriers that have limited the clinical impact of PMO-based exon skipping therapies for DMD to date.

Materials and Methods Animals. The Institutional Animal Care and Use Committee (IACUC) of the Children’s

National Research Institute (CNRI) reviewed and approved all animal procedures. The mouse strains were used,

which harbor a nonsense point mutation in Dmd exon 23. We also used the DBA/2J wild type strain to confirm the efficacy of PMO-mediated Tgfbl knockdown. Mice were purchased from the

Jackson Laboratory (Bar Harbor, ME) and bred in-house for experiments. Mice were housed in a sterile barrier facility and were maintained under normal, ambient conditions (-21% O2, -22 °C}

1°C, 12h light/12 h dark) with continuous access to food and water throughout the studies. For experiments, mice were randomized to cohorts based on sex and body weight. However, in the chronic dual PMO trial, we used strictly 3 -week-old D2-mdx male mice to standardize functional testing measures. For the tissue harvesting, mice were euthanized at designated timeframes with

CO2 inhalation and cervical dislocation, and muscles were collected and mounted on cork in tragacanth gum and flash-frozen in liquid nitrogen-chilled isopentane for storage at -80°C.

BrdU labeling. 5-bromo-2-deoxyuridine (BrdU) (Sigma-Aldrich, B9285) was administered ad libitum in drinking water (0.8 mg/ml) for a period of 7d after systemic injection of DPMO ¹⁹’³¹. Water containing BrdU was kept protected from light during administration.

Immunofluorescence. The muscles' transverse cryosections (8 μm thick) were prepared using a

CM3050S cryostat (Leica Biosystems) and stored at -80°C. For immunofluorescence procedures as previously described ^19,31,32. Sectioned muscle samples were stained with primary and secondary antibodies as described in Table SI.

Table SI. Primary and secondary antibodies for immunofluorescence.

TGF-β1 EUSA. RAW 264.7 macrophage cell line (ATCC, VA) were cultured in DMEM, high glucose (4500 mg/L), GlutaMAX supplemented with 10% heat-inactivated FBS, and 1% penicillin/streptomycin (100 U/mL) (Thermo Fisher, MA) at 37°C with 5% CO2. Cells were plated at 12-well plates with a density of 3.0x105 cells per well, stimulated with LPS (100 ng/ml) to induce TGF-β1 expression, and treated with each TPMO for 48h. The amount of active TGF-β1 was determined in whole-cell lysates by a Quantikine ELISA mouse TGF-β 1 immunoassay (R&D

Systems, Bio-Techne, MB100B) according to the manufacturer’s recommendations. Final values were normalized to total protein concentration.

PMO antisense sequences and delivery. Dystrophin-restoring PMO targeting the exon- intron boundary of exon 23 of the Dmd pre-mRNA (+7-18; 5 -GGC CAA ACC TCG GCT TAC CTGAAA T-3 '(SEQ ID NO: 13) (murine DPMO) and a modified PMO conjugated to fluorescein (+7-18; 5'-GGC CAA ACC TCG GCT TAC CTG AAA T-3') (SEQ ID NO:

14)(murine -Fluorescein: F-DPMO) were synthesized (GeneTools, LLC).

To suppress the expression of TGF-β1 using PMO, we synthesized PMOs based on murine sequences to target six different regions of Tgfbl pre-mRNA, including the 5’ UTR, the start codon, and the exon-intron junction (FIG. 3A);

The murine PMOs above were diluted in saline and incubated at 50°C for 15 min before administration.

Mice were anesthetized with 3% isoflurane, and PMOs were administered IM in the tibialis anterior muscle or systemically via the retro-orbital sinus (200 mg/kg or 400 mg/kg, <100 μl total volume) using an insulin syringe. Saline or random control oligo 25-N (GeneTools, LLC) was administered at equivalent volumes as a control.

TaqMan RT-qPCR. Total RNA was extracted from sections of muscle samples using

TRIzol reagent (Thermo Fisher, MA, 15596018) according to the manufacturer’s recommendations. cDNA was synthesized from RNA (1000 ng) using the High-Capacity cDNA

Reverse Transcription kit (Thermo Fisher, 368813) following the manufacturer’s protocol. cDNA

(50 ng) was loaded for each triplicate reaction in 384-well plates and analyzed using mouse- specific Taqman probes (Thermo Fisher) and Taqman Fast Advanced Master Mix (Thermo Fisher,

4444557) on the 7900HT Fast Real-time PCR system (Applied Biosystems). Taqman probes are listed in Table S2.

Table S2. Taqman qPCR gene expression assays.

The comparative Ct method (AACt) quantified relative amounts of mRNA. All mRNA values were normalized to those of Hprt. To calculate the exon skipping of Dmd rate, we used the skipped Dmd product (AIODQL; Thermo Fisher, splice junction spanning Dmd exon 22-24) and non-skipped Dmd product (Mm01216492_ml (Thermo Fisher) amplifies the region spanning

Dmd exons 2-3). Percent exon skipping was calculated as previously described 19,56.

Forelimb grip strength. A grip strength meter (Columbus Instruments) was used to assess the forelimb grip strength test daily for three consecutive days according to Treat NMD protocols

(DMD M.2.2.001). Data are represented as averaged grip strength of maximum daily values over three days. Timed inverted hang. For the timed wire hanging test, mice were placed in a homemade box covered with wire mesh (1 x 1 cm grid), flipped over, and placed ~35 cm above a cage with soft bedding. Hang time was recorded, with 600 seconds used as a cutoff.

