Methods for treating muscular dystrophy

Abstract

To provide methods for treating muscular dystrophy.SOLUTION: Provided are methods for treating muscular dystrophy by administering a pharmaceutical composition comprising about 80 to about 300 mg / kg of an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein, in one embodiment, the composition is administered less frequently than occurs in existing methods of treatment, and Duchenne muscular dystrophy (DMD) is a severe, progressively debilitating, ultimately fatal X-linked neuromuscular disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 863,456, filed June 19, 2019, which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listings submitted electronically via EFS-WEB

[0003] The contents of the electronically submitted Sequence Listing (Name: 4140_032PC01_Seqlisting_ST25; Size: 12,950 bytes and Creation Date: June 17, 2020) are incorporated herein by reference in their entirety.

[0004] The present disclosure relates to methods of treating muscular dystrophy in a human subject. [Background technology]

[0005] Duchenne muscular dystrophy (DMD) is a severe, progressively debilitating, and ultimately fatal X-linked neuromuscular disease. DMD is caused by mutations in the dystrophin gene, characterized by the absence or near-absence of functional dystrophin protein, resulting in the absence of dystrophin, a critical part of the protein complex that connects the cytoskeleton of muscle fibers to the extracellular matrix. In the absence of dystrophin, patients with DMD experience a predictable disease course. Affected patients, typically boys, develop muscle weakness within the first few years of life, lose the ability to walk during childhood, and usually require respiratory assistance in their late teens. Loss of functional abilities leads to a loss of independence and increased caregiver burden. Once lost, these abilities cannot be regained. Despite improvements in standard treatments, such as the use of glucocorticoids, DMD is ultimately fatal, with patients dying of respiratory or cardiac failure in their mid- to late twenties.

[0006] The progressive loss of muscle tissue and function in DMD is caused by the absence or near absence of functional dystrophin, a protein that plays a key role in muscle cell structure and function. Potential therapeutic approaches for the treatment of DMD are suggested by Becker muscular dystrophy (BMD), a milder form of dystrophinopathy. Both dystrophinopathies are caused by mutations in the DMD gene. In DMD, mutations that disrupt the pre-mRNA reading frame, referred to as "out-of-frame" mutations, prevent the production of functional dystrophin. In BMD, "in-frame" mutations do not disrupt the reading frame and result in the production of an internally truncated functional dystrophin protein.

[0007] An important approach to restoring these "out-of-frame" mutations is to use antisense oligonucleotides to exclude or skip molecular mutations in the DMD gene (dystrophin gene). The DMD or dystrophin gene is one of the largest genes in the human body, consisting of 79 exons. Antisense oligonucleotides (AONs) are specifically designed to target specific regions of pre-mRNA, typically exons, to induce skipping of DMD gene mutations, thereby restoring these out-of-frame mutations in frame and allowing the production of an internally truncated but functional dystrophin protein.

[0008] Exondys 51® (eteplirsen) is a phosphorodiamidate morpholino oligomer (PMO) designed to skip exon 51 of the human dystrophin gene in patients with DMD, where exon 51 skipping restores the reading frame and produces a functional truncated form of the dystrophin protein. The U.S. Food and Drug Administration (FDA) approved EXONDYS 51® (eteplirsen, SEQ ID NO: 1) in 2016 for the treatment of Duchenne muscular dystrophy (DMD) in patients with confirmed mutations in the DMD gene who are amenable to exon 51 skipping. The recommended dose of EXONDYS 51® is 30 mg / kg administered once weekly as a 35- to 60-minute intravenous infusion.

[0009] However, there remains a need for improved methods of treating muscular dystrophies, such as DMD and BMD, in patients. Summary of the Invention

[0010] The present disclosure provides a method for treating DMD in a human subject having a mutation in the DMD gene amenable to exon skipping, comprising administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, casimersen is administered at a dose of about 80 to about 300 mg / kg.

[0011] In one embodiment, the present disclosure provides a method for treating DMD in a human subject having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. In one embodiment of the method, eteplirsen, golodirsen, or casimersen is administered at a dose of about 100 mg / kg. In one embodiment of the method, eteplirsen is administered at a dose of about 100 mg / kg. In one embodiment of the method, golodirsen is administered at a dose of about 100 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 100 mg / kg.

[0012] In another embodiment, the present disclosure provides a method for treating DMD in a human subject having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. In one embodiment of the method, eteplirsen, golodirsen, or casimersen is administered at a dose of about 100 mg / kg. In one embodiment of the method, eteplirsen is administered at a dose of about 100 mg / kg. In one embodiment of the method, golodirsen is administered at a dose of about 100 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 100 mg / kg.

[0013] In one embodiment, the present disclosure provides a method for treating DMD in a human subject having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 200 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 200 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 200 mg / kg. In one method embodiment, casimersen is administered at a dose of about 200 mg / kg.

[0014] In another embodiment, the present disclosure provides a method for treating DMD in a human subject having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 200 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 200 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 200 mg / kg. In one method embodiment, casimersen is administered at a dose of about 200 mg / kg.

[0015] In one embodiment, the present disclosure provides a method for treating DMD in a human subject having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 300 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 300 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 300 mg / kg. In one method embodiment, casimersen is administered at a dose of about 300 mg / kg.

[0016] In another embodiment, the present disclosure provides a method for treating DMD in a human subject having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. In one embodiment of the method, eteplirsen, golodirsen, or casimersen is administered at a dose of about 300 mg / kg. In one embodiment of the method, eteplirsen is administered at a dose of about 300 mg / kg. In one embodiment of the method, golodirsen is administered at a dose of about 300 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 300 mg / kg.

[0017] The present disclosure also relates to a method for restoring the mRNA reading frame and inducing dystrophin production in a human subject having a mutation in the dystrophin gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 80 to about 300 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 80 to about 300 mg / kg.

[0018] In one embodiment, the present disclosure provides a method for restoring the mRNA reading frame and inducing dystrophin production in a human subject having a mutation in the dystrophin gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. In one embodiment of the method, eteplirsen, golodirsen, or casimersen is administered at a dose of about 100 mg / kg. In one embodiment of the method, eteplirsen is administered at a dose of about 100 mg / kg. In one embodiment of the method, golodirsen is administered at a dose of about 100 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 100 mg / kg.

[0019] In another embodiment, the present disclosure provides a method for restoring the mRNA reading frame and inducing dystrophin production in a human subject having a mutation in the dystrophin gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. In one embodiment of the method, eteplirsen, golodirsen, or casimersen is administered at a dose of about 200 mg / kg. In one embodiment of the method, eteplirsen is administered at a dose of about 200 mg / kg. In one embodiment of the method, golodirsen is administered at a dose of about 200 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 200 mg / kg.

[0020] In one embodiment, the present disclosure provides a method for restoring the mRNA reading frame and inducing dystrophin production in a human subject having a mutation in the dystrophin gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. In one embodiment of the method, eteplirsen, golodirsen, or casimersen is administered at a dose of about 300 mg / kg. In one embodiment of the method, eteplirsen is administered at a dose of about 300 mg / kg. In one embodiment of the method, golodirsen is administered at a dose of about 300 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 300 mg / kg.

[0021] The present disclosure also relates to a method for excluding exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA during mRNA processing in a human subject having a mutation in the dystrophin gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 80 to about 300 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 80 to about 300 mg / kg.

[0022] In one embodiment, the present disclosure also relates to a method for excluding exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA during mRNA processing in a human subject having a mutation in the dystrophin gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 100 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 100 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 100 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 100 mg / kg.

[0023] In another embodiment, the present disclosure also relates to a method for excluding exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA during mRNA processing in a human subject having a mutation in the dystrophin gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 200 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 200 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 200 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 200 mg / kg.

[0024] In another embodiment, the present disclosure also relates to a method for excluding exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA during mRNA processing in a human subject having a mutation in the dystrophin gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 300 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 300 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 300 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 200 mg / kg.

[0025] In another aspect, the present disclosure also relates to a method for ligating exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA in a human subject having a mutation in the dystrophin gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 80 to about 300 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 80 to about 300 mg / kg. In one embodiment of the method, golodirsen is administered at a dose of about 80 to about 300 mg / kg.In one embodiment of the method, casimersen is administered at a dose of about 80 to about 300 mg / kg.

[0026] In one embodiment, the present disclosure also relates to a method for ligating exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA in a human subject having a mutation in the dystrophin gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 100 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 100 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 100 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 100 mg / kg.

[0027] In one embodiment, the present disclosure also relates to a method for ligating exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA in a human subject having a mutation in the dystrophin gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 200 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 200 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 200 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 200 mg / kg.

[0028] In one embodiment, the present disclosure also relates to a method for ligating exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA in a human subject having a mutation in the dystrophin gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. In one method embodiment, eteplirsen, golodirsen, or casimersen is administered at a dose of about 300 mg / kg. In one method embodiment, eteplirsen is administered at a dose of about 300 mg / kg. In one method embodiment, golodirsen is administered at a dose of about 300 mg / kg. In one embodiment of the method, casimersen is administered at a dose of about 300 mg / kg.

[0029] In certain embodiments, the methods of the present disclosure include administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 80 to about 300 mg / kg. In some embodiments, the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg. In some embodiments, the antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg. In some embodiments, the antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg. In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof (e.g., eteplirsen), is administered at a dose of about 100 mg / kg. In other embodiments, the antisense oligomer (eg, eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg.

[0030] In some embodiments, the methods of the present disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is administered once weekly. In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg, and the pharmaceutical composition is administered once weekly. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg, and the pharmaceutical composition is administered once weekly. In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg, and the pharmaceutical composition is administered once weekly. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg.

[0031] In some embodiments, the methods of the present disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is administered once weekly. In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg, and the pharmaceutical composition is administered once weekly. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg, and the pharmaceutical composition is administered once weekly. In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg, and the pharmaceutical composition is administered once weekly. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg.

[0032] In some embodiments, the methods of the present disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is administered once weekly. In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg, and the pharmaceutical composition is administered once weekly. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg, and the pharmaceutical composition is administered once weekly. In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg, and the pharmaceutical composition is administered once weekly. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg.

[0033] In some embodiments, the methods of the present disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, for up to about 24 weeks, up to about 48 weeks, up to about 60 weeks, up to about 80 weeks, up to about 100 weeks, up to about 120 weeks, up to about 140 weeks, up to about 150 weeks, up to about 160 weeks, up to about 180 weeks, or up to about 200 weeks. In some embodiments, the pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for up to about 144 weeks. In some embodiments, the composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for at least 24 weeks, at least 36 weeks, at least 48 weeks, at least 120 weeks, at least 144 weeks, or at least 164 weeks. In yet other embodiments, the pharmaceutical composition comprising the antisense oligomer (eg, eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for the duration of the disease.

[0034] In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof for up to about 24 weeks, wherein the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof for up to about 48 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg. In yet another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof for up to about 144 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg.

[0035] In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof for up to about 24 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof for up to about 48 weeks, wherein the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg. In yet another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof for up to about 144 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg.

[0036] In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof for up to about 24 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof for up to about 48 weeks, wherein the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg. In yet another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof for up to about 144 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg.

[0037] In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof for up to about 24 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof for up to about 48 weeks, wherein the antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg. In yet another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof for up to about 144 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg.

[0038] In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof for up to about 24 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof for up to about 48 weeks, wherein the antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg. In yet another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof for up to about 144 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg.

[0039] In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof for up to about 24 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof for up to about 48 weeks, wherein the antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg. In yet another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof for up to about 144 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg.

[0040] In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof for up to about 24 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof for up to about 48 weeks, wherein the antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg. In yet another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof for up to about 144 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg.

[0041] In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof for up to about 24 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof for up to about 48 weeks, wherein the antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg. In yet another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof for up to about 144 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg.

[0042] In one embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof for up to about 24 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg. In another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof for up to about 48 weeks, wherein the antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg. In yet another embodiment, the method comprises administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof for up to about 144 weeks, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg / kg.

[0043] In some embodiments, a pharmaceutical composition comprising the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is formulated for systemic administration. In some embodiments, a composition comprising the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered intravenously. In some embodiments, a composition comprising the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered as an intravenous infusion. In some embodiments, a pharmaceutical composition comprising the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered as an intravenous infusion over 35 to 60 minutes. In other embodiments, a composition comprising the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered intramuscularly. In yet other embodiments, a composition comprising the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered orally.

[0044] In some embodiments, the methods of the present disclosure comprise administering to a male human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 80 to about 300 mg / kg. In one embodiment, the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg. In another embodiment, the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of 200 mg / kg. In another embodiment, the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of 300 mg / kg. In some embodiments, the human subject is between 7 and 13 years of age (inclusive).

[0045] In some embodiments, the methods of the present disclosure comprise administering to a male human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 80 to about 300 mg / kg. In one embodiment, the antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg. In another embodiment, the antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of 200 mg / kg. In another embodiment, the antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of 300 mg / kg. In some embodiments, the human subject is between 7 and 13 years of age (inclusive).

[0046] In some embodiments, the methods of the present disclosure comprise administering to a male human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 80 to about 300 mg / kg. In one embodiment, the antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg. In another embodiment, the antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof is administered at a dose of 200 mg / kg. In another embodiment, the antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof is administered at a dose of 300 mg / kg. In some embodiments, the human subject is between 7 and 13 years of age (inclusive).

[0047] In some embodiments, the pharmaceutical composition comprising the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a saline solution, including a phosphate buffer, e.g., phosphate buffered saline.

[0048] In some embodiments, the method of the present disclosure increases the number of dystrophin-positive fibers in a human subject. In other embodiments, a human subject treated with the method of the present disclosure achieves a higher North American Outpatient Assessment (NSAA) total score. In some embodiments, a higher NSAA score is achieved at about 24 weeks, about 48 weeks, about 60 weeks, about 80 weeks, about 100 weeks, about 120 weeks, about 140 weeks, about 150 weeks, about 160 weeks, about 180 weeks, or about 200 weeks compared to baseline. In one embodiment, a higher NSAA score is achieved at about 144 weeks compared to baseline. In another embodiment, a higher NSAA score is achieved at about 164 weeks compared to baseline.

[0049] In some embodiments, the method of the present disclosure reduces the loss of locomotor activity in human subjects compared to baseline, as measured by 6-minute walking test (6MWT).In some embodiments, locomotor activity is maintained compared to baseline.In other embodiments, locomotor activity is improved compared to baseline.

[0050] In still other embodiments, the methods of the present disclosure reduce loss of lung function in a human subject compared to baseline. Loss of lung function can be measured as the % annual decline in forced vital capacity (FVC).

[0051] In some embodiments, the methods of the present disclosure include administering to the subject another therapeutic agent, such as a steroid.