In vivo isometric torque. Mice were anesthetized with 2% isoflurane-mixed 02, and the foot was attached to the dual-mode lever and maintained at 90 degrees for isometric torque assessment (Aurora Scientific) as previously described ^37>58. Contraction of the plantar flexor was controlled by stimulation of the tibial nerve. Isometric muscle contractions were stimulated at 1.0-

2.0 mA using Pt-Ir needle electrodes inserted percutaneously adjacent to the tibial nerve. Peak isometric torque was measured in response to varying tetanic stimulations (20, 40, 80, 100, 120,

140, 160, 180, and 200 Hz), providing a 60s rest period between stimuli.

Capillary western immunoassay (Wes). Muscles were dissected and frozen in liquid- nitrogen cooled isopentane. 8 μm sections were lysed in 5% SDS buffer containing lOmM EDT A

(pH 8.0), 75mM Tris-HCL (pH 6.8), and protease inhibitors. Capillary western immunoassay

(Wes) analysis was performed according to the manufacturer’s instructions using 66-440 kDa

Separation Modules (ProteinSimple, #SM-W008). In each capillary, 0.2 mg/mL protein was loaded for analysis with antibodies to dystrophin (Abeam # ab 154168, dilution 1:15) or alpha actinin (Abeam #ab68167, dilution 1:100), and anti-rabbit secondary (ProteinSimple #042-206).

Compass for SW software was used to quantify chemiluminescence data. To measure the expression level of dystrophin after dual PMO treatment to D2-mdx mice, dystrophin in protein lysate of WT gastrocnemius was set to 100% and mixed with protein lysate of D2- mdxgastrocnemius to create a standard curve. A standard curve was generated using the mixture ofWT lysate/ T)2-mdx lysate denoting 20%, 10%, 5%, 2.5%, 1.25%, and0%WT dystrophin levels. For example, in the case of 5% WT dystrophin, protein lysate for Wes was made by mixing 95%

D2-mdx lysate and 5% WT lysate ^56,57.

Statistical analyses. Data were analyzed using Prism GraphPad software (9.2.0). Statistical analysis was performed using non-parametric Mann-Whitney test or one-way ANOVA. All p values less than 0.05 were considered statistically significant; *p < 0.05, **p < 0.01, ***p <0.001, and ****p < 0.0001. Data plots were reported as scatter plots with mean and SD.

REFERENCE TO PROVISIONAL DISCLOSURE

The entire contents of the priority document U.S. Provisional Application No. 63/591,431, filed October 18, 2023, are incorporated by reference. Specific reference is made to the data and accompanying text in Figure 1, Macrophage-mediated PMO delivery; Figures 2A and 2B, D2- mdx macrophages retaining large quantities of PMO at the site of muscle damage; Figures 3 A-3G,

D2-mdx satellite cells show limited myogenicity after spontaneous and NTX-induced injury;

Figures 4A-4H, Increase in TGF-β levels drives D2-mdx muscle degeneration at disease onset;

Figures 5A-5C, development of PMO-mediated TGF-β knockdown In vitro,- Figures 6A and 6B, In vivo efficacy of PMO-mediated TGF-β knockdown; Figures 1 A and B (Report 1), Identification of macrophage and fibroblast genes responsive to TGF-β signaling; Figures 2A and 2B (Report 1),

Macrophage genotype does not affect expression of TGF-β-responsive genes; Figures 3 A and 3B

(Report 1), Macrophage genotype affects production and secretion of active TGF-β in response to inflammatory signaling; Figures 4A and 4B (lower three panels; Report 1), Effect of injury on in vivo expression of TGF-β-responsive genes and on muscle regeneration; and Figures 5 A and 5B

(Report 1), Assessment of muscle regeneration in juvenile and adult mdx and WT mice; Figures

1A-1C (Report 2), Confirmation of PMO-medicated TGF-β knockdown through expression of TGF-β target genes in TA muscle; Figures 2A and 2B (Report 2), Evaluation of PMO-mediated inhibition of TGF-β activity through the express of pSmad3, the downstream signal of TGF-β;

Figures 3 A-3C (Report 2), Assessment of muscle regeneration of anti-TGF-β PMO in TA muscle;

Figures 4A-4D (Report 2), The effectiveness of anti-TGF-β (AO2 or AO6) and dystrophin PMO

(DPMO); Figures 5A and 5B (Report 2), Analysis of muscle regeneration of anti-TGF-β(AO2 or

AO6) and dystrophin PMO (DPMO) combination in TA muscle of D2 mdx mice; Figure 6 (Report

2), Assessment of dystrophin expression of anti-TGF-β (AO2 or AO6) . Each of these figures and the accompanying background, materials and methods, remarks, analysis, and discussion is incorporated by reference.

ADDITIONAL TECHNOLOGICAL DESCRIPTION

The term “subject" typically refers to human subjects and any of the embodiments or claims described herein may be applied to treatment of humans. In some embodiments, the methods disclosed herein are applied to non-human mammalian subjects, for example, in experimental models of human disease.

TGF-f-related musculoskeletal disorders include but are not limited to such disorders as

Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Limb-girdle muscular dystrophy (LGMD), Congenital Muscular Dystrophies (CMD), Emery-Dreifuss

Muscular Dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Osteogenesis

Imperfecta, Marfan Syndrome (MFS), and conditions such as Cachexia and Sarcopenia. Such disorders are described by and incorporated by reference to Limback, D., et al.. (2022). A

Comprehensive Review of Duchenne Muscular Dystrophy. BIOTECHNOLOGY JOURNAL

INTERNATIONAL, 26(6), 1-31 and Burks, T.N. and Cohn, RD. (2011). Role of TGF-β signaling in inherited and acquired myopathies. SKELETAL MUSCLE, 1, 19. Dystrophin. A sequence of full-length mRNA transcript encoding normal dystrophin and a corresponding amino acid sequence for dystrophin are given by GenBank Accession:

NM 004006.1 and by GenBank Accession: NP 004007. The GenBank accession number for the normal human dystrophin is NM 004006.1. Here are some other GenBank accession numbers for dystrophin: NM_004007:The cDNA for human dystrophin; D32048: The genomic DNA for human dystrophin; NP 003998: The protein for human dystrophin. The dystrophin gene is responsible for encoding a large protein that forms part of the dystrophin-glycoprotein complex

(DGC). The DGC connects the extracellular matrix to the inner cytoskeleton. Mutations in the dystrophin gene can cause Duchenne muscular dystrophy (DMD), Becker muscular dystrophy

(BMD), or cardiomyopathy.

Micro-dystrophins are shorter, engineered forms of dystrophin which are easier to fit into vectors for gene therapy. They typically contain a dystrophin N-terminal domain, a reduced number of spectrin-like repeats from the central rod domain, a cysteine-rich domain and/or dystrophin C-terminal domain. Further description of micro-dystrophins is found in, and incorporated by reference to, Ramos, et al., Development of Novel Micro-dystrophins with

Enhanced Functionality, MOL. TllER 2019 27(3): 623-635 or WO 2021 108755A2,

Microdystrophin gene therapy and uses thereof Micro-dystrophins and therapy with micro- dystrophins may be used in the methods described herein of gene therapy.

TGF-beta. The TGF-beta gene encodes a secreted ligand of the TGF-beta (transforming growth factor-beta) superfamily of proteins. Ligands of this family bind various TGF-beta receptors leading to recruitment and activation of SMAD family transcription factors that regulate gene expression. TGF-beta 1 is a common form, but in some embodiments TGF-beta 2 and TGF- beta may also be targeted with antisense or other gene silencing procedures. Accession numbers for human TGF-beta 1 : mRNA: NM 000660.7 and for Protein: NP 000651.3. The mRNA sequence may be employed to design antisense oligonucleotides as described herein.

Gene therapy to enhance dystrophin expression or restoration include gene addition, gene correction, exon skipping procedures or treatment with exon-skipping drugs such as etepliresn, goldirsen, viltolarsen and casimersen, multi exon skipping. Such drugs may use small synthetic pieces of DNA or RNA called antisense oligonucleotides (AONs) which bind to specific exons in pre-mRNA, causing them to be "skipped" during the splicing process thus promoting expression of more highly functional dystrophins. Preferred procedures for increasing dystrophin expression or levels include exon skipping drugs and gene therapy.

Orthologs and variants. When a non-human mammalian sense or antisense oligonucleotide is disclosed, a corresponding human ortholog is expressly contemplated. Such orthologs may be identified by at least 70, 80, 90, 95, and up to 99% sequence homology or similarity to the corresponding non-human mammalian genes or by their ability to bind to mRNA sequences complementary to the non-human antisense oligonucleotides disclosed herein and/or to inhibit expression of genes via the human ortholog of the AO. The inventors expressly contemplate human orthologs to the murine DPMO and TPMO sequences disclosed here as well as variants of the human antisense sequences disclosed here.

Vectors useful for expressing dystrophin or truncated forms of dystrophin include adenovirus, adeno-associated virus, HSV, retrovirus, and lentivirus vectors. Polynucleotide encoding dystrophin may also be introduced into cells using , cationic lipids, cationic polymers, electroporation, or encapsulation in nanoparticles.

Gene silencing includes partial as well as full silencing of a gene or transcribed mRNA, such as mRNA encoding TGF-beta. Various modes of silencing include use of antisense oligonucleotides that target TFG-beta mRNA; use of RNA interference (RNAi), small interfering

RNAs (siRNAs, e.g. about 20-25 nucleotides in length) or short hairpin RNAs (shRNAs) that bind to or cleave complementary RNA sequences such as those encoding TGF-beta. Smad7

Overexpression to attenuated TGF-beta effects in its targeted tissues. A preferred procedure for decreasing TGF-beta expression or levels involve administering antisense oligonucleotide drugs.

A phosphorodiamidate morpholino oligomer (PMO) is a synthetic molecule used to modify gene expression. Its structure comprises DNA bases attached a backbone of methylene morpholine rings linked by phosphorodiamidate groups. It is resistant to degradation, substantially non-toxic, water soluble and can specifically bind to complementary nucleic acid strands.

Phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that inhibit gene expression in a sequence-dependent manner. PMOs of various lengths (7 to 20 bases) were tested for inhibition of luciferase expression in Escherichia coli. Shorter PMOs generally inhibited luciferase greater than longer PMOs. See also ANTIMICROB AGENTS CHEMOTHER.2005 Jan; 49(1):

249-255. doi: 10.1128/AAC.49.1.249-255.2005 which is incorporated by reference for its description of PMO structure and function.