[0052] In certain embodiments, the disclosed method comprises administering a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, via intravenous infusion at a dose of about 100 mg / kg once weekly for up to 24 weeks to a human subject with DMD having a mutation in the DMD gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping. In one embodiment, the human subject is a male between the ages of 7 and 13 (inclusive). In one method embodiment, the antisense oligomer is eteplirsen, golodirsen, or casimersen.

[0053] In certain other embodiments, the disclosed method comprises administering to a human subject with DMD having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, via intravenous infusion at a dose of about 100 mg / kg once weekly for up to 48 weeks. In one embodiment, the human subject is a male aged 7 to 13 years (inclusive). In one method embodiment, the antisense oligomer is eteplirsen, golodirsen, or casimersen.

[0054] In certain embodiments, the disclosed method comprises administering a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, via intravenous infusion at a dose of about 100 mg / kg once weekly for up to 144 weeks to a human subject with DMD having a mutation in the DMD gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping. In one embodiment, the human subject is a male aged 7 to 13 years (inclusive). In one method embodiment, the antisense oligomer is eteplirsen, golodirsen, or casimersen.

[0055] In certain embodiments, the disclosed method comprises administering to a human subject with DMD having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, administered intravenously at a dose of about 200 mg / kg once weekly for up to 24 weeks. In one embodiment, the human subject is a male aged 7 to 13 years (inclusive). In one method embodiment, the antisense oligomer is eteplirsen, golodirsen, or casimersen.

[0056] In certain other embodiments, the disclosed method comprises administering to a human subject with DMD having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, a pharmaceutical composition comprising an antisense oligonucleotide administered as an intravenous infusion at a dose of about 200 mg / kg once weekly for up to 48 weeks. In one embodiment, the human subject is a male aged 7 to 13 years (inclusive). In one method embodiment, the antisense oligomer is eteplirsen, golodirsen, or casimersen.

[0057] In certain other embodiments, the disclosed method comprises administering to a human subject with DMD having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, a pharmaceutical composition comprising an antisense oligonucleotide administered as an intravenous infusion at a dose of about 300 mg / kg once weekly for up to 48 weeks. In one embodiment, the human subject is a male between the ages of 7 and 13 (inclusive). In one method embodiment, the antisense oligomer is eteplirsen, golodirsen, or casimersen.

[0058] In certain embodiments, the disclosed method comprises administering to a human subject with DMD having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, administered intravenously at a dose of about 200 mg / kg once weekly for up to 144 weeks. In one embodiment, the human subject is a male aged 7 to 13 years (inclusive). In one method embodiment, the antisense oligomer is eteplirsen, golodirsen, or casimersen.

[0059] In certain embodiments, the disclosed method comprises administering to a human subject with DMD having a mutation in the DMD gene amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, administered intravenously at a dose of about 300 mg / kg once weekly for up to 144 weeks. In one embodiment, the human subject is a male aged 7 to 13 years (inclusive). In one method embodiment, the antisense oligomer is eteplirsen, golodirsen, or casimersen. [Brief explanation of the drawings]

[0060] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0061] [Figure 1] FIG. 1 shows the increase in % exon 51 skipping in hDMD Δ52 mdx mice after administration of high doses of eteplirsen.

[0062] [Figure 2] FIG. 2 shows dystrophin production in hDMD Δ52 mdx mice after administration of high doses of eteplirsen.

[0063] [Figure 3] FIG. 3 shows the results of grip strength testing in hDMD Δ52 mdx mice after administration of high doses of eteplirsen.

[0064] [Figure 4] Figure 4 shows exon skipping in non-human primates (NHPs) in the quadriceps, heart, and diaphragm after administration of high doses of eteplirsen.

[0065] [Figure 5] Figure 5 compares exon skipping in NHPs in the quadriceps muscle after intravenous or subcutaneous administration of high-dose eteplirsen.

[0066] [Figure 6] FIG. 6 provides images of stained myotube cultures (including myosin heavy chain in red and dystrophin in green) treated with increasing concentrations of golodirsen.

[0067] [Figure 7] Figure 7 provides a high-content analysis of dystrophin staining intensity measurements at different treatment concentrations. DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention relates to an improved method for treating muscular dystrophy, such as DMD and BMD, by administering antisense compounds specifically designed to induce exon skipping in human dystrophin gene.Dystrophin plays an important role in muscle function, and various muscle-related diseases are characterized by the mutant form of this gene.Therefore, in certain embodiments, the improved method described herein can be used to induce exon skipping in the mutant form of human dystrophin gene, such as the mutant dystrophin gene found in DMD and BMD.

[0069] Due to the abnormal mRNA splicing events caused by mutation, these mutant human dystrophin genes either express defective dystrophin protein or do not express any measurable dystrophin, which is the condition that causes various forms of muscular dystrophy.To treat this condition, the antisense compounds of the present invention hybridize to the selected region of the pre-processed RNA of mutant human dystrophin gene, and induce exon skipping and differential splicing in otherwise abnormally spliced ​​dystrophin mRNA, thereby allowing muscle cells to produce the mRNA transcript that codes for functional dystrophin protein.In certain embodiments, the resulting dystrophin protein is not necessarily " wild type " form of dystrophin, but rather is internally truncated but functional or semi-functional form of dystrophin.

[0070] By increasing the level of functional dystrophin protein in muscle cells, these and related embodiments are useful for the prevention and treatment of muscular dystrophies, particularly those forms of muscular dystrophies such as DMD and BMD, which are characterized by defective expression of dystrophin protein due to abnormal mRNA splicing.The methods described herein further provide improved treatment options for patients with muscular dystrophies and offer significant and practical advantages over alternative methods of treating related forms of muscular dystrophies.For example, in some embodiments, the improved methods involve administering antisense compounds to induce exon skipping in the human dystrophin gene at higher doses and / or for longer periods than previous approaches.

[0071] Thus, the present invention relates to an improved method for treating muscular dystrophies, such as DMD and BMD, by inducing exon skipping in patients. In some embodiments, exon skipping is induced by administering an effective amount of a composition comprising a neutrally charged phosphorodiamidate morpholino oligomer (PMO), such as eteplirsen, that selectively binds to a target sequence in an exon of dystrophin pre-mRNA. In some embodiments, the present invention relates to a method for treating DMD or BMD, wherein an effective amount of a composition, for example, about 80 mg / kg to about 300 mg / kg, comprises an antisense oligomer described herein, such as eteplirsen, or a pharmaceutically acceptable salt thereof, for a period sufficient to treat the disease.

[0072] In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, is complementary to one or more exons, or portions thereof, in the transcript. In certain embodiments, the one or more exons, or portions thereof, are selected from the group consisting of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53, and any combination thereof. In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 51, exon 45, or exon 53 of the dystrophin transcript.

[0073] In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 53 of the dystrophin transcript. In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 45 of the dystrophin transcript. In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 44 of the dystrophin transcript. In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 50 of the dystrophin transcript. In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 51 of the dystrophin transcript. In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 52 of the dystrophin transcript.

[0074] In some embodiments, the methods of the disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is complementary to one or more exons, or portions thereof, in a dystrophin transcript. In some embodiments, the one or more exons, or portions thereof, are selected from the group consisting of exon 44, exon 45, exon 50, exon 51, exon 52, and exon 53. In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 51, exon 45, or exon 53 of the dystrophin transcript. In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 51 of the dystrophin transcript. In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 45 of the dystrophin transcript. In some embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 53 of the dystrophin transcript.

[0075] Various mutations in the dystrophin gene are suitable for exon 51 skipping. Non-limiting examples of mutations in the following exons that are suitable for exon 51 skipping include, for example, 45-50, 47-50, 48-50, 49-50, 50, 52, and 52-63 (Leiden Duchenne Muscular Dystrophy Mutation Database, Leiden University Medical Center, The Netherlands). It is well within the understanding of those skilled in the art to determine whether a patient has a mutation in the DMD gene that is suitable for exon skipping (see, for example, Aartsma-Rus et al. (2009) Hum Mut 30:293-299).

[0076] In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 51 of the dystrophin transcript. In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises a base sequence that is complementary to a target region of exon 51 of the dystrophin transcript designated as an annealing site, wherein the base sequence and the annealing site are selected from: [Table 1] Each T and U in SEQ ID NOs: 1-6 is thymine or uracil, respectively. In certain embodiments, both T and U in the antisense oligomer are thymine. In certain embodiments, both T and U in the antisense oligomer are uracil. In certain embodiments, the annealing site is H51A(+66+95).

[0077] In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 53 of the dystrophin transcript. In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises a base sequence that is complementary to a target region of exon 53 of the dystrophin transcript designated as an annealing site, wherein the base sequence and the annealing site are selected from: [Table 2] Each T and U in SEQ ID NOs: 7-16 is thymine or uracil, respectively. In certain embodiments, both T and U in the antisense oligomer are thymine. In certain embodiments, both T and U in the antisense oligomer are uracil. In certain embodiments, the annealing site is H53A(+36+60). In certain embodiments, the annealing site is H53A(+36+56).

[0078] In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 45 of the dystrophin transcript. In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises a base sequence that is complementary to a target region of exon 45 of the dystrophin transcript designated as an annealing site, wherein the base sequence and the annealing site are selected from: [Table 3] Each T and U in SEQ ID NOs: 18-34 is thymine or uracil, respectively. In certain embodiments, both T and U in the antisense oligomer are thymine. In certain embodiments, both T and U in the antisense oligomer are uracil. In certain embodiments, the annealing site is H45A(-03+19).

[0079] In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 44 of the dystrophin transcript. In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises a base sequence that is complementary to a target region of exon 44 of the dystrophin transcript designated as an annealing site, wherein the base sequence and the annealing site are selected from: [Table 4] In each of SEQ ID NOs: 35-41, T is thymine or uracil. In certain embodiments, T in the antisense oligomer is thymine. In certain embodiments, T in the antisense oligomer is uracil.

[0080] In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 50 of the dystrophin transcript. In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises a base sequence that is complementary to a target region of exon 50 of the dystrophin transcript designated as an annealing site, wherein the base sequence and the annealing site are selected from: [Table 5] In each of SEQ ID NOs: 42-50, T is thymine or uracil. In certain embodiments, T in the antisense oligomer is thymine. In certain embodiments, T in the antisense oligomer is uracil.

[0081] In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises skipping of exon 52 of the dystrophin transcript. In certain embodiments, the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises a base sequence that is complementary to a target region of exon 52 of the dystrophin transcript designated as an annealing site, wherein the base sequence and the annealing site are selected from: [Table 6] T in SEQ ID NO: 51 is thymine or uracil. In certain embodiments, T in the antisense oligomer is thymine. In certain embodiments, T in the antisense oligomer is uracil.

[0082] In some embodiments, the antisense oligomer is described by formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein each Nu is a nucleobase that, together with T, forms a targeting sequence; and wherein each Nu from 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of the following: [Table 7-1] [Table 7-2] Each U and T in the antisense oligomer is independently thymine or uracil. In some embodiments, each T and U in the antisense oligomer is thymine.

[0083] In one embodiment of the method, the annealing site is H51A(+66+95), H53A(+36+60), H53A(+36+56), or H45A(-03+19).

[0084] The T moiety attached to the 5' end of the antisense oligomer of formula (I) is [ka] wherein R 1 is C1-C6 alkyl.

[0085] In one embodiment, the method comprises administering eteplirsen [sequence 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3'] (SEQ ID NO: 1), a PMO designed to skip exon 51 of the human dystrophin gene in patients with DMD who are amenable to exon 51 skipping to restore the reading frame and produce a functional shorter form of dystrophin protein. Eteplirsen is registered under CAS Registry Number 1173755-55-9. Chemical names include: RNA, [P-deoxy-P-(dimethylamino)](2',3'-dideoxy-2',3'-imino-2',3'-seco)(2'a→5')(C-m5U-CCAACA-m5U-CAAGGAAGA-m5U-GGCA-m5U-m5U-m5U-C-m5U-AG), 5'-[P-[4-[[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]carbonyl]-1-piperazinyl]-N, N-dimethylphosphonamidate] and P,2',3'-trideoxy-P-(dimethylamino)-5'-O-{P-[4-(10-hydroxy-2,5,8-trioxadecanoyl)piperazin-1-yl]-N,N-dimethylphosphonamidyl}-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino- 2',3'-Secothymidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2' ',3'-Secoadenylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-Secoadenylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-( 2'a→5')-P,2',3'-Trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoguanylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoguanylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P ,2',3'-Trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoguanylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoguanylyl-(2'a→5')-P,2',3'-trideoxy si-P-(dimethylamino)-2',3'-imino-2',3'-secoguanylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-Imino-2',3'-secothymidylyl-(2'a→5')-P, 3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P, 2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P, 3 '-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-2',3'-dideoxy-2',3'-imino-2',3'-secoguanosine. The structure of eteplirsen is shown below. [ka]

[0086] In one embodiment, the method comprises administering golodirsen, also known by its code name "SRP-4053." Golodirsen is a PMO having the base sequence 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 7). Golodirsen is registered under CAS Registry Number 1422959-91-8. Chemical names include ol-P-ambo-[P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-seco](2'a→5')(GTTGCCTCCGGTTCTGAAGGT-GTTC)5'-[4-({2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}carbonyl)-N,N-dimethylpiperazine-1-phosphonamidate]. The structure of golodirsen is shown below: [ka]

[0087] In one embodiment, the antisense oligomer is casimersen, a PMO also known by its code name "SPR-4045" and having the base sequence 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 17). Casimersen is registered under CAS Registry Number 1422959-91-8. Chemical names include ol-P-ambo-[P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-seco](2'a→5')(CAATGCCATCCTGGAGTTCCT-G)5'-[4-({2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}carbonyl)-N,N-dimethylpiperazine-1-phosphonamidate].

[0088] The structure of casimersene is shown below: [ka]

[0089] For clarity, the structures of the present disclosure, e.g., the above structures of eteplirsen, golodirsen, and casimersen, are continuous from 5' to 3' and include various diagrammatic breaks labeled "Break A" and "Break B" for the convenience of depicting the entire structure in a compact form. As will be understood by those skilled in the art, for example, each "Break A" designation indicates a continuation of the diagram of the structure at that point. Those skilled in the art will understand that the same is true for each instance of "Break B" in the above structures. However, none of the diagrammatic breaks are intended to indicate an actual discontinuity in the above structures, and those skilled in the art would not understand them to mean so.

[0090] The present disclosure relates to a method for treating muscular dystrophy, such as DMD and BMD, by administering antisense oligomer or its pharmaceutically acceptable salt by itself or as pharmaceutical composition.Dystrophin plays an important role in muscle function, and various muscle-related diseases are characterized by the mutant form of this gene.Therefore, in certain embodiments, the method described herein can be used to induce exon skipping in the mutant form of human dystrophin gene, such as the mutant dystrophin gene found in DMD and BMD.