Modes of administration. The antisense oligonucleotides disclosed herein may be administered by any mode that brings them into contact with target cells or tissues. These include in situ inj ection into muscle, tissue, or other target site; intracardiac or intramuscular administration, intravenous administration or infusion, intrathecal administration, intrapulmonary administration, intranasal administering or administration into or onto a mucous membrane, parenteral administration, subcutaneous administration, oral or sub buccal administration. Isabels or tags. The PMOs described herein may be tagged, for example, with a fluorescent label, fluorescent base analogue, external fluorophore, radioactive label, biotin, or other small chemical group.

Carrier groups. The antisense oligonucleotides disclosed herein may be conjugated to other chemical groups that increase their stability in vivo or promote their uptake by cells. These include covalently attaching polyethylene glycol (PEG) chains; conjugation to a lipid, conjugation to a cell penetrating peptide, conjugation to GalNAc, or incorporation into a nanocarrier.

PMO backbone modifications which are in preclinical or clinical development and which may be used in conjunction with the products and methods disclosed herein include peptide or lipid modified or encapsulated PMOs.

Antisense DNA design. Antisense DNA may be designed by methods known in the art, for example, by siDirect version 2.1 highly effective, target specific siRNA online design site; incorporated by reference to Ui-Tei et al. , Nucleic Acids Res 32, 936-948 (2004); Reynolds et al. ,

Nat Biotechnol 22, 326-330 (2004) or Amarzguioui etal., BBRC 316, 1050-1058 (2004).

DMD\ Duchenne Muscular Dystrophy

PM0\ Phosphorodiamidate Morpholino Oligomer.

DPMO: dystrophin-restoring PMO.

TPMO: TGF-β1 -targeting PMO.

TGFB1-TARGETING ANTISENSE OLIGONUCLEOTIDES (TPMO) SEQUENCES

TPMO described in this application and in FIG 3A are detailed below in their 5’-3’ orientation (murine, SEQ ID NOS: 1, 3, 5, 7, 9 and 11, together with their Tgfbl mRNA target sequence (murine, SEQ ID NOS: 2, 4, 6, 8, 10 and 12). Tgfbl-AO-1 (TPMO1). AO sequence: 5’- TCCTGAATAATTTGAGGTTGAGGGA-3’

(SEQ ID NO: 1);

Tgfbl mRNA sense sequence: 5’-TCCCTCAACCTCAAATTATTCAGGA-3’ (SEQ ID

NO: 2

Tgfbl-AO-2 (TPMO2). AO sequence: 5’- GGTCTCCCAAGGAAAGGTAGGTGAT-3’

(SEQ ID NO: 3);

Tgfbl mRNA sense sequence: 5’-ATCACCTACCTTTCCTTGGGAGACC-3’ (SEQ ID

NO: 4)

Tgfbl-AO-3 (TPMO3). AO sequence: 5’-TTCGGAGAGCGGGAACCCTCGGCAA-3*

(SEQ ID NO: 5);

Tgfbl mRNA sense sequence: 5’-TTGCCGAGGGTTCCCGCTCTCCGAA-3’(SEQ ID

NO: 6)

Tgfbl-AO-4 (TPMO4). AO sequence: 5’-CAGTAGCCGCAGCCCCGAGG-3’(SEQ ID

NO: 7);

Tgfbl mRNA sense sequence: 5’-CCTCGGGGCTGCGGCTACTG-3’ (SEQ ID NO: 8)

Tgfbl-AO-5 (TPMO5).

AO sequence: 5’- GGCTCAAAGCCTTACCTGGTAGAGT-3’ (SEQ ID NO: 9);

Tgfbl mRNA sense sequence: 5’- ACTCTACCAGgtaaggctttgagcc-3’(SEQ ID NO: 10)

Tgfbl-AO-6 (TPMO6). AO sequence: 5’-ACAAAGACAAGCAATCTCACCTCCT-3’

(SEQ ID NO: 11);

Tgfbl mRNA sense sequence: 5’-AGGAGgtgagattgcttgtctttgt-3’(SEQ ID NO: 12. Variants or orthologs of the TPMOs described above (or other AO s described herein) may contain 1, 2, 3 or 4 mismatches with the corresponding TPMO sequences and bind to the mRNA complements of the TPMOs described above.

The two murine sequences below, SEQ ID NOS: 13 and 14 are relevant only in context of a DMD mouse model that harbors an exon 23 mutation.

Murine dystrophin-restoring PMO targeting the exon-intron boundary of exon 23 of the

Dmd pre-mRNA (+7-18; 5'-GGC CAA ACC TCG GCT TAC CTGAAA T-3'(SEQ ID NO: 13) :

DPMO) and a modified PMO conjugated to fluorescein (+7-18; 5 -GGC CAA ACC TCG GCT

TAC CTG AAA T-3'(SEQ ID NO: 14).

Human TPMO sequences corresponding to the sequences above are disclosed below.

TPMO1 (human): 5’- TCGAGGGAAAGCTGAGGTCCTCAGGGA-3’ (SEQ ID NO:

15);

TPMO2 (human): 5’- GGTCTCCCGGCAAAAGGTAGGAGGG-3’(SEQ ID NO: 16);

TPM03 (human): 5’- TGCCGAGAGCGCGAACAGGGCTGG-3’ (SEQ ID NO: 17);

TPMO4 (human): 5’- CAGCAGCCGCAGCCCGGAGG-3’(SEQ ID NO: 18);

TPMO5 (human): 5’- ggctcatgtcctcacCTGGTACAGCT-3’(SEQ ID NO: 19);

TPMO6 (human): 5’-gacacacaagtaatcctcacCTCCA-3’(SEQ ID NO: 20).

Reference is made to the 100% complements of SEQ ID NOS: 15-20 which mRNAs can be used to identify TPMO variants having 1, 2, 3, or 4 mismatches with the human TPMO1-

TPMO6 that bind to these complementary human mRNA target sequences.

Dystrophin-targeting antisense oligonucleotides (DPMO). A combinational oligonucleotide approach can be applied to any such dystrophin-targeting antisense oligonucleotide drug currently in preclinical or clinical development for DMD, or that has received regulatory approval for clinical use in DMD. These include, but at not limited to: Eteplirsen

(Sarepta; DMD exon 51 skipping); Golodirsen (Sarepta; DMD exon 53 skipping); Casimersen

(Sarepta; DMD exon 45 skipping); Viltolarsen (NS Pharma; DMD exon 53).

Eteplirsen (SEQ ID NO: 21):

Goldirsen (SEQ ID NO: 22):

Casimersen (SEQ ID NO: 23):

Viltolarsen (SEQ ID NO: 24:

TERMINOLOGY

Terminology. Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The headings (such as "Background" and "Summary") and sub-headings used herein are intended only for general organization of topics within the present invention and are not intended to limit the disclosure of the present invention or any aspect thereof. In particular, subject matter disclosed in the "Background" may include novel technology and may not constitute a recitation of prior art.

Subject matter disclosed in the "Summary" is not an exhaustive or complete disclosure of the entire scope of the technology or any embodiments thereof. Classification or discussion of a material within a section of this specification as having a particular utility is made for convenience, and no inference should be drawn that the material must necessarily or solely function in accordance with its classification herein when it is used in any given composition.

The terms “we” and “our” refer to one, two or more of the inventors.

As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof.

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 “/”.

As used herein in the specification, phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), +/- 15% of the stated value (or range of values), +/- 20% of the stated value (or range of values), etc. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

A range encompasses its endpoints as well as values inside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2, 3, 4, <5 and 5; and a range of 1-10 includes all intermediate values and subranges, for example, 1-9, 1-8, 1-7, 2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-

10, 8-10 or 9-10.

As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the technology.

As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word "include," and its variants, is intended to be non- limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms "can" and "may" and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present invention that do not contain those elements or features.

Although the terms “first” and “second” may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

The description and specific examples, while indicating embodiments of the technology, are intended for purposes of illustration only and are not intended to limit the scope of the technology. Moreover, recitation of multiple embodiments having stated features is not intended to exclude other embodiments having additional features, or other embodiments incorporating different combinations of the stated features. Specific examples are provided for illustrative purposes of how to make and use the compositions and methods of this technology and, unless explicitly stated otherwise, are not intended to be a representation that given embodiments of this technology have, or have not, been made or tested.

All publications and patent applications mentioned in this specification 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.

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.

CITATIONS

1. Crisafulli S, Sultana J, Fontana A, Salvo F, Messina S, Trifiro G. Global epidemiology of Duchenne muscular dystrophy: an updated systemaMc review and meta-analysis. Orphanet J Rare Dis. 2020;15(l):141.

2. Blake DJ, Weir A, Newey SE, Davies KE. FuncMon and geneMcs of dystrophin and dystrophin-related proteins in muscle. Physiological reviews. 2002;82(2):291-329.

3. ErvasM JM, Campbell KP. Membrane organizaMon of the dystrophin-glycoprotein complex. Cell. 1991;66(6):1121-31.

4. Duan D, Goemans N, Takeda S, Mercuri E, Aartsma-Rus A. Duchenne muscular dystrophy. Nat Rev Dis Primers. 2021 ;7(1): 13.

5. Mercuri E, Bonnemann CG, Muntoni F. Muscular dystrophies. Lancet. 2019;394(10213):2025-38.

6. Duan D. Systemic AAV Micro-dystrophin Gene Therapy for Duchenne Muscular Dystrophy. Mol Ther. 2018;26(10):2337-56.

7. Ramos JN, Hollinger K, Bengtsson NE, Allen JM, Hauschka SD, Chamberlain JS. Development of Novel Micro-dystrophins with Enhanced FuncMonality. Mol Ther. 2019;27(3):623-35.

8. Rind DM. The FDA and Gene Therapy for Duchenne Muscular Dystrophy. JAMA. 2024;331(20): 1705-6.

30

9. Alter J, Lou F, Rabinowitz A, Yin H, Rosenfeld J, Wilton SD, et al. Systemic delivery of morpholino oligonucleoMde restores dystrophin expression bodywide and improves dystrophic pathology. Nat Med. 2006; 12(2): 175-7.

10. Lu QL, Rabinowitz A, Chen YC, Yokota T, Yin H, Alter J, et al. Systemic delivery of anMsense oligoribonucleoMde restores dystrophin expression in body-wide skeletal muscles. Proceedings of the NaMonal Academy of Sciences of the United States of America. 2005; 102(1): 198-203.

11. Yokota T, Lu QL, Partridge T, Kobayashi M, Nakamura A, Takeda S, et al. Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs. Annals of neurology. 2009;65(6):667-76.

12. Mendell JR, Khan N, ShaN, Eliopoulos H, McDonald CM, Goemans N, et al. Comparison of Long-term Ambulatory FuncMon in PaMents with Duchenne Muscular Dystrophy Treated with Eteplirsen and Matched Natural History Controls. JNeuromuscul Dis. 2021;8(4):469-79.