[0091] Due to the abnormal mRNA splicing events caused by mutation, these mutant human dystrophin genes either express defective dystrophin protein or do not express any measurable dystrophin, which causes various forms of muscular dystrophy.To treat this condition, antisense oligomers hybridize to exon 44, exon 45, exon 50, exon 51, exon 52 or exon 53 of the pre-processed RNA of mutant human dystrophin gene, and induce exon skipping and differential splicing in otherwise abnormally spliced ​​dystrophin mRNA, thereby allowing muscle cells to produce the mRNA transcripts encoding functional dystrophin protein.In certain embodiments, the resulting dystrophin protein is not necessarily a "wild-type" form of dystrophin, but rather a truncated but functional or semi-functional form of dystrophin.

[0092] By increasing the level of functional dystrophin protein in muscle cells, these and related embodiments are useful for the prevention and treatment of muscular dystrophies, particularly those forms of muscular dystrophies such as DMD and BMD, which are characterized by defective expression of dystrophin protein due to abnormal mRNA splicing. The methods described herein provide additional treatment options for patients with muscular dystrophies and offer significant and practical advantages over alternative methods of treating related forms of muscular dystrophies. For example, in some embodiments, the methods involve administering antisense oligomers, or pharmaceutically acceptable salts thereof, to induce exon skipping in the human dystrophin gene at higher doses and / or for longer periods than previous approaches.

[0093] Accordingly, the present disclosure relates to methods for treating muscular dystrophies, such as DMD and BMD, in a human subject by inducing exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping. In some embodiments, exon skipping is induced by administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg / kg. In some embodiments, the present disclosure relates to a method for treating DMD or BMD, comprising administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg. In some embodiments, the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg. In other embodiments, the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg / kg. In other embodiments, the antisense oligomer (eg, eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg.

[0094] In some embodiments, exon skipping is induced by administering a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 80 to about 300 mg / kg. In some embodiments, the present disclosure relates to a method of treating DMD or BMD comprising administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg. In some embodiments, the antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. In other embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. In other embodiments, the antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg.

[0095] In some embodiments, exon skipping is induced by administering a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 80 to about 300 mg / kg. In some embodiments, the present disclosure relates to a method of treating DMD or BMD comprising administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg. In some embodiments, the antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. In other embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. In other embodiments, the antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg.

[0096] In some embodiments, the methods of the present disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof once weekly. In some embodiments, the composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered twice weekly. In yet other embodiments, the composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered once every two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or twelve weeks. In some embodiments, the pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered once monthly.

[0097] In some embodiments, the methods of the present disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof once weekly. In some embodiments, the composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof is administered twice weekly. In yet other embodiments, the composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof is administered once every two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or twelve weeks. In some embodiments, the pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof is administered once monthly.

[0098] In some embodiments, the methods of the present disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof once weekly. In some embodiments, the composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof is administered twice weekly. In yet other embodiments, the composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof is administered once every two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or twelve weeks. In some embodiments, the pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof is administered once monthly.

[0099] In some embodiments, the method comprises administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, for up to about 24 weeks, up to about 48 weeks, up to about 60 weeks, up to about 80 weeks, up to about 100 weeks, up to about 120 weeks, up to about 140 weeks, up to about 150 weeks, up to about 160 weeks, up to about 180 weeks, or up to about 200 weeks. In some embodiments, the pharmaceutical composition comprising the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for up to about 144 weeks. In some embodiments, the composition comprising the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for at least 24 weeks, at least 36 weeks, at least 48 weeks, at least 120 weeks, at least 144 weeks, or at least 164 weeks. In still other embodiments, the pharmaceutical composition comprising the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for the duration of the disease.

[0100] In some embodiments, the method comprises administering a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, for up to about 24 weeks, up to about 48 weeks, up to about 60 weeks, up to about 80 weeks, up to about 100 weeks, up to about 120 weeks, up to about 140 weeks, up to about 150 weeks, up to about 160 weeks, up to about 180 weeks, or up to about 200 weeks. In some embodiments, the pharmaceutical composition comprising the antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, is administered for up to about 144 weeks. In some embodiments, the composition comprising the antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, is administered for at least 24 weeks, at least 36 weeks, at least 48 weeks, at least 120 weeks, at least 144 weeks, or at least 164 weeks. In still other embodiments, the pharmaceutical composition comprising the antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, is administered for the duration of the disease.

[0101] In some embodiments, the method comprises administering a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, for up to about 24 weeks, up to about 48 weeks, up to about 60 weeks, up to about 80 weeks, up to about 100 weeks, up to about 120 weeks, up to about 140 weeks, up to about 150 weeks, up to about 160 weeks, up to about 180 weeks, or up to about 200 weeks. In some embodiments, the pharmaceutical composition comprising the antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered for up to about 144 weeks. In some embodiments, the composition comprising the antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered for at least 24 weeks, at least 36 weeks, at least 48 weeks, at least 120 weeks, at least 144 weeks, or at least 164 weeks. In still other embodiments, the pharmaceutical composition comprising the antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered for the duration of the disease.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are described. For the purposes of this disclosure, the following terms are defined below.

[0103] I. Definition "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0104] The terms "antisense oligomer," "antisense compound," and "antisense oligonucleotide" are used interchangeably and refer to a sequence of circular subunits joined by intersubunit linkages that each have base-pairing portions that hybridize to a target sequence in a nucleic acid (typically RNA) by Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex within the target sequence.

[0105] The term "morpholino oligomer" or "PMO" (phosphoramidate- or phosphorodiamidate morpholino oligomer) refers to an oligonucleotide analog composed of morpholino subunit structures, (i) which are linked together by a 1-3 atom long, preferably 2 atom long, preferably uncharged or cationic, phosphorus-containing linkage connecting the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring yields a purine or pyrimidine base pairing moiety available for binding to a base in a polynucleotide by base-specific hydrogen bonding. The synthesis, structure, and binding properties of morpholino oligomers are described in detail in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337, 8,076,476, 8,299,206, and 7,943,762 (cationic linkages), all of which are incorporated herein by reference.

[0106] "Exon" refers to a defined section of a nucleic acid that encodes a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after any portion of a preprocessed (or precursor) RNA has been removed by splicing. A mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as rRNA or tRNA. The human dystrophin gene has approximately 79 exons.

[0107] "Intron" refers to a region of nucleic acid (within a gene) that is not translated into protein. Introns are non-coding sections that are transcribed into precursor mRNA (pre-mRNA) and then spliced ​​out during formation of the mature RNA.

[0108] "Exon skipping" generally refers to the process by which an entire exon, or a portion thereof, is removed from a given pre-processed RNA, thereby excluding it from being present in a mature RNA, such as a mature mRNA, that is translated into a protein. Thus, the portion of the protein encoded by the skipped exon is not present in the expressed protein form, typically producing a modified, though still functional, form of the protein.

[0109] Dystrophin is a rod-shaped cytoplasmic protein that is an integral part of a protein complex that connects the cytoskeleton of muscle fibers through the cell membrane to the surrounding extracellular matrix. Dystrophin contains multiple functional domains. For example, it contains an actin-binding domain at approximately amino acids 14–240 and a central rod domain at approximately amino acids 253–3040. This large central domain is formed by 24 spectrin-like triple-helical elements of approximately 109 amino acids that share homology with α-actinin and spectrin. Repeats are typically interrupted by four proline-rich non-repeat segments, also known as hinge regions. Repeats 15 and 16 are separated by an 18-amino acid stretch that appears to provide the primary site for dystrophin proteolytic cleavage. Sequence identity between most repeats ranges from 10–25%. Each repeat contains three α-helices: 1, 2, and 3. α-helices 1 and 3 are each formed by seven helical turns, which likely interact as a coiled coil via a hydrophobic interface. α-helix 2 has a more complex structure, formed by segments of four and three helical turns separated by glycine or proline residues. Each repeat is encoded by two exons, typically interrupted by an intron between amino acids 47 and 48 in the first part of α-helix 2. Other introns are usually found at different positions within the repeat, scattered across helix 3. Dystrophin also contains a cysteine-rich domain at approximately amino acids 3080–3360, including a cysteine-rich segment (i.e., 15 cysteines out of 280 amino acids), which shows homology to the C-terminal domain of Dictyostelium discoideum α-actinin. The carboxy-terminal domain is located at approximately amino acids 3361–3685.

[0110] The amino terminus of dystrophin binds to F-actin, and the carboxy terminus binds to the dystrophin-associated protein complex (DAPC) in the sarcolemma. DAPC includes dystroglycan, sarcoglycan, integrin, and caveolin, and mutations in any of these components cause autosomal inherited muscular dystrophies. DAPC is destabilized in the absence of dystrophin, resulting in decreased levels of its member proteins and leading to progressive fiber damage and membrane leakage. In various forms of muscular dystrophy, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), muscle cells produce altered and functionally defective forms of dystrophin or no dystrophin at all, primarily due to mutations in the gene sequence that result in incorrect splicing. Predominant expression of defective dystrophin protein or the complete lack of dystrophin or dystrophin-like proteins leads to the rapid progression of muscle degeneration, as described above. In this regard, a "defective" dystrophin protein may be characterized by the form of dystrophin produced in a particular subject with DMD or BMD, or by the absence of detectable dystrophin, as known in the art.

[0111] "Suitable for exon 53 skipping," as used herein with respect to a subject or patient, is intended to include subjects and patients with one or more mutations in the dystrophin gene such that lack of skipping of exon 53 of the dystrophin gene results in an out-of-frame reading frame, thereby preventing translation of the pre-mRNA and rendering the subject or patient unable to produce dystrophin. Non-limiting examples of mutations in the following exons of the dystrophin gene that are suitable for exon 53 skipping include, for example, exons 3-52, 4-52, 5-52, 6-52, 9-52, 10-52, 11-52, 13-52, 14-52, 15-52, 16-52, 17-52, 19-52, 21-52, 23-52, 24-52, 25-52, 26-52, 27-52, 28-52, and 29-52. Examples of mutations that may be amenable to exon skipping include deletions of exon 52, 9-52, 30-52, 31-52, 32-52, 33-52, 34-52, 35-52, 36-52, 37-52, 38-52, 39-52, 40-52, 41-52, 43-52, 42-52, 45-52, 47-52, 48-52, 49-52, 50-52, 54-58, 54-61, 54-63, 54-64, 54-66, 54-76, 54-77, or deletions of exon 52. Determining whether a patient has a mutation in the dystrophin gene that is amenable to exon skipping is well within the purview of one of ordinary skill in the art (see, e.g., Aartsma-Russell, (See et al. (2009) Hum Mutat. 30:293-299, Gurvich et al., Hum Mutat. 2009;30(4)633-640, and Fletcher et al. (2010) Molecular Therapy 18(6)1218-1223).

[0112] "Suitable for exon 45 skipping," as used herein with respect to a subject or patient, is intended to include subjects and patients with one or more mutations in the dystrophin gene in which lack of skipping of exon 45 in the dystrophin pre-mRNA results in an out-of-frame reading frame, thereby preventing translation of the pre-mRNA and preventing the subject or patient from producing functional or semi-functional dystrophin. Examples of mutations in the dystrophin gene that are suitable for exon 45 skipping include, for example, mutations in exons 7-44, 12-44, 18-44, 44, 46, 46-47, 46-48, 46-49, 46-51, 46-53, 46-55, 46-57, 46-59, 46-60, 46-67, 46-69, 46-75, and 46-78 (Leiden Duchenne Muscular Dystrophy Mutation Database, Leiden University Medical Center, The Netherlands). It is well within the purview of one skilled in the art to determine whether a patient has a mutation in the dystrophin gene that is amenable to exon skipping (see, e.g., Aartsma-Rus et al. (2009) Hum Mutat. 30:293-299, Gurvich et al., Hum Mutat. 2009;30(4)633-640, and Fletcher et al. (2010) Molecular Therapy 18(6)1218-1223).

[0113] As used herein, the terms "function" and "functionality" and the like refer to biological, enzymatic, or therapeutic functions.

[0114] A "functional" dystrophin protein generally refers to a dystrophin protein that has sufficient biological activity to reduce the progressive degradation of muscle tissue, which is otherwise characteristic of muscular dystrophy, compared to the "altered" or "missing" forms of dystrophin protein present in a particular subject with DMD or BMD. In certain embodiments, a functional dystrophin protein may have approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers therebetween) of the in vitro or in vivo biological activity of wild-type dystrophin, as measured according to conventional techniques in the art. As an example, dystrophin-related activity in in vitro muscle cultures can be measured according to myotube size, myofibril organization (or disorganization), contractile activity, and spontaneous clustering of acetylcholine receptors (see, for example, Brown et al., Journal of Cell Science. 112:209-216, 1999). Animal models are also valuable resources for studying disease pathogenesis and provide a means to test dystrophin-related activities. Two of the most widely used animal models for DMD research are the mdx mouse and the Golden Retriever Muscular Dystrophy (GRMD) dog, both of which are dystrophin-negative (e.g., Collins & Morgan, Int. J. Med. 2004). J Exp Pathol 84: 165-172, 2003). These and other animal models can be used to measure the functional activity of various dystrophin proteins, including truncated forms of dystrophin, such as those produced by certain exon-skipping antisense compounds of the present disclosure.

[0115] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, substances, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0116] As used herein, the phrase "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Some examples of materials which may serve as pharmaceutically acceptable carriers, according to the judgment of the formulator, include sugars such as lactose, glucose, sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol esters, for example, ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free distilled water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; release agents; coating agents; sweetening agents; flavoring agents; fragrances; preservatives; and antioxidants.

[0117] The term "restoration" of dystrophin synthesis or production generally refers to the production of dystrophin protein, including truncated dystrophin, in a patient with muscular dystrophy after treatment with an antisense oligonucleotide described herein. In some embodiments, the treatment results in new dystrophin production in the patient. In some embodiments, the treatment increases the number of normal dystrophin-positive fibers in the subject. The proportion of dystrophin-positive fibers in a patient after treatment can be determined by muscle biopsy using known techniques. For example, a muscle biopsy can be taken from a suitable muscle, such as the patient's biceps.

[0118] The analysis of the proportion of positive dystrophin fibers can be performed before and / or after treatment, or at time points throughout the course of treatment. In some embodiments, the post-treatment biopsy is taken from the muscle contralateral to the pre-treatment biopsy. Pre-treatment and post-treatment dystrophin expression analysis can be performed using any suitable dystrophin assay. In one embodiment, immunohistochemical detection is performed on tissue sections from muscle biopsies using an antibody that is a marker for dystrophin, such as a monoclonal or polyclonal antibody. For example, the MANDYS106 antibody, which is a highly sensitive marker for dystrophin, can be used. Any suitable secondary antibody can be used.

[0119] In some embodiments, the percentage of dystrophin-positive fibers is calculated by dividing the number of positive fibers by the total number of fibers counted. Normal muscle samples have 100% dystrophin-positive fibers. Therefore, the percentage of dystrophin-positive fibers can be expressed as a percentage of normal. To control for the presence of trace levels of dystrophin in pre-treatment muscle as well as revertant fibers, a baseline can be established using pre-treatment muscle sections from each patient when counting dystrophin-positive fibers in post-treatment muscle. This can be used as a threshold for counting dystrophin-positive fibers in that patient's post-treatment muscle sections. In other embodiments, antibody-stained tissue sections can also be used for dystrophin quantification using Bioquant image analysis software (Bioquant Image Analysis Corporation, Nashville, TN). Total dystrophin fluorescent signal intensity can be reported as a percentage of normal. Furthermore, the percentage of dystrophin-positive fibers can be determined using Western blot analysis using monoclonal or polyclonal anti-dystrophin antibodies. For example, the anti-dystrophin antibody NCL-Dys1 from Novacastra can be used. The proportion of dystrophin-positive fibers can also be analyzed by determining the expression of components of the sarcoglycan complex (β, γ) and / or neuronal NOS.

[0120] "Treatment" of an individual (e.g., a mammal, such as a human subject) or cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be performed prophylactically or after the onset of a pathological event or contact with a pathogen. Treatment includes any desired effect on the symptoms or pathology of a disease or condition associated with a dystrophin protein, such as in certain forms of muscular dystrophy, and may include, for example, a minimal change or improvement in one or more measurable markers of the disease or condition being treated. Also included is "prophylactic" treatment, which may be aimed at slowing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0121] As used herein, the term "alkyl," unless otherwise specified, refers to a saturated straight-chain or branched hydrocarbon. In certain embodiments, an alkyl group is a primary, secondary, or tertiary hydrocarbon. In certain embodiments, an alkyl group is a group having 1 to 10 carbon atoms, i.e., C1 to C6. 10and alkyl. In certain embodiments, alkyl groups contain 1 to 6 carbon atoms, i.e., C1-C6 alkyl. This term includes both substituted and unsubstituted alkyl groups, including halogenated alkyl groups. In certain embodiments, the alkyl group is a fluorinated alkyl group. Non-limiting examples of moieties with which the alkyl group may be substituted are selected from the group consisting of halogen (fluoro, chloro, bromo, or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected or optionally protected, as known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, incorporated herein by reference. In certain embodiments, the alkyl group is selected from the group consisting of methyl, CF3, CCl3, CFCl2, CF2Cl, ethyl, CH2CF3, CF2CF3, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

[0122] The term "aryl," used alone or as part of a larger moiety, such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to an aromatic ring group having 6 to 14 ring atoms, such as phenyl, 1-naphthyl, 2-naphthyl, 1-anthracyl, and 2-anthracyl. The "aryl" ring may contain one or more substituents. The term "aryl" may be used interchangeably with the term "aryl ring." "Aryl" also includes fused polycyclic aromatic ring systems in which an aromatic ring is fused to one or more rings. Non-limiting examples of useful aryl ring groups include phenyl, hydroxyphenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl, trialkoxyphenyl, alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro, etc., as well as 1-naphthyl, 2-naphthyl, 1-anthracyl, and 2-anthracyl. Also included within the scope of the term "aryl," as used herein, are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as, for example, indanyl, phenanthridinyl, or tetrahydronaphthyl, where the radical or point of attachment is on the aromatic ring.

[0123] The term "acyl" refers to a C(O)R group, where R is H, alkyl, or aryl, as defined herein. Examples of acyl groups include formyl, acetyl, benzoyl, phenylacetyl, and similar groups.

[0124] The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each containing a nucleobase sequence) related to each other by Watson-Crick base pairing rules, or between an oligomer and a target gene sequence. For example, the nucleobase sequence "TGA(5'→3')" is complementary to the nucleobase sequence "ACT(3'→5')." Complementarity may be "partial," in which fewer than all nucleobases of a given nucleobase sequence are matched with another nucleobase sequence according to the base pairing rules. For example, in some embodiments, the complementarity between a given nucleobase sequence and another nucleobase sequence may be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Alternatively, continuing the example, there may be "complete" or "perfect" (100%) complementarity between a given nucleobase sequence and another nucleobase sequence. The degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between the sequences.

[0125] In some embodiments, treatment with the methods of the present disclosure increases new dystrophin production and delays or reduces the lameness predicted without treatment. For example, treatment may stabilize, maintain, improve, or increase a subject's walking ability (e.g., gait stabilization). In some embodiments, treatment maintains or increases a patient's stable walking distance, as measured by the 6-minute walk test (6MWT), for example, as described by McDonald, et al. (Muscle Nerve, 2010; 42:966-74, incorporated herein by reference). Changes in 6-minute walk distance (6MWD) can be expressed as absolute values, percentage changes, or percent predicted changes. A DMD patient's performance in the 6MWT compared to the typical performance of healthy counterparts can be determined by calculating the percent predicted value. For example, the percent predicted 6MWD for men can be calculated using the following equation: 196.72 + (39.81 × age) - (1.36 × age) 2) + (132.28 × height in meters). For women, % predicted 6MWD can be calculated using the following equation: 188.61 + (51.50 × age) - (1.86 × age) 2 ) + (86.10 × height in meters) (Henricson et al. PLoS Curr., 2012, version 2, incorporated herein by reference). In some embodiments, treatment with an antisense oligonucleotide increases a patient's stable walking distance from baseline.

[0126] The loss of muscle function in DMD patients may occur against the background of normal childhood growth and development. Indeed, children with DMD may demonstrate an increase in walking distance during the 6MWT over the course of approximately one year, despite progressive muscle impairment. In some embodiments, 6MWD from DMD patients is compared to existing normative data from typically developing control subjects and age- and sex-matched subjects. In some embodiments, normal growth and development may be accounted for using an equation based on age and height fitted to normative data. Such an equation can be used to convert the 6MWD in subjects with DMD into a percent predicted (% predicted) value. In certain embodiments, analysis of % predicted 6MWD data represents a way to account for normal growth and development, and may show that increases in function at younger ages (e.g., 7 years or younger) represent a stable rather than improved performance in DMD patients (Henricson et al. PLoS Curr., 2012, version 2, incorporated herein by reference).

[0127] In some embodiments, treatment with an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof of the present disclosure slows or reduces the progressive respiratory muscle dysfunction and / or failure in DMD patients that would be expected without treatment. In some embodiments, treatment with an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof can reduce or eliminate the need for ventilatory support that would be expected without treatment. In some embodiments, measurements of respiratory function to track the course of the disease and evaluate potential therapeutic interventions include maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), and forced vital capacity (FVC). MIP and MEP measure the pressure levels a person can generate during inhalation and exhalation, respectively, and are sensitive measures of respiratory muscle strength. MIP is a measure of diaphragmatic weakness.

[0128] In some embodiments, MEP may decline before changes in other pulmonary function tests, including MIP and FVC. In certain embodiments, MEP may be an early indicator of respiratory dysfunction. In certain embodiments, FVC may be used to measure the total volume of air expelled during forced expiration after maximal inspiration. In DMD patients, FVC increases concurrently with physical growth until the early teens. However, as growth slows or becomes poorer with disease progression and muscle weakness progresses, vital capacity enters a decline phase, declining at an average rate of approximately 8 to 8.5 percent per year after age 10 to 12. In certain embodiments, percent predicted MIP (MIP adjusted for weight), percent predicted MEP (MEP adjusted for age), and percent predicted FVC (FVC adjusted for age and height) are supportive analyses.

[0129] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to administration of a compound, drug, or other material other than by direct administration into the central nervous system, thereby entering the patient's system and thus being subject to metabolism and other similar processes, e.g., subcutaneous administration.

[0130] II. Treatment method The present disclosure relates to improved methods for treating muscular dystrophies, such as DMD and BMD, in human subjects by inducing exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping. In these methods, exon skipping is induced by administering an antisense oligomer, or a pharmaceutically acceptable salt thereof, per se or as a pharmaceutical composition, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg / kg.

[0131] In some embodiments, the methods of the present disclosure include administering a pharmaceutical composition containing an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 mg to about 300 mg per kilogram of body weight per day, or about 100 mg to about 200 mg per kilogram of body weight per day. In some cases, a dose greater than 300 mg / kg may be required. In some embodiments, the dose for administration is about 80 mg to 300 mg / kg. In some embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, about 105 mg / kg, about 110 mg / kg, about 120 mg / kg, about 125 mg / kg, about 130 mg / kg, about 135 mg / kg, about 140 mg / kg, about 145 mg / kg, about 150 mg / kg, about 155 mg / kg, about 160 mg / kg, about 165 mg / kg, about 170 mg / kg, about 175 mg / kg, about 180 mg / kg , about 185 mg / kg, about 190 mg / kg, about 195 mg / kg, about 200 mg / kg, about 205 mg / kg, about 210 mg / kg, about 215 mg / kg, about 220 mg / kg, about 225 mg / kg, about 230 mg / kg, about 235 mg / kg, about 240 mg / kg, about 245 mg / kg, about 250 mg / kg, about 255 mg / kg, about 260 mg / kg, about 265 mg / kg, about 270 mg / kg, about 275 mg / kg, about 280 mg / kg, about 285 mg / kg, about 290 mg / kg, about 295 mg / kg, or about 300 mg / kg. In one embodiment, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. In another embodiment, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. In another embodiment, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. In some embodiments, administration is via intravenous (iv) infusion.

[0132] In some embodiments, the methods of the present disclosure include administering a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 80 mg to about 300 mg per kilogram of body weight per day or about 100 mg to about 200 mg per kilogram of body weight per day. In some cases, a dose greater than 300 mg / kg may be required. In some embodiments, the dose for administration is about 80 mg to 300 mg / kg. In some embodiments, the antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, about 105 mg / kg, about 110 mg / kg, about 120 mg / kg, about 125 mg / kg, about 130 mg / kg, about 135 mg / kg, about 140 mg / kg, about 145 mg / kg, about 150 mg / kg, about 155 mg / kg, about 160 mg / kg, about 165 mg / kg, about 170 mg / kg, about 175 mg / kg, about 180 mg / kg , about 185 mg / kg, about 190 mg / kg, about 195 mg / kg, about 200 mg / kg, about 205 mg / kg, about 210 mg / kg, about 215 mg / kg, about 220 mg / kg, about 225 mg / kg, about 230 mg / kg, about 235 mg / kg, about 240 mg / kg, about 245 mg / kg, about 250 mg / kg, about 255 mg / kg, about 260 mg / kg, about 265 mg / kg, about 270 mg / kg, about 275 mg / kg, about 280 mg / kg, about 285 mg / kg, about 290 mg / kg, about 295 mg / kg, or about 300 mg / kg. In one embodiment, the antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. In another embodiment, the antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. In another embodiment, the antisense oligomer (e.g., golodirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. In some embodiments, administration is via intravenous (iv) infusion.

[0133] In some embodiments, the methods of the present disclosure include administering a pharmaceutical composition containing an antisense oligomer (e.g., casimersen) or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer or a pharmaceutically acceptable salt thereof is administered at a dose of about 80 mg to about 300 mg per kilogram of body weight per day or about 100 mg to about 200 mg per kilogram of body weight per day. In some cases, a dose greater than 300 mg / kg may be required. In some embodiments, the dose for administration is about 80 mg to 300 mg / kg. In some embodiments, the antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, about 105 mg / kg, about 110 mg / kg, about 120 mg / kg, about 125 mg / kg, about 130 mg / kg, about 135 mg / kg, about 140 mg / kg, about 145 mg / kg, about 150 mg / kg, about 155 mg / kg, about 160 mg / kg, about 165 mg / kg, about 170 mg / kg, about 175 mg / kg, about 180 mg / kg, The antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 185 mg / kg, about 190 mg / kg, about 195 mg / kg, about 200 mg / kg, about 205 mg / kg, about 210 mg / kg, about 215 mg / kg, about 220 mg / kg, about 225 mg / kg, about 230 mg / kg, about 235 mg / kg, about 240 mg / kg, about 245 mg / kg, about 250 mg / kg, about 255 mg / kg, about 260 mg / kg, about 265 mg / kg, about 270 mg / kg, about 275 mg / kg, about 280 mg / kg, about 285 mg / kg, about 290 mg / kg, about 295 mg / kg, or about 300 mg / kg. In one embodiment, the antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. In another embodiment, the antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. In another embodiment, the antisense oligomer (e.g., casimersen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. In some embodiments, administration is via intravenous (iv) infusion.

[0134] If desired, the effective daily dose of the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, may be administered as two, three, four, five, six, or more subdoses, either by itself or as a pharmaceutical composition, administered separately at appropriate intervals throughout the day, week, month, or year. In some embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, may be administered once every year, two years, three years, four years, or five years. In certain circumstances, the dosage is a single daily administration. In certain embodiments, administration is performed every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, as needed to maintain the desired expression of functional dystrophin protein. In some embodiments, administration is performed once a week. In some embodiments, administration is performed once every two weeks. In various embodiments, administration is performed once or a plurality of times monthly. In still other embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered once every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In some embodiments, a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered once every month. For other embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, may be administered once every 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0135] In some embodiments, a pharmaceutical composition containing an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof is administered at regular intervals, e.g., once daily, once every two days, once every three days, once every three to seven days, once every three to ten days, once every seven to ten days, once weekly, once every two weeks, or once monthly. For example, the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof may be administered weekly by intravenous infusion. In another example, the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof may be administered monthly by intravenous infusion. The antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof may also be administered intermittently over a longer period, e.g., over weeks, months, or years. The treatment regimen may be adjusted (dosage, frequency, route, etc.) as indicated based on the results of immunoassays, other biochemical tests, and physiological tests of the subject being treated.

[0136] In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered weekly, either by itself or as a pharmaceutical composition, at a dose of about 100 mg / kg. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered weekly at a dose of about 125 mg / kg. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered weekly at a dose of about 150 mg / kg. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered weekly at a dose of about 175 mg / kg. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered weekly at a dose of about 200 mg / kg. In various other embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered weekly at a dose of about 300 mg / kg. In some embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered weekly via intravenous infusion, either by itself or as a pharmaceutical composition, at a dose of about 100 mg / kg. In some embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered weekly via intravenous infusion, either by itself or as a pharmaceutical composition, at a dose of about 200 mg / kg. As used herein, weekly is understood to have the art-accepted meaning of once a week.