13. McDonald CM, Shieh PB, Abdel-Hamid HZ, Connolly AM, Ciafaloni E, Wagner KR, et al. Open-Label EvaluaMon of Eteplirsen in PaMents with Duchenne Muscular Dystrophy Amenable to Exon 51 Skipping: PROMOVI Trial. JNeuromuscul Dis. 2021;8(6):989-1001. 14. Iff J, Done N, Tuhle E, Zhong Y, Wei F, Darras BT, et al. Survival among paMents receiving eteplirsen for up to 8 years for the treatment of Duchenne muscular dystrophy and contextualizaMon with natural history controls. Muscle & nerve. 2024;70(l):60-70.

15. Clemens PR, Rao VK, Connolly AM, Harper AD, Mah JK, Smith EC, et al. Safety, Tolerability, and Efficacy of Viltolarsen in Boys With Duchenne Muscular Dystrophy Amenable to Exon 53 Skipping: A Phase 2 Randomized Clinical Trial. JAMA Neurol. 2020;77(8):982-91.

16. Arora V, Devi GR, Iversen PL. Neutrally charged phosphorodiamidate morpholino anMsense oligomers: uptake, efficacy and pharmacokineMcs. Curr Pharm Biotechnol. 2004;5(5):431-9.

17. van de Steeg E, Lappchen T, Aguilera B, Jansen HT, Muilwijk D, Vermue R, et al. Feasibility of SPECT-CT Imaging to Study the PharmacokineMcs of AnMsense OligonucleoMdes in a Mouse

Model of Duchenne Muscular Dystrophy. Nucleic Acid Ther. 2017;27(4):221-31.

18. Aartsma-Rus A, Krieg AM. FDA Approves Eteplirsen for Duchenne Muscular Dystrophy: The Next Chapter in the Eteplirsen Saga. Nucleic Acid Ther. 2017;27(l):l-3.

19. Novak JS, Hogarth MW, Boehler JF, Nearing M, Vila MC, Heredia R, et al. Myoblasts and macrophages are required for therapeuMc morpholino anMsense oligonucleoMde delivery to dystrophic muscle. Nature communicaMons. 2017;8(l):941.

20. Novak JS, Jaiswal JK, Partridge TA. The macrophage as a Trojan horse for anMsense oligonucleoMde delivery. Expert Opin Ther Targets. 2018;22(6):463-6.

21. Sacco A, MourkioM F, Tran R, Choi J, Llewellyn M, Kral P, et al. Short telomeres and stem cell exhausMon model Duchenne muscular dystrophy in mdx/mTRmice. Cell. 2010;143(7):1059- 71.

22. Blau HM, Webster C, Pavlath GK. DefecMve myoblasts idenMfied in Duchenne muscular dystrophy. Proceedings of the NaMonal Academy of Sciences of the United States of America. 1983;80(15):4856-60.

23. Ismaeel A, Kim JS, Kirk JS, Smith RS, Bohannon WT, Koutakis P. Role of Transforming Growth Factor-beta in Skeletal Muscle Fibrosis: A Review. Int J Mol Sci. 2019;20(10).

31

24. Song Y, Yao S, Liu Y, Long L, Yang H, Li Q, et al. Expression levels of TGF-betal and CTGF are associated with the severity of Duchenne muscular dystrophy. Exp Ther Med.

2017; 13(4): 1209-14.

25. Pescatori M, Broccolini A, Minen C, BerMni E, Bruno C, D'Amico A, et al. Gene expression profiling in the early phases of DMD: a constant molecular signature characterizes DMD muscle from early postnatal life throughout disease progression. FASEB journal : official publicaMon of the FederaMon of American SocieMes for Experimental Biology. 2007;21(4): 1210-26. 26. Girardi F, Taleb A, Ebrahimi M, Datye A, Gamage DG, Peccate C, et al. TGFbeta signaling curbs cell fusion and muscle regeneraMon. Nature communicaMons. 2021;12(l):750.

27. Kemaladewi DU, Pasteuning S, van der Meulen JW, van Heiningen SH, van Ommen GJ, Ten Dijke P, et al. TargeMng TGF-beta Signaling by AnMsense OligonucleoMde-mediated Knockdown of TGF-beta Type I Receptor. Molecular therapy Nucleic acids. 2014;3(4):el56.

28. Osseni A, Ravel-Chapuis A, Belon E, ScionM I, Gangloff YG, Moncollin V, et al. Pharmacological inhibiMon of HDAC6 improves muscle phenotypes in dystrophin-deficient mice by downregulaMng TGF-beta via Smad3 acetylaMon. Nature communicaMons. 2022;13(l):7108.

29. Nawaz A, Bilal M, Fujisaka S, Kado T, Aslam MR, Ahmed S, et al. DepleMon of CD206(+) M2-like macrophages induces fibro-adipogenic progenitors acMvaMon and muscle regeneraMon. Nature communicaMons. 2022; 13(1): 7058.

30. Massague J, Sheppard D. TGF-beta signaling in health and disease. Cell. 2023;186(19):4007-37.

31. Mazala DA, Novak JS, Hogarth MW, Nearing M, Adusumalli P, Tully CB, et al. TGF- betadriven muscle degeneraMon and failed regeneraMon underlie disease onset in a DMD mouse model. JCI Insight. 2020;5(6).

32. Mazala DAG, Hindupur R, Moon YJ, Shaikh F, Gamu IH, Alladi D, et al. Altered muscle niche contributes to myogenic deficit in the D2-mdx model of severe DMD. Cell Death Discov. 2023;9(l):224.

33. Contreras O, Cruz-Soca M, Theret M, Soliman H, Tung LW, Groppa E, et al. Cross-talk between TGF-beta and PDGFRalpha signaling pathways regulates the fate of stromal fibroadipogenic progenitors. Journal of cell science. 2019; 132(19).

34. Hogarth MW, Uapinyoying P, Mazala DAG, Jaiswal JK. Pathogenic role and therapeuMc potenMal of fibro-adipogenic progenitors in muscle disease. Trends Mol Med. 2022;28(l):8-l 1.

35. Joe AW, Yi L, Natarajan A, Le Grand F, So L, Wang J, et al. Muscle injury acMvates resident fibro/adipogenic progenitors that facilitate myogenesis. Nat Cell Biol. 2010;12(2):153-63.

36. Lemos DR, Babaeijandaghi F, Low M, Chang CK, Lee ST, Fiore D, et al. NiloMnib reduces muscle fibrosis in chronic muscle injury by promoMng TNF-mediated apoptosis of fibro/adipogenic progenitors. Nat Med. 2015;21(7):786-94.

37. Juban G, Saclier M, Yacoub-Youssef H, Kemou A, Arnold L, Boisson C, et al. AMPK AcMvaMon Regulates LTBP4-Dependent TGF-betal SecreMon by Pro-inflammatory Macrophages and Controls Fibrosis in Duchenne Muscular Dystrophy. Cell reports. 2018;25(8):2163-76 e6.

38. Theret M, Saclier M, Messina G, Rossi FMV. Macrophages in Skeletal Muscle Dystrophies, An Entangled Partner. J Neuromuscul Dis. 2022;9(l):l-23.

39. Meng XM, Nikolic-Paterson DJ, Lan HY. TGF-beta: the master regulator of fibrosis. Nat Rev Nephrol. 2016;12(6):325-38.

32

40. Batlie E, Massague J. Transforming Growth Factor-beta Signaling in Immunity and Cancer. Immunity. 2019;50(4):924-40.

41. Yokota T, Lu QL, Morgan JE, Davies KE, Fisher R, Takeda S, et al. Expansion of revertant fibers in dystrophic mdx muscles reflects acMvity of muscle precursor cells and serves as an index of muscle regeneraMon. Journal of cell science. 2006;119(Pt 13):2679-87.

42. Shah MNA, Yokota T. Cardiac therapies for Duchenne muscular dystrophy. Ther Adv Neurol Disord. 2023;16:17562864231182934.

43. Vila MC, Klimek MB, Novak JS, Rayavarapu S, Uaesoontrachoon K, Boehler JF, et al. Elusive sources of variability of dystrophin rescue by exon skipping. Skeletal muscle. 2015;5:44.

44. Benny Klimek ME, Vila MC, Edwards K, Boehler J, Novak J, Zhang A, et al. Effects of Chronic, Maximal Phosphorodiamidate Morpholino Oligomer (PMO) Dosing on Muscle FuncMon and Dystrophin RestoraMon in a Mouse Model of Duchenne Muscular Dystrophy. J Neuromuscul Dis. 2021;8(s2):S369-S81.

45. Flanigan KM, Ceco E, Lamar KM, Kaminoh Y, Dunn DM, Mendell JR, et al. LTBP4 genotype predicts age of ambulatory loss in Duchenne muscular dystrophy. Annals of neurology. 2013;73(4):481-8.

46. Chen YW, Nagaraju K, Bakay M, McIntyre O, Rawat R, Shi R, et al. Early onset of inflammaMon and later involvement of TGFbeta in Duchenne muscular dystrophy. Neurology. 2005;65(6):826-34.

47. Ishitobi M, Haginoya K, Zhao Y, Ohnuma A, Minato J, Yanagisawa T, et al. Elevated plasma levels of transforming growth factor betal in paMents with muscular dystrophy. Neuroreport. 2000; 11(18):4033-5.

48. Amantana A, Moulton HM, Cate ML, Reddy MT, Whitehead T, Massinger JN, et al. PharmacokineMcs, biodistribuMon, stability and toxicity of a cell-penetraMng pepMdemorpholino oligomer conjugate. Bioconjugate chemistry. 2007;18(4):1325-31.

49. Burki U, Keane J, Blain A, O'Donovan L, Gait MJ, Laval SH, et al. Development and ApplicaMon of an UltrasensiMve HybridizaMon-Based ELISA Method for the DeterminaMon of PepMde-Conjugated Phosphorodiamidate Morpholino OligonucleoMdes. Nucleic Acid Ther. 2015;25(5):275-84.

50. Ratnayake D, Nguyen PD, Rossello FJ, Wimmer VC, Tan JL, Galvis LA, et al. Macrophages provide a transient muscle stem cell niche via NAMPT secreMon. Nature. 2021;591(7849):281-7.

51. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Bamabe-Heider F, Walsh S, et al. Evidence for cardiomyocyte renewal in humans. Science. 2009;324(5923):98-102.

52. Willems E, Cabral-Teixeira J, Schade D, Cai W, Reeves P, Bushway PJ, et al. Small molecule-mediated TGF-beta type II receptor degradaMon promotes cardiomyogenesis in embryonic stem cells. Cell Stem Cell. 2012;lI(2):242-52.