[0137] In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered per se or as a pharmaceutical composition at a dose of about 100 mg / kg every other week. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 125 mg / kg every other week. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 150 mg / kg every other week. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 175 mg / kg every other week. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg every other week. In some embodiments, the antisense oligomer (e.g., eteplirsen) or its pharmaceutically acceptable salt is administered by itself or as a pharmaceutical composition via intravenous infusion at a dose of about 100 mg / kg every other week. In some embodiments, the antisense oligomer (e.g., eteplirsen) or its pharmaceutically acceptable salt is administered by itself or as a pharmaceutical composition via intravenous infusion at a dose of about 200 mg / kg every other week. As used herein, every other week is understood to have the generally accepted meaning in the art of every two weeks.

[0138] In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered per se or as a pharmaceutical composition at a dose of about 100 mg / kg every two weeks. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 125 mg / kg every two weeks. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 150 mg / kg every two weeks. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 175 mg / kg every two weeks. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg every two weeks. In some embodiments, the antisense oligomer (e.g., eteplirsen) or its pharmaceutically acceptable salt is administered by itself or as a pharmaceutical composition via intravenous infusion at a dose of about 100 mg / kg every two weeks. In some embodiments, the antisense oligomer (e.g., eteplirsen) or its pharmaceutically acceptable salt is administered by itself or as a pharmaceutical composition via intravenous infusion at a dose of about 200 mg / kg every two weeks. As used herein, every two weeks is understood to have the generally accepted meaning in the art of once every three weeks.

[0139] In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered monthly, either by itself or as a pharmaceutical composition, at a dose of about 100 mg / kg. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered monthly at a dose of about 125 mg / kg. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered monthly at a dose of about 150 mg / kg. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered monthly at a dose of about 175 mg / kg. In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered monthly at a dose of about 200 mg / kg. In some embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered monthly via intravenous infusion at a dose of about 100 mg / kg, either by itself or as a pharmaceutical composition. In some embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered monthly via intravenous infusion at a dose of about 200 mg / kg, either by itself or as a pharmaceutical composition. As used herein, monthly is understood to have the commonly accepted meaning in the art of monthly.

[0140] As is understood in the art, weekly, biweekly, triweekly, or monthly administration may be in single or multiple administrations or sub-doses as discussed herein.

[0141] In certain embodiments, the intravenous infusion time is from about 15 minutes to about 4 hours. In some embodiments, the infusion time is from about 30 minutes to about 3 hours. In some embodiments, the infusion time is from about 30 minutes to about 2 hours. In some embodiments, the infusion time is from about 1 hour to about 2 hours. In some embodiments, the infusion time is from about 30 minutes to about 1 hour. In some embodiments, the infusion time is about 60 minutes. In some embodiments, the infusion time is from about 35 to 60 minutes.

[0142] In some embodiments, the methods of the disclosure include administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, wherein the composition is administered for up to about 24 weeks, up to about 25 weeks, up to about 26 weeks, up to about 27 weeks, up to about 28 weeks, up to about 29 weeks, up to about 30 weeks, up to about 31 weeks, up to about 32 weeks, up to about 33 weeks, up to about 34 weeks, up to about 35 weeks, up to about 36 weeks, up to about 37 weeks, up to about 38 weeks, up to about 39 weeks, up to about 40 weeks, Up to approximately 41 weeks, up to approximately 42 weeks, up to approximately 43 weeks, up to approximately 44 weeks, up to approximately 45 weeks, up to approximately 46 weeks, up to approximately 47 weeks, up to approximately 48 weeks, up to approximately 49 weeks, up to approximately 50 weeks, up to approximately 51 weeks, up to approximately 52 weeks, up to approximately 53 weeks, up to approximately 54 weeks, up to approximately 55 weeks, up to approximately 56 weeks, up to approximately 57 weeks, up to approximately 58 weeks, up to approximately 59 weeks, up to approximately 60 weeks, up to approximately 61 weeks, up to approximately 62 weeks, up to approximately 63 weeks, up to approximately 64 weeks, up to approximately 65 weeks, up to approximately 66 weeks, up to approximately 67 weeks, up to approximately 68 weeks, up to approximately 69 weeks, up to approximately 70 weeks, up to approximately 71 weeks, approximately 72 weeks, up to approximately 73 weeks, up to approximately 74 weeks, up to approximately 75 weeks, up to approximately 76 weeks, up to approximately 77 weeks, up to approximately 78 weeks, up to approximately 79 weeks, up to approximately 80 weeks, up to approximately 81 weeks, up to approximately 82 weeks, up to approximately 83 weeks, up to approximately 84 weeks, up to approximately 85 weeks, up to approximately 86 weeks, up to approximately 87 weeks, up to approximately 88 weeks, up to approximately 89 weeks, up to approximately 90 weeks, up to approximately 91 weeks, approximately 92 weeks, up to approximately 93 weeks, up to approximately 94 weeks, up to approximately 95 weeks, The antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for up to about 96 weeks, up to about 97 weeks, up to about 98 weeks, up to about 100 weeks, up to about 105 weeks, up to about 110 weeks, up to about 115 weeks, up to about 120 weeks, up to about 125 weeks, up to about 130 weeks, up to about 135 weeks, up to about 140 weeks, up to about 145 weeks, up to about 150 weeks, up to about 155 weeks, up to about 160 weeks, up to about 165 weeks, up to about 170 weeks, up to about 175 weeks, up to about 180 weeks, up to about 185 weeks, up to about 190 weeks, up to about 195 weeks, or up to about 200 weeks. In some embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for up to about 24 weeks.In some embodiments, a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for up to about 48 weeks. In some embodiments, a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for up to about 144 weeks. In some embodiments, a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for up to about 168 weeks.

[0143] In some embodiments, the methods of the disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, wherein the composition provides a therapeutically effective treatment for at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, at least about 31 weeks, at least about 32 weeks, at least about 33 weeks, at least about 34 weeks, at least about 35 weeks, at least about 36 weeks, at least about 37 weeks, at least about 38 weeks, at least about 39 weeks, at least about 40 weeks, at least about 41 weeks, at least about 42 weeks, at least about 43 weeks, at least about 44 weeks, at least about 45 weeks, at least about 46 weeks, at least about 47 weeks, at least about 48 weeks, at least about 49 weeks, at least about 50 weeks, at least about 51 weeks, at least about 52 weeks, at least about 53 weeks, at least about 54 weeks, at least about 55 weeks, at least about 56 weeks, at least about 57 weeks, at least about 58 weeks, at least about 59 weeks, at least about 60 weeks, at least about 61 weeks, at least about 62 weeks, at least about 63 weeks, at least about 64 weeks, at least about 65 weeks, at least about 66 weeks, at least about 67 weeks, at least about 68 weeks, at least about 69 weeks, at least about 70 weeks, at least about 71 weeks, at least about 72 weeks, at least about 73 weeks, at least about 74 weeks, at least about 75 weeks, at least about 76 weeks, at least about 77 weeks, at least about 78 weeks, at least about 79 weeks, at least about 80 weeks, at least about at least about 39 weeks, at least about 40 weeks, at least about 41 weeks, at least about 42 weeks, at least about 43 weeks, at least about 44 weeks, at least about 45 weeks, at least about 46 weeks, at least about 47 weeks, at least about 48 weeks, at least about 49 weeks, at least about 50 weeks, at least about 51 weeks, at least about 52 weeks, at least about 53 weeks, at least about 54 weeks, at least about 55 weeks, at least about 56 weeks, at least about 57 weeks, at least about 58 weeks, at least about 59 weeks, at least about 60 weeks, at least about 61 weeks, 1 week, at least about 62 weeks, at least about 63 weeks, at least about 64 weeks, at least about 65 weeks, at least about 66 weeks, at least about 67 weeks, at least about 68 weeks, at least about 69 weeks, at least about 70 weeks, at least about 71 weeks, about 72 weeks, at least about 73 weeks, at least about 74 weeks, at least about 75 weeks, at least about 76 weeks, at least about 77 weeks, at least about 78 weeks, at least about 79 weeks, at least about 80 weeks, at least about 81 weeks, at least about 82 weeks, at least about 83 weeks, at least about 84 weeks, 4 weeks, at least about 85 weeks, at least about 86 weeks, at least about 87 weeks, at least about 88 weeks, at least about 89 weeks, at least about 90 weeks, at least about 91 weeks, about 92 weeks, at least about 93 weeks, at least about 94 weeks, at least about 95 weeks, at least about 96 weeks, at least about 97 weeks, at least about 98 weeks, at least about 100 weeks, at least about 105 weeks, at least about 110 weeks, at least about 115 weeks, at least about 120 weeks, at least about 125 weeks, at least about 130 weeks, at least about 135 weeks,The composition is administered for at least about 140 weeks, at least about 145 weeks, at least about 150 weeks, at least about 155 weeks, at least about 160 weeks, at least about 165 weeks, at least about 170 weeks, at least about 175 weeks, at least about 180 weeks, at least about 185 weeks, at least about 190 weeks, at least about 195 weeks, or up to about 200 weeks. In some embodiments, the composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is administered for at least 24 weeks, at least 36 weeks, at least 48 weeks, at least 120 weeks, at least 144 weeks, or at least 168 weeks. In yet other embodiments, the pharmaceutical composition is administered for the duration of the disease.

[0144] Several literature reports have suggested an association in DMD patients between the loss of the ability to independently rise from the floor and the subsequent loss of ambulation over the next year (Bushby and Connor (2011) Clin Investig (Lond.) 1(9):1217-1235 and Henricson et al. (2013) Muscle Nerve 48(1):55-67). Thus, any of the methods described herein may include treating a patient who has lost the ability to independently rise from a supine position. In some embodiments of any of the methods described herein, the patient has lost the ability to independently rise from a supine position at least one year prior to treatment with the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof. In some embodiments of any of the methods described herein, the patient has lost the ability to independently rise from a supine position within one year of initiating treatment with the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof. In some embodiments of any of the methods described herein, the patient loses the ability to sit up independently from a supine position within two years of initiating treatment.

[0145] In some embodiments, any of the methods described herein include continuing treatment with the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, even if the patient loses the ability to rise from a supine position during treatment with the antisense oligomer, or a pharmaceutically acceptable salt thereof.

[0146] In some embodiments of any of the methods described herein, the patient has a rise time of greater than 10 seconds. In some embodiments of any of the methods described herein, the patient has a rise time of greater than 15 seconds. In some embodiments of any of the methods described herein, the patient has a rise time of greater than 20 seconds.

[0147] Clinical outcomes for analyzing the effectiveness of the disclosed methods, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, comprises exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, include percentage of dystrophin-positive fibers (PDPF), 6-minute walk test (6MWT), loss of ambulation (LOA), North Star Ambulation Assessment (NSAA), pulmonary function tests (PFTs), ability to stand (from a supine position) without external support, de novo dystrophin production, and other functional measures.

[0148] In some embodiments, the methods of the present disclosure slow the progression of disease in a human subject treated with the method, as measured by the 6-minute walk test (6MWT).

[0149] In some embodiments, the method of the present disclosure can maintain lung function or reduce the loss of lung function in the human subject treated with the method.In some embodiments of any of the methods described herein, lung function is measured as maximum expiratory pressure (MEP).In some embodiments, lung function is measured as maximum inspiratory pressure (MIP).In some embodiments, lung function is measured as forced vital capacity (FVC).

[0150] In some embodiments, the disclosed method restores the mRNA reading frame and induces dystrophin protein production in a human subject with DMD. Protein production can be measured by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, or immunohistochemistry (IHC). In some embodiments, tissues from different muscle groups (e.g., quadriceps, diaphragm, biceps, skin, heart, etc.) can be homogenized (e.g., by TissueLyser), RNA isolated, and processed for PCR (e.g., droplet digital PCR, "ddPCR") to determine the level of exon skipping. In some embodiments, Western blot analysis can be performed as described in Schnell et al., "Challenges in Interpreting Dystrophin Content by Western Blot," US Neurology, 2019;15(1):40-6, the disclosure of which is incorporated herein in its entirety.

[0151] In some embodiments, the human subject to be treated by the methods of the present disclosure is male. In some embodiments, the human subject (e.g., male) is about 6 months to about 4 years old (inclusive). In some embodiments, the human subject is at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 36) months old. In some embodiments, the human subject is 4 years old or younger. In some embodiments, the male human subject is 7 to 13 years old (inclusive). In other embodiments, the male human subject is less than 7 years old. In other embodiments, the male human subject is more than 13 years old.

[0152] The methods of the present disclosure also include administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, in combination with another therapeutic agent. The additional therapeutic agent may be administered prior to, simultaneously with, or subsequent to administration of the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof. The additional therapeutic agent may be formulated in the same composition as the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, or may be in a different composition. For example, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, may be administered in combination with a steroid and / or an antibiotic. The steroid may be a glucocorticoid or prednisone. Other agents that may be administered include those described in patient cells and mouse models of DMD (G. Kendall et al., J. Med. Chem. Soc. 2004). al. Sci Tranl Med 4 164ra160 (2012), incorporated herein by reference), antagonists of the ryanodine receptor, such as dantrolene, have been shown to enhance antisense-mediated exon skipping.

[0153] In some embodiments, the methods of the present disclosure include co-administering an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, as a pharmaceutical composition with a carbohydrate, either in the same composition or a separate composition, as provided in Han et al., Nat. Comms. 7, 10981 (2016), the entire contents of which are incorporated herein by reference. In some embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, may be co-administered with 5% hexose carbohydrate. For example, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, may be co-administered with 5% glucose, 5% fructose, or 5% mannose. In certain embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, may be co-administered with 2.5% glucose and 2.5% fructose. In some embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, may be co-administered with a carbohydrate selected from arabinose present in an amount of 5% by volume, glucose present in an amount of 5% by volume, sorbitol present in an amount of 5% by volume, galactose present in an amount of 5% by volume, fructose present in an amount of 5% by volume, xylitol present in an amount of 5% by volume, mannose present in an amount of 5% by volume, a combination of glucose and fructose, each present in an amount of 2.5% by volume, and a combination of glucose present in an amount of 5.7% by volume, fructose present in an amount of 2.86% by volume, and xylitol present in an amount of 1.4% by volume.

[0154] In various embodiments, the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, is co-administered with a therapeutically effective amount of a nonsteroidal anti-inflammatory compound, either by itself or as a pharmaceutical composition. In some embodiments, the nonsteroidal anti-inflammatory compound is an NF-kB inhibitor. For example, in some embodiments, the NF-kB inhibitor can be CAT-1004 or a pharmaceutically acceptable salt thereof. In various embodiments, the NF-kB inhibitor can be a conjugate of salicylate and DHA. In some embodiments, the NF-kB inhibitor is CAT-1041 or a pharmaceutically acceptable salt thereof. In certain embodiments, the NF-kB inhibitor is a conjugate of salicylate and EPA. In various embodiments, the NF-kB inhibitor is [ka] or a pharmaceutically acceptable salt thereof.

[0155] In some embodiments, the nonsteroidal anti-inflammatory compound is a TGF-b inhibitor. For example, in certain embodiments, the TGF-b inhibitor is HT-100.

[0156] III. Preparations The method according to the present disclosure includes administering an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof as a pharmaceutical composition containing the antisense oligomer or a pharmaceutically acceptable salt thereof. Methods for the delivery of nucleic acid molecules are described, for example, in Akhtar et al., 1992, Trends Cell Bio., 2:139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar; Sullivan et al., PCT WO94 / 02595. These and other protocols can be used to deliver virtually any nucleic acid molecule, including eteplirsen.

[0157] In certain embodiments, the present disclosure provides methods comprising administering a pharmaceutically acceptable composition comprising a therapeutically effective amount of an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, formulated together with one or more pharmaceutically acceptable carriers (excipients) and / or diluents.

[0158] In some embodiments, the pharmaceutical composition comprises about 0.1 to about 99% antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises about 1 to about 90% antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises about 5 to about 70%, about 5 to about 60%, about 5 to about 50%, about 5 to about 40%, or about 5 to about 30% antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof. In other embodiments, the pharmaceutical composition comprises about 10 to about 80%, about 10 to about 70%, about 10 to about 60%, about 10 to about 50%, about 10 to about 40%, about 10 to about 30%, or about 10 to about 20% antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof. In other embodiments, the concentration of the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, in the pharmaceutical composition is about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, or about 65 mg / ml. , about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml, about 110 mg / ml, about 120 mg / ml, about 130 mg / ml, about 140 mg / ml, about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, or about 200 mg / ml. In one embodiment, the concentration of the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, in the pharmaceutical composition is about 50 mg / ml.

[0159] Pharmaceutical compositions suitable for administration in the methods of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., as an immersion solution (aqueous or non-aqueous solution or suspension), tablet, e.g., buccal, sublingual, or tablet targeted for systemic absorption, bolus, powder, granule, paste for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension or sustained release formulation; (3) topical application, e.g., as a cream, ointment, or controlled-release patch or spray for application to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam; (5) sublingual administration; (6) intraocular administration; (7) transdermal administration; or (8) nasal administration.

[0160] Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) glycols, such as protease inhibitors. (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free distilled water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffers, (21) polyesters, polycarbonates, and / or polyanhydrides, and (22) other non-toxic compatible materials used in pharmaceutical formulations.

[0161] Additional non-limiting examples of agents suitable for formulation with an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, include PEG-conjugated nucleic acids, phospholipid-conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (Pluronic Acid Inhibitors), which can enhance drug entry into various tissues. P85, etc.); biodegradable polymers, such as poly(DL-lactide-co-glycolide) microspheres for sustained delivery after implantation (Emerich, DF et al., 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such as those made of polybutyl cyanoacrylate, which can deliver drugs across the blood-brain barrier and alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).

[0162] The present disclosure also features the use of compositions comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, branched, and unbranched, or a combination thereof, or long-circulating or stealth liposomes). These formulations provide a method for increasing drug accumulation in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear cell phagocytic system (MPS or RES), thereby allowing for extended blood circulation time and enhanced tissue exposure of encapsulated drugs (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to selectively accumulate in tumors, likely due to extravasation and entrapment in vascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA compared with conventional cationic liposomes, which are known to accumulate in tissues, particularly in MPS (Liu et al.). (See, e.g., Choi et al., International PCT Publication No. WO 96 / 10391; Ansell et al., International PCT Publication No. WO 96 / 10390; Holland et al., International PCT Publication No. WO 96 / 10392). Long-circulating liposomes also likely protect drugs from nuclease degradation to a greater extent than cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

[0163] In a further embodiment, the present disclosure includes an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, composition prepared for delivery, as described in U.S. Patent Nos. 6,692,911, 7,163,695, and 7,070,807. In this regard, in one embodiment, the present disclosure provides an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof (described in U.S. Patent Nos. 7,163,695, 7,070,807, and 6,692,911) in a composition comprising a copolymer of lysine and histidine (HK), either alone or in combination with PEG (e.g., branched or unbranched PEG, or a mixture of both), in combination with PEG and a targeting moiety, or in combination with a crosslinker. In certain embodiments, the present disclosure provides eteplirsen in a composition comprising gluconate-modified polyhistidine or gluconyl-polyhistidine / transferrin-polylysine.Those skilled in the art will also recognize that amino acids with properties similar to His and Lys can be substituted in the composition.

[0164] In some embodiments, antisense oligomers (e.g., eteplirsen) can be administered as pharmaceutically acceptable salts. In this context, the term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic acid addition salts of antisense oligomers. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J.Pharm.Sci.66:1-19). Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of compounds from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids, such as hydrochloride, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; as well as salts prepared from organic acids, such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid (salicyclic acid), sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothionic acid, and the like. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like. (See, for example, Berge et al., supra.)

[0165] Wetting agents, emulsifying agents, and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0166] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0167] Formulations suitable for use in the methods of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0168] Methods of preparing pharmaceutical compositions comprising an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, include the step of bringing the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, into association with the carrier and, optionally, one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately bringing into association the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0169] Formulations of the present disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (with a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (with an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof as an active ingredient. The antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof may also be administered as a bolus, electuary, or paste.

[0170] In solid dosage forms of the present disclosure for oral administration (capsules, tablets, pills, dragees, powders, granules, lozenges, etc.), the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, may be incorporated into one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, especially sorbitol; (5) dissolution retarders, such as paraffin; (6) absorption enhancers, such as quaternary ammonium compounds and surfactants, such as poloxamers and sodium lauryl sulfate; (7) wetting agents, such as cetyl alcohol, glycerol monostearate, and nonionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) coloring agents; and (11) release-controlling agents, such as crospovidone or ethylcellulose. For capsules, tablets, and pills, pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard-shell gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0171] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents, or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0172] Tablets and other solid dosage forms of the pharmaceutical compositions of the present disclosure, such as dragees, capsules, pills, and granules, can be optionally obtained or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, using, for example, hydroxypropylmethylcellulose in various proportions to provide the desired release profile, other polymer matrices, liposomes, and / or microspheres. They can also be formulated for rapid release, such as by lyophilization. They can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents and can be compositions that release the active ingredient(s) only, or preferentially, in a certain portion of the digestive tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0173] Oral administration liquid dosage forms of antisense oligomer (such as eteplirsen) or its pharmaceutically acceptable salts include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active ingredients, liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0174] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0175] In addition to the active compound, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0176] Formulations for rectal or vaginal administration may be presented as suppositories, which may be prepared by mixing the antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, which are solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active compound.

[0177] Preparations or dosage forms for topical or transdermal administration of antisense oligomers (e.g., eteplirsen) or its pharmaceutically acceptable salts include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.Antisense oligomers (e.g., eteplirsen) or its pharmaceutically acceptable salts can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.Ointments, pastes, creams, and gels can contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof, in addition to antisense oligomers (e.g., eteplirsen) or its pharmaceutically acceptable salts.

[0178] Powders and sprays can contain, in addition to the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons such as butane and propane.

[0179] Transdermal patches have the additional advantage of providing controlled delivery of antisense oligomers (such as eteplirsen) or its pharmaceutically acceptable salts into the body.Such dosage forms can be prepared by dissolving or dispersing the oligomer in a suitable medium.Absorption enhancers can also be used to increase the flux of the drug across the skin.The rate of such flux can be controlled by providing a rate-controlling membrane or dispersing the drug in a polymer matrix or gel, among other methods known in the art.

[0180] Pharmaceutical compositions suitable for parenteral administration may contain an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions. These may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In one embodiment, the pharmaceutical composition comprises phosphate-buffered saline.

[0181] These pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action on the subject oligomers can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. Furthermore, prolonged absorption of the injectable dosage form can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0182] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injection in order to prolong the effect of drugs.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility, among other methods known in the art.Then, the absorption rate of drug depends on its dissolution rate, which in turn depends on crystal size and crystalline form.Alternatively, the delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in oil vehicle.

[0183] Injectable depot forms can be made by forming microencapsule matrices of an antisense oligomer (e.g., eteplirsen) or its pharmaceutically acceptable salt in a biodegradable polymer such as polylactide-polyglycolide. The rate of release can be controlled depending on the ratio of the antisense oligomer (e.g., eteplirsen) or its pharmaceutically acceptable salt to the polymer and the properties of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0184] The antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, can be administered to cells by a variety of methods known to those skilled in the art, including, but not limited to, iontophoresis, or incorporation into other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, as described herein and known in the art. In certain embodiments, microemulsification technology can be utilized to improve the bioavailability of lipophilic (water-insoluble) pharmaceutical agents. Examples include trimetrine (Dordunoo, SK, et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991, and REV 5901 (Sheen, PC, et al., J Pharm Sci 80(7), 712-714, 1991). Among other benefits, microemulsification provides enhanced bioavailability by preferentially directing absorption into the lymphatic system instead of the circulatory system, thereby bypassing the liver and preventing breakdown of the compound in the hepatobiliary circulation.

[0185] Cyclodextrins are cyclic oligosaccharides consisting of six, seven, or eight glucose units, designated by the Greek letters α, β, or γ, respectively. The glucose units are linked by α-1,4-glucosidic bonds. As a result of the chair conformation of the sugar units, all secondary hydroxyl groups (at C-2 and C-3) are located on one side of the ring, while all primary hydroxyl groups at C-6 are located on the other. As a result, the exterior is hydrophilic, making cyclodextrins water-soluble. In contrast, the cavities of cyclodextrins are hydrophobic because they are lined by hydrogen atoms at C-3 and C-5, as well as by ether-like oxygens. These matrices allow complexation with a variety of relatively hydrophobic compounds, including steroid compounds such as 17α-estradiol (see, e.g., van Uden et al., Plant Cell Tiss. Org. Cult. 38:1-3-113 (1994)). Complexation occurs through van der Waals interactions and hydrogen bond formation. For a general review of the chemistry of cyclodextrins, see Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822 (1994).

[0186] The physicochemical properties of cyclodextrin derivatives depend largely on the type and degree of substitution. For example, their water solubility ranges from insoluble (e.g., triacetyl-beta-cyclodextrin) to 147% soluble (w / v) (G-2-beta-cyclodextrin). Furthermore, they are soluble in many organic solvents. The properties of cyclodextrins allow for the control of the solubility of various formulation components by increasing or decreasing their solubility.

[0187] Numerous cyclodextrins and methods for their preparation have been described. For example, Parmeter (I) et al. (U.S. Pat. No. 3,453,259) and Gramera et al. (U.S. Pat. No. 3,459,731) described electrically neutral cyclodextrins. Other derivatives include cyclodextrins with cationic character [Parmeter (II), U.S. Pat. No. 3,453,257], insoluble crosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), and cyclodextrins with anionic character [Parmeter (III), U.S. Pat. No. 3,426,011]. Among cyclodextrin derivatives with anionic character, carboxylic acids, phosphorous acids, phosphinic acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfinic acids, and sulfonic acids are added to the parent cyclodextrin [Parmeter (III), see above]. Additionally, sulfoalkyl ether cyclodextrin derivatives have been described by Stella et al. (US Pat. No. 5,134,127).

[0188] In one aspect, the formulation contains micelles formed from an antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, and at least one amphiphilic carrier, wherein the micelles have an average diameter of less than about 100 nm. More preferred embodiments provide micelles having an average diameter of less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter of less than about 30 nm or even less than about 20 nm.

[0189] While any suitable amphiphilic carrier is contemplated, currently preferred carriers are generally those that have a generally recognized as safe (GRAS) status and are capable of both solubilizing the compounds of the present disclosure and microemulsifying them at a later stage when the solution comes into contact with a complex aqueous phase (such as that found in the human gastrointestinal tract). Amphiphilic components that meet these requirements typically have an HLB (hydrophilic-lipophilic balance) value of 2-20, and their structures contain straight-chain aliphatic radicals ranging from C-6 to C-20. Examples include polyethylene glycolated fatty acid glycerides and polyethylene glycol.

[0190] Examples of amphiphilic carriers include saturated and monounsaturated polyethylene glycolated fatty acid glycerides, such as those obtained from various fully or partially hydrogenated vegetable oils. Such oils can advantageously consist of tri-, di-, and mono-fatty acid glycerides and di- and mono-polyethylene glycol esters of the corresponding fatty acids; particularly preferred fatty acid compositions include capric acid 4-10%, capric acid 3-9%, lauric acid 40-50%, myristic acid 14-24%, palmitic acid 4-14%, and stearic acid 5-15%. Another useful class of amphiphilic carriers includes partially esterified sorbitan and / or sorbitol with saturated or monounsaturated fatty acids (SPAN® series) or the corresponding ethoxylated analogs (TWEEN® series).

[0191] Commercially available amphiphilic carriers, including the Gelucire series, Labrafil, Labrasol, or Lauroglycol (all manufactured and distributed by Gattefosse Corporation, Saint-Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono- and di-laurate, lecithin, polysorbate 80, and the like (manufactured and distributed by numerous companies in the United States and around the world), may be particularly useful.

[0192] In certain embodiments, delivery of antisense oligomers (e.g., eteplirsen), or pharmaceutically acceptable salts thereof, can occur by using liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. to introduce the compositions of the present disclosure into suitable host cells. In particular, the compositions of the present disclosure can be formulated for delivery by being encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, or the like. The formulation and use of such delivery vehicles can be carried out using known and conventional techniques.

[0193] Liposomes consist of at least one lipid bilayer membrane surrounding an aqueous interior compartment. Liposomes can be characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and typically range in diameter from 0.02 to 0.05 μm, while large unilamellar vesicles (LUVs) are typically larger than 0.05 μm. Large oligolamellar vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 μm. Liposomes with several non-concentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are called multivesicular vesicles.

[0194] One aspect of the present disclosure relates to a formulation comprising liposomes containing an antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof, wherein the liposome membrane is formulated to provide liposomes with increased loading capacity. Alternatively, or in addition, the antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof can be contained within or adsorbed onto the liposome bilayer of the liposome. The antisense oligomer (e.g., eteplirsen) or a pharmaceutically acceptable salt thereof can be aggregated with a lipid surfactant and loaded within the interior space of the liposome. In these cases, the liposome membrane is formulated to resist the disruptive effect of the active agent-surfactant aggregates.

[0195] According to one embodiment of the present disclosure, the lipid bilayer of the liposome contains lipids derivatized with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the interior space enclosed by the liposome and from the exterior of the lipid bilayer into the surrounding environment.