53. Chen WP, Liu YH, Ho YJ, Wu SM. Pharmacological inhibiMon of TGFbeta receptor improves Nkx2.5 cardiomyoblast-mediated regeneraMon. Cardiovasc Res. 2015;105(l):44-54.

54. Goldstein JA, Kelly SM, LoPresM PP, Heydemann A, Earley JU, Ferguson EL, et al. SMAD signaling drives heart and muscle dysfuncMon in a Drosophila model of muscular dystrophy. Human molecular geneMcs. 2011;20(5):894-904.

55. Goldstein JA, Bogdanovich S, Beiriger A, Wren LM, Rossi AE, Gao QQ, et al. Excess SMAD signaling contributes to heart and muscle dysfuncMon in muscular dystrophy. Human molecular geneMcs. 2014;23(25):6722-31.

33

56. Novak JS, Spathis R, Dang UJ, Fiorillo AA, Hindupur R, Tully CB, et al. InterrogaMon of Dystrophin and Dystroglycan Complex Protein Turnover Aler Exon Skipping Therapy. J Neuromuscul Dis. 2021;8(s2):S383-S402.

57. Heier CR, McCormack NM, Tully CB, Novak JS, Newell-Stamper BL, Russell AJ, et al. The X-linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deleMon of murine dystrophin exons 45-47. J Cachexia Sarcopenia Muscle. 2023.

58. Coley WD, Bogdanik L, Vila MC, Yu Q, Van Der Meulen JH, Rayavarapu S, et al. Effect of geneMc background on the dystrophic phenotype in mdx mice. Human molecular geneMcs. 2016;25(l): 130-45.

Claims

1. A method for treating a subject having an elevated TGF-beta level which comprises administering to the subject at least one TGF-beta lowering antisense oligonucleotide for a time and under conditions that inhibit TGF-beta expression.

2. The method of claim 1, wherein the subject has an elevated TGF-beta driven myogenic deficit or tissue fibrosis, such as Duchenne Muscular Dystrophy (DMD), wherein the method comprises administering to a subject in need thereof at least one TGF-beta lowering antisense oligonucleotide, optionally in combination with at least one dystrophin-restoring antisense oligonucleotide for a time and under conditions that inhibit TGF-beta expression and, when the at least one dystrophin restoring antisense oligonucleotide is administered, under conditions that increase dystrophin expression; wherein, optionally, the antisense oligonucleotides comprise TPM02 (SEQ ID NO: 16) which targets human mRNA encoding TGF-beta- 1 and DPMO Eteplirsen which targets human mRNA encoding dystrophin.

3. The method of claim 1, wherein the subject has a single exon deletion or duplication of a dystrophin gene; or wherein the subject has a multi -exon deletion or duplication of a dystrophin gene.

4. The method of claim 1, wherein the subject has a nonsense or missense mutation in the dystrophin gene, has a small deletion, duplication, or insertion in a dystrophin gene.

5. The method of claim 1, wherein the subject has a lower level of dystrophin compared to a healthy control.

6. The method of claim 1, wherein the subject has elevated TGF-beta 1 as compared to a healthy control.

7. The method of claim 1, wherein the subject is no more than five years old.

8. The method of claim 1, wherein the subject is more than five years old.

9. The method of claim 1, wherein a dystrophin-restoring phosphorodiamidate morpholino oligomer (DPMO) and a TGF-beta 1 targeting phosphorodiamidate morpholino oligomer (TPMO) is locally administered to muscle or to damaged muscle lesions in the subject.

10. The method of claim 9, wherein the muscle is skeletal muscle.

11. The method of claim 9, wherein the muscle is cardiac muscle.

12. The method of claim 1, wherein a dystrophin-restoring phosphorodiamidate morpholino oligomer (DPMO) and a TGF-beta 1 targeting phosphorodiamidate morpholino oligomer (TPMO) are systemically administered to the subject.

13. The method of claim 1, wherein the TGF-beta 1 targeting phosphorodiamidate morpholino oligomer (TPMO) is TPMO2 (SEQ ID NO: 16), or a corresponding orthologous sequence.

14. The method of claim 1, wherein the TGF-beta 1 targeting phosphorodiamidate morpholino oligomer (TPMO) is human TPMO 1, 3, 4, 5 or 6, or a corresponding orthologous sequences.

15. The method of claim 1, wherein the dystrophin-restoring PMO (DPMO) comprises etepliresn, goldirsen, viltolarsen or casimersen and the TPMO is at least one of human

TPMO 1, 2, 3, 4, 5, or 6 (SEQ ID NOS: 15-20).

16. The method of claim 1, further comprising administering an agent that increases macrophages infiltrating the damaged and inflamed regions of dystrophic muscle.

17. The method of claim 1, further comprising a TCF-beta targeting antibody, small molecule or other agent other than a TPMO that reduces a level of, or expression of, phospho-

SMAD3 (pSMAD3).

18. A method for treating a subject in need of muscle regeneration or repair or a muscular disease or condition other than Duchenne Muscular Dystrophy (DMD) comprising administering to a subject in need thereof a combination of TGF-beta and dystrophin antisense oligonucleotides for a time and under conditions that restore or increase dystrophin expression and inhibit TGF-beta expression.

19. A pharmaceutical composition comprising a combination of TGF-beta and dystrophin antisense oligonucleotides.

20. The pharmaceutical composition of claim 19, further comprising an agent that reduces SMAD3 expression other than the TGF-beta antisense oligonucleotides and/or an agent that promotes macrophage infiltration in and around damaged muscle tissue.