[0196] Aggregates of surfactant and active agent (such as emulsions or micelles containing the active agent of interest) can be entrapped within the interior space of liposomes to disperse and / or solubilize antisense oligomers (e.g., eteplirsen) or their pharmaceutically acceptable salts. The surfactant can be selected from any suitable aliphatic, alicyclic, or aromatic surfactant, including, but not limited to, biocompatible lysophosphatidylcholines (LPGs) of various chain lengths (e.g., about C14 to about C20). Polymer-derivatized lipids, such as PEG-lipids, can also be used for micelle formation, as they act to inhibit micelle / membrane fusion and the addition of polymers to the surfactant molecule reduces the CMC of the surfactant, aiding in micelle formation. Surfactants with CMCs in the micromolar range are preferred, and higher CMC surfactants can be used to prepare micelles entrapped within the liposomes of the present disclosure.

[0197] Liposomes according to the present disclosure can be prepared by any of a variety of techniques known in the art. See, for example, U.S. Patent No. 4,235,871, published PCT application WO96 / 14057, New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104; Lasic DD, Liposomes from physics to applications, Elsevier Science Publishers BV, Amsterdam, 1993. For example, liposomes according to the present disclosure can be prepared by diffusing a lipid derivatized with a hydrophilic polymer into preformed liposomes at a lipid concentration corresponding to the final molar percentage of the desired derivatized lipid in the liposome, e.g., by exposing the preformed liposomes to micelles composed of lipid-grafted polymers. Liposomes containing hydrophilic polymers can also be formed by homogenization, lipid field hydration, or extrusion techniques, as known in the art.

[0198] In another exemplary formulation procedure, antisense oligomer (e.g., eteplirsen) or its pharmaceutically acceptable salt is first dispersed by sonication in lysophosphatidylcholine or other low CMC surfactants (containing polymer-grafted lipids) that easily solubilize hydrophobic molecules.The resulting micelle suspension of antisense oligomer (e.g., eteplirsen) or its pharmaceutically acceptable salt is then used to rehydrate a dried lipid sample containing an appropriate mole percentage of polymer-grafted lipids or cholesterol.The lipid and antisense oligomer suspension is then formed into liposomes using extrusion techniques known in the art, and the resulting liposomes are separated from the unencapsulated solution by standard column separation.

[0199] In one aspect of the present disclosure, liposomes are prepared to have a substantially uniform size within a selected size range. One effective sizing method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes with a selected uniform pore size, the pore size of which roughly corresponds to the largest size of liposomes that will be produced by extrusion through the membrane. See, for example, U.S. Patent No. 4,737,323 (April 12, 1988). In certain embodiments, reagents such as DharmaFECT® and Lipofectamine® can be used to introduce polynucleotides or proteins into cells.

[0200] The release characteristics of the disclosed formulations depend on the encapsulation material, the concentration of the encapsulated drug, and the presence of release modifiers. For example, release can be engineered to be pH-dependent, using a pH-sensitive coating that releases only at low pH, such as found in the stomach, or at high pH, ​​such as found in the intestine. Enteric coatings can be used to prevent release from occurring until after passage through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach, followed by a later release in the intestine. Release can also be engineered by including salts or pore-forming agents, which can increase water uptake or drug release by diffusion from the capsule. Excipients that modify the solubility of the drug can also be used to control the release rate. Agents that enhance matrix degradation or release from the matrix can also be incorporated. They can be added to the drug, added as a separate phase (i.e., as microparticles), or co-dissolved in the polymer phase, depending on the compound. In most cases, the amount should be between 0.1 and 30 percent (w / w polymer). Types of degradation accelerators include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine, as well as surfactants such as Tween® and Pluronic®. Pore-forming agents (i.e., water-soluble compounds such as inorganic salts and sugars) that add microstructure to the matrix are added as microparticles. The range is typically 1 to 30 percent (w / w polymer).

[0201] Hydrophilic polymers suitable for use in liposomes are those that are readily water-soluble, can be covalently attached to vesicle-forming lipids, and are tolerated in vivo without toxic effects (i.e., are biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic acid (also called polylactide), polyglycolic acid (also called polyglycolide), polylactic acid-polyglycolic acid copolymers, and polyvinyl alcohol. In certain embodiments, the polymer has a molecular weight of about 100 or 120 daltons up to about 5,000 or 10,000 daltons, or about 300 daltons to about 5,000 daltons. In other embodiments, the polymer is polyethylene glycol having a molecular weight of about 100 to about 5,000 daltons, or about 300 to about 5,000 daltons. In certain embodiments, the polymer is 750 dalton polyethylene glycol (PEG(750)). Polymers can also be defined by the number of monomers therein, with preferred embodiments of the present disclosure utilizing polymers of at least about three monomers, such PEG polymers consisting of three monomers (about 150 daltons).

[0202] Other hydrophilic polymers that may be suitable for use in the present disclosure include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethylcellulose.

[0203] In certain embodiments, the formulations of the present disclosure comprise a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

[0204] Uptake can also be manipulated by changing the residence time of the particles in the intestine. This can be achieved, for example, by coating the particles with a mucoadhesive polymer or by selecting it as the encapsulating material. Examples include most polymers with free carboxyl groups, such as chitosan, cellulose, and especially polyacrylates (as used herein, polyacrylate refers to polymers containing acrylate groups and modified acrylate groups such as cyanoacrylate and methacrylate).

[0205] The antisense oligomer (e.g., eteplirsen), or a pharmaceutically acceptable salt thereof, can be formulated to be contained in or adapted to be released by a surgical or medical device or implant. In certain embodiments, the implant can be coated with the oligomer or otherwise treated. For example, the composition of the present disclosure can be coated onto the implant using a hydrogel or other polymer, such as a biocompatible and / or biodegradable polymer (i.e., the composition can be adapted for use with a medical device by using a hydrogel or other polymer). Polymers and copolymers for coating medical devices with drugs are well known in the art. Examples of implants include, but are not limited to, stents, drug-eluting stents, sutures, prosthetic organs, vascular catheters, dialysis catheters, vascular grafts, artificial heart valves, cardiac pacemakers, implantable cardioverter-defibrillators, IV needles, bone and osteogenic devices, such as pins, screws, plates, and other devices, and artificial tissue matrices for wound healing.

[0206] The routes of administration described are intended as a guide only, as one skilled in the art can readily determine the optimal route of administration. Several approaches have been attempted to introduce functional new genetic material into cells, both in vitro and in vivo (Friedmann (1989) Science, 244:1275-1280). These approaches include incorporating the gene to be expressed into a modified retrovirus (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl.:5074S-5079S), incorporating the gene into a non-retroviral vector (e.g., an adeno-associated virus vector) (Rosenfeld, et al. al. (1992) Cell, 68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434), or liposome-mediated delivery of transgenes linked to heterologous promoter-enhancer elements (Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281; ​​Nabel, et al. (1990) Science, 249:1285-1288; Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855), ligand-specific, binding to cation transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624), or using naked DNA expression vectors (Nabel et al. (1990) supra), Wolff et al. (1990) Science, 247:1465-1468). Direct injection of transgenes into tissues results in only local expression (Rosenfeld (1992) supra, Rosenfeld et al. (1991) supra, Brigham et al. (1989) supra, Nabel (1990) supra, and Hazinski et al. (1991) supra). The group of Brigham et al. (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) has reported only in vivo transfection of mouse lungs after either intravenous or intratracheal administration of DNA-liposome complexes. An example of a review article on human gene therapy procedures is Anderson, Science (1992) 256:808-813. [Example]

[0207] Although the above disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art, in light of the teachings of the present disclosure, that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only, and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield substantially similar results.

[0208] Example 1. High-dose eteplirsen treatment in the hDMD Δ52 mdx mouse model Eteplirsen was tested in a humanized DMD mdx mouse model, in which a human DMD carrying an exon 52 deletion-YAC transgene (hDMD Δ52 mice) was integrated into mouse chromosome 5. This model is described in Hoen et al., "Generation and Characterization of Transgenic Mice with the Full-Length Human DMD Gene," J. Biol. Chem., 2008, 283(9):5899-5907, the disclosure of which is incorporated herein in its entirety.

[0209] The deletion of exon 52 in the human DMD gene results in an out-of-frame pre-mRNA. hDMD Δ52 mice are suitable for exon 51 skipping drugs. As shown in DMD patients, eteplirsen is designed to bind to exon 51 of the dystrophin pre-mRNA in hDMD Δ52 mice, resulting in the exon being excluded during mRNA processing. Skipping exon 51 with eteplirsen is expected to restore the reading frame of the dystrophin mRNA during protein translation. This is intended to enable the production of an internally truncated dystrophin protein that localizes to the muscle membrane and can protect muscles from injury and improve force in dystrophic muscle fibers.

[0210] Experimental design hDMD Δ52 mice were treated with intravenous injections of vehicle or eteplirsen for 1 (500 or 960 mg / kg), 4 (500 or 750 mg / kg), and 8 weeks (500 or 750 mg / kg) or once weekly (QW) for 8 weeks. Plasma exposures at the 500, 750, and 960 mg / kg doses in mice are equivalent to plasma exposures at 100, 150, and 192 mg / kg in humans. This is based on a 5:1 dose / exposure relationship between mice and humans.

[0211] result The results of exon 51 skipping, dystrophin production, and grip strength testing in hDMD Δ52 mice after 4 and 8 weekly doses of eteplirsen at exposures in the same range as 100-200 mg / kg in patients are shown in Figures 1-3.

[0212] The percent exon 51 skipping in hDMD Δ52 mdx mice (n=6) was measured using droplet digital PCR (ddPCR). Briefly, quadriceps, diaphragm, biceps, skin, and heart were homogenized using a TissueLyser, and RNA was isolated and processed for ddPCR to determine the level of exon skipping. Figure 1 shows that eteplirsen increases the percent exon 51 skipping in hDMD Δ52 mice.

[0213] Dystrophin production in hDMD Δ52 mdx mice (n=6) was measured according to the following method. Protein lysates prepared in NCH buffer were diluted to 0.2 μg / μl in the 0.1× sample buffer provided with the Jess kit. A wild-type standard curve was prepared using pooled wild-type lysates diluted in pooled dystrophic lysates at a concentration of 0.2 μg / μl in 0.1× sample buffer. 4 μl of each sample and 1 μl of the prepared fluorescent 5× master mix were added to a PCR plate. The plate was then incubated at 95°C for 5 minutes and then placed on ice. 3 μl of sample was then added to the Jess plate in the order indicated by the manufacturer. The remainder of the plate was prepared according to the manufacturer's instructions using milk-free antibody diluent.

[0214] The primary antibody cocktail was prepared by diluting dystrophin antibody (ab154168) 1:1000 and α-actinin (ab254074) 1:50 together in milk-free antibody diluent. The secondary antibody cocktail consisted of 20× NIR anti-mouse antibody (obtained from Protein simplex; Beekman et al., "Use of capillary Western immunoassay(Wes)for quantification of dystrophin levels in skeletal muscles of healthy controls and "Individuals with Becker and Duchenne muscular dystrophy," PLOS One, Apr. 11, 2018, the contents of which are incorporated herein in their entirety, were treated with anti-rabbit chemiluminescent antibody (Protein The plate was prepared by diluting in 100% ethanol (simple). Once loaded, it was placed in the Jess instrument, and the 66-440 kDa 25-capillary module was inserted into the instrument. The assay was then run using both chemiluminescence and fluorescence programs. After running, dystrophin was normalized to actinin by dividing the dystrophin (308 kDa) peak area by the actinin (106 kDa) peak area for each well. A line was fitted to the standard curve, and the equation for that line was used to calculate percent dystrophin compared to humanized DMD WT mice. Figure 2 shows that eteplirsen increases dystrophin production in hDMD Δ52 mdx mice.

[0215] The grip strength test measures the maximum peak force exerted by the rodent. Measured in grams, the peak force is obtained by the operator drawing the mouse along a straight line across the platform guided by the attached sensor. The animal is released at the edge of the platform to obtain a maximum force measurement. Three measurements are taken and averaged for each testing day to obtain results. Results are normalized to the animal's weight. Mice are tested on the first day of the study to determine a baseline and allow the mice to become accustomed to the apparatus. Figure 3 shows that eteplirsen improves function in hDMD Δ52 mdx mice.

[0216] Example 2. Safety and efficacy of high-dose eteplirsen in non-human primates This example evaluates the safety and efficacy of eteplirsen administered either intravenously (IV) or subcutaneously (SC) to cynomolgus monkeys once weekly for 12 weeks at doses of 5, 40, and 320 mg / kg per injection.

[0217] Experimental design A total of 60 male and female cynomolgus monkeys were used in the study, with 12 per dose group (6 males and 6 females), as follows: All animals received either intravenous bolus (IV) infusions or subcutaneous injections (SC) weekly for 12 weeks, and then the pharmacodynamics (PD) exhibited by exon skipping was assessed at the end of the study.

[0218] Animals in Group 1 received the vehicle control. Groups 2-4 received eteplirsen as an IV bolus at dose levels 5-320 mg / kg. Group 5 received eteplirsen as an SC dose of 320 mg / kg. A dose of 320 mg / kg in humans significantly reduced the AUC and C produced by 320 mg / kg in NHPs. max AUC and C that are equivalent to PK max Based on the linear dose-exposure relationship observed in NHPs, the human 200 mg / kg dose is expected to produce similar AUC and C max I predict it will be a penalty kick. [Table 8]

[0219] All animals were monitored throughout the study using clinical observations and body weight measurements. Clinical pathology evaluations on blood samples were performed on all animals at pre-examination, week 4, and before terminal and recovery necropsies. Urine samples for clinical pathology evaluation were performed on all animals at pre-examination and before terminal and recovery necropsies. Blood samples (approximately 4.8-5.8 mL) were collected from the femoral vein. Samples were collected into tubes containing K3EDTA for evaluation of hematological parameters and sodium citrate for evaluation of coagulation parameters. Serum separators were used for clinical chemistry samples. Urine samples were collected using a steel pan placed under the cage for approximately 16 hours.

[0220] result Quadriceps, heart, and diaphragm tissues were homogenized and processed for RT-PCR analysis to determine the level of exon 51 skipping. No exon skipping was detected in the vehicle-treated group. As shown in Figure 4, exon skipping was detected at all doses in the three muscle groups. A dose-dependent increase in exon skipping was observed in quadriceps, heart, and diaphragm. Both IV and SC administration resulted in measurable exon skipping levels, as shown in Figure 5. The bioavailability of SC administration on day 1 was 104%.

[0221] In conclusion, administration of eteplirsen by intravenous infusion for 12 doses was clinically well tolerated under the conditions of this study in cynomolgus monkeys. Based on the absence of adverse findings, the no-adverse-effect level for AVI-4658 in male and female cynomolgus monkeys administered 12 weekly IV boluses was 320 mg / kg (IV and SC), with a 10-20 hour response rate on Day 71 of the study. * mg / mL (IV) and 1250 hours * The mean AUC of 10 mg / mL (SC) corresponded to the mean AUC of 10 mg / mL (SC). The bioavailability of SC administration on day 1 was 104%, indicating that eteplirsen can be administered using both IV and SC administration. Both IV and SC administration resulted in measurable levels of exon skipping.

[0222] Example 3. Treatment of human patients with high-dose eteplirsen patient Eligible patients were boys aged 7–13 years (inclusive) with an out-of-frame deletion in the DMD gene that could be corrected by skipping exon 51. Patients achieved a mean 6-minute walk test (6MWT) distance of ≥ 300 and ≤ 450 meters (unassisted) at both the screening and baseline visits. Patients were required to maintain a sufficient body mass (1 cm) before and during treatment. 3Patients will have intact right and left biceps (preferred biopsy sites) or alternative brachialis muscle groups to obtain muscle biopsies. Patients will also be on a stable dose or equivalent of oral corticosteroids for at least 24 weeks prior to randomization, with the dose expected to remain constant throughout the study (except for modifications to accommodate weight changes). Patients will also have stable pulmonary function (≥50% predicted ventilation, no need for nocturnal ventilation), with pulmonary function unlikely to significantly decompensate during the study.

[0223] Study Drug Eteplirsen [sequence 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3'] (SEQ ID NO: 1) is supplied by Sarepta Therapeutics, Inc. in a single-use vial of phosphate-buffered saline (100 mg / ml). Eteplirsen is reconstituted with 150 ml of normal saline and infused over 60 minutes. A placebo is administered for the first 24 weeks of the study. The placebo is supplied in an identical vial of phosphate-buffered saline and is administered in the same manner as eteplirsen.

[0224] Test Design Open-label dose escalation: The objectives of this study were to evaluate the safety and tolerability of weekly IV administration of 100 and 200 mg / kg eteplirsen and to evaluate the pharmacokinetics of 100 and 200 mg / kg eteplirsen.

[0225] Cohorts of four eligible DMD patients will be treated with weekly eteplirsen IV at doses of 100 mg / kg followed by 200 mg / kg for four weeks at each dose level, with each four-week treatment period potentially extended based on patient preference. After each of the four patients completes their two-week treatment period, safety and tolerability data will be reviewed to determine whether the patient will continue treatment for an additional two weeks. Cohorts may be expanded to two more patients at each dose level, who will begin treatment after two weeks of treatment within the applicable four-week treatment period. Available data will be evaluated to determine whether weekly IV administration of 100 mg / kg, or both 100 mg / kg and 200 mg / kg, is sufficiently safe and tolerable in DMD patients to allow the double-blind portion of the study to begin. Patients in the open-label dose escalation phase will continue treatment at the selected higher dose as a separate cohort.

[0226] Double-blind: This is a randomized, double-blind, dose-ranging, and dose-comparison evaluation of the safety and efficacy of one or two higher doses of eteplirsen, 100 mg / kg and 200 mg / kg, and 30 mg / kg, administered intravenously once weekly in approximately 114 patients with Duchenne muscular dystrophy (DMD) and a genetically confirmed deletion mutation suitable for treatment with exon 51 skipping. Randomization will be stratified by baseline NSAA total score (≤22 or >22). Patients will be randomized 1:1:1 to three dose groups: 30, 100, and 200 mg / kg, with the latter two doses determined to be safe and tolerable through open-label dose escalation. Upon completion of dose-ranging, patients receiving either 100 mg / kg or 200 mg / kg will continue on their selected higher dose, while patients already randomized to 30 mg / kg will continue on that dose.

[0227] If only the 100 mg / kg dose is deemed acceptable for dose escalation, no titration will be performed and patients will be randomized in a 1:2 ratio to either the 30 or 100 mg / kg dose for dose comparison.

[0228] All patients will undergo a baseline muscle biopsy prior to treatment. Each patient will undergo one additional muscle biopsy at either week 12, week 24, or week 48 (24 patients at week 12, 45 patients at week 24, and 45 patients at week 48). An interim analysis of the week 12 muscle biopsy data will be performed to assess dystrophin expression for high-dose selection, with additional interim analyses at week 24 and, if high-dose selection is not required, week 48. If high-dose selection is required, patients will enter the dose comparison portion of the study at either week 12, week 24, or week 48. If no interim analysis for high-dose selection is required at week 24 or week 48, assessment of dystrophin expression at those weeks will be performed as part of the dose comparison. Both PD (dystrophin expression endpoint) and safety endpoints will be considered for high-dose selection.

[0229] Interim analysis To allow for high dose selection of 100 mg / kg to 200 mg / kg during the double-blind portion, planned muscle biopsies will be taken at these time points and measured for dystrophin expression using Western blot, as described above, followed by interim analyses of muscle biopsy data at weeks 12, 24, and 48 to assess dystrophin expression. To maintain the integrity of the study, an independent, external, unblinded statistical group will conduct unblinded muscle biopsy interim analyses. Dose selection decisions will occur at either of the muscle biopsy interim analyses. If dose selection decisions occur at an earlier time point (i.e., week 12 or week 24), a subsequent muscle biopsy interim analysis will be conducted for dose comparison on the dystrophin expression endpoint.

[0230] Example 4. DMD Patient Myotube Assay with High-Dose Golodirsen The pharmacological activity of high-dose golodirsen was evaluated in a well-characterized in vitro cellular model of Duchenne muscular dystrophy.

[0231] Experimental design The cell model used was isolated from a DMD patient with an exon 52 deletion (DMD Del52), a method shown to preserve essential key skeletal muscle characteristics of the cells (Mamchaoui et al., “Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders,” Skeletal Muscle, 2011, 1(1):34; Thorley et al., “Skeletal muscle characteristics are The muscle cell line was immortalized by the Institute of Myology using a mouse myogenic cell line “preserved in hTERT / cdk4 human myogenic cell lines,” Skeletal Muscle, 2016, 6(1):43; the contents of both references are incorporated herein in their entireties.

[0232] Cell lines and culture conditions Myoblasts were isolated from the paravertebral muscles of a 16-year-old healthy donor and a 16-year-old DMD patient with a deletion in exon 52 and were analyzed as previously described (Mamchaoui et al., "Immortalized pathological human myoblasts: towards a universal tool for the study"). Cells were immortalized by ectopic expression of hTERT and CDK4 at the Institute of Myology, as described in “The Journal of Neuromuscular Disorders,” Skeletal Muscle, 2011, 1(1):34. Cells were maintained at 37°C for 3 hours at 50 μl / cm2 in growth medium containing 1 volume of Medium 199, 4 volumes of Dulbecco's Modified Eagle's Medium (DMEM), 20% fetal bovine serum, 50 μg / ml gentamicin, 25 μg / ml fetuin, 0.5 μg / ml bFGF, 5 ng / ml EGF, 0.2 μg / ml dexamethasone, and 5 μg / ml insulin in tissue culture plates coated with 1% collagen I and 0.5% MaxGel (Sigma-Aldrich E0282).

[0233] In vitro assays The golodirsen lot used was synthesized by Bachem and assessed to be 92% pure. A positive and negative control compound were included on each plate for assay quality control. The positive control was 30 μM SRP-5051, lot number RD00128-17, and the negative control was 30 μM RC-1001 (MZ-194-170). All compounds were dissolved in sterile water, and concentrations were confirmed spectrophotometrically prior to assay. Myoblasts were cultured in growth medium at 6000 cells / well in 50 μl / well for 3 hours at 37°C in 96-well clear-bottom imaging plates (Perkin Elmer) coated with 1% collagen I and 0.5% MaxGel (Sigma-Aldrich E0282). Cells were seeded in a 100% PBS-containing medium (#6055300). 24 hours after seeding, cultures were switched to differentiation medium containing DMEM, 2% heat-inactivated FBS, 50 μg / ml gentamicin, and 10 μg / ml insulin. 48 hours after switching to differentiation medium, oligonucleotides were added, and cultures were incubated for an additional 4 days before analysis.

[0234] Cell staining and high-content image analysis Cultures were washed once with PBS, fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature, and rinsed once with PBS. For staining, cultures were blocked and permeabilized with 3% bovine serum albumin (BSA) and 0.2% Triton® X-100 in PBS for 1 hour at room temperature. Cells were stained with primary antibodies rabbit anti-MyoD (1:100, Fisher Scientific, #NC0819717), mouse anti-dystrophin MANDRA1 (IgG1(7A10), Santa Cruz, #sc-47760), mouse anti-dystrophin (IgG2ak, MANDYS106, EMD Millipore, #MABT827), mouse anti-myosin heavy chain (IgG2B, 1:1000, R&D System, #MAB4470) and secondary antibodies Alexa Fluor 488 donkey anti-rabbit (1:1000, Life Technologies, #A21206), Alexa Fluor 555 goat anti-mouse IgG2a (1:1000, Life Technologies, #A21137), Alexa Fluor 555 goat anti-mouse IgG1 (1:1000, Life Technologies, # Cells were stained with Alexa Fluor 647 goat anti-mouse IgG2b (1:1000, Life Technologies, #A21127) and Alexa Fluor 647 goat anti-mouse IgG2b (1:1000, Life Technologies, #A21242). Nuclei were stained with DAPI (1 μg / ml in PBS) for 20 minutes at room temperature before imaging. Imaging and analysis were performed using GE InCell 2200 and 6600 instruments and the InCell Investigator software package.

[0235] result The production of dystrophin protein in the model was assessed by immunofluorescence staining after 4 days of continuous exposure to the compounds and measured by high-content image analysis. Dystrophin protein was detected at concentrations >10 μM SRP-4053 / golodirsen (Figure 6). Emax was achieved at 40 μM, allowing the assignment of an EC50 of approximately 30 μM for dystrophin production in this assay (Figure 7).

[0236] Example 5. Safety of high-dose golodirsen in non-human primates Golodirsen was administered to male cynomolgus monkeys by IV bolus injection once weekly for 12 weeks at dose levels of 0 (vehicle), 5, 40, or 320 mg / kg, followed by a 4-week recovery period. The 320 mg / kg dose in humans significantly increased the AUC and C produced by 320 mg / kg in NHPs. max AUC and C that are equivalent to PK max Based on the linear exposure relationship observed in NHPs, the 200 mg / kg human dose is expected to produce similar AUC and C max I predict it will be a penalty kick.

[0237] Golodirsen plasma exposure (AUC0-t, Cmax) was consistently high throughout the dosing phase and increased with increasing dose, with no evidence of plasma accumulation after 12 weeks of treatment. There were no golodirsen-related effects on clinical signs, BW, cardiovascular parameters (including QT interval), ophthalmologic examination, or clinical pathology parameters. Transient increases in Bb and C3a complement fragments were observed, and sporadic increases in C5a also occurred but did not show a consistent pattern, suggesting that golodirsen did not significantly affect the terminal pathway of complement activation. A statistically significant increase in testis size (approximately 50% higher testis weight:BW ratio than controls) was observed at 320 mg / kg, with slightly lower luteinizing hormone concentrations ≥ 5 mg / kg and follicle-stimulating hormone concentrations ≥ 40 mg / kg in men compared to controls, but testosterone was unaffected. These reversible findings were considered non-adverse, as there were no relevant effects on other reproductive endpoints (sperm count, sperm motility, and morphology) and no histopathological correlation to organ weight changes. The only golodirsen-related histopathological finding noted was minimal diffuse follicular cell hypertrophy in the thyroid gland of one animal at 320 mg / kg, which was also considered non-adverse. The NOAEL was considered to be the highest dose level tested, 320 mg / kg (Cmax = 1790 μg / mL, AUC0 t = 2550 μg·hr / mL). *********************

[0238] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In certain embodiments, for example, the following are provided: (Item 1) 1. A method of treating Duchenne muscular dystrophy (DMD) in a human subject having a mutation in the DMD gene that is amenable to exon skipping, comprising administering to the human subject an antisense oligomer, or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 mg / kg to about 300 mg / kg. (Item 2) 2. The method of item 1, wherein the antisense oligomer induces skipping of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 target region of dystrophin pre-mRNA. (Item 3) The target region of the antisense oligonucleotide is designated as an annealing site, and the base sequence and the annealing site are as follows: [Table 9-1] [Table 9-2] or a pharmaceutically acceptable salt thereof, wherein each T and U in each of SEQ ID NOs: 1 to 51 is thymine or uracil. (Item 4) Item 4. The method according to Item 3, wherein each T and U in the base sequence is thymine. (Item 5) 5. The method according to item 3 or 4, wherein the annealing site is H51A(+66+95). (Item 6) 5. The method according to item 3 or 4, wherein the annealing site is H53A(+36+60) or H53A(+36+56). (Item 7) 5. The method according to item 3 or 4, wherein the annealing site is H45A(-03+19). (Item 8) The antisense oligomer has the formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: each Nu is a nucleobase that together form a targeting sequence; T in formula (I) is [ka] is a moiety selected from; R 1 is a C1-C6 alkyl, and each Nu in 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of SEQ ID NOs: 1 to 51. (Item 9) Item 9. The method according to item 8, wherein each T and U in the base sequence is thymine. (Item 10) The T moiety in the antisense oligonucleotide conjugate of formula (I) is [ka] 10. The method according to item 8 or 9, wherein (Item 11) 11. The method of any one of items 1 to 10, wherein the antisense oligomer is in free base form. (Item 12) 12. The method of any one of items 1 to 11, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 50 mg / kg. (Item 13) 12. The method of any one of items 1 to 11, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. (Item 14) 12. The method of any one of items 1 to 11, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 150 mg / kg. (Item 15) 12. The method of any one of items 1 to 11, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. (Item 16) 12. The method of any one of items 1 to 11, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 250 mg / kg. (Item 17) 12. The method of any one of items 1 to 11, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. (Item 18) 18. The method of any one of items 1 to 17, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered once a week. (Item 19) 19. The method of any one of items 1 to 18, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered for up to about 24 weeks. (Item 20) 19. The method of any one of items 1 to 18, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered for up to about 48 weeks. (Item 21) 19. The method of any one of items 1 to 18, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered for up to about 144 weeks. (Item 22) 22. The method of any one of items 1 to 21, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is formulated for systemic administration. (Item 23) 23. The method of any one of items 1 to 22, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered intravenously. (Item 24) 24. The method of any one of items 1 to 23, wherein the human subject is male. (Item 25) 25. The method of any one of items 1 to 24, wherein the human subject is 7 to 13 years old. (Item 26) 26. The method of any one of items 1 to 25, wherein said treating increases the number of dystrophin-positive fibers in said human subject. (Item 27) 27. The method of any one of items 1 to 26, wherein the human subject achieves a higher North American Ambulatory Assessment (NSAA) total score at week 24 compared to baseline. (Item 28) 28. The method of any one of items 1 to 27, wherein the human subject achieves a higher North American Ambulatory Assessment (NSAA) total score at week 48 compared to baseline. (Item 29) 28. The method of any one of items 1 to 27, wherein the human subject achieves a higher North American Ambulatory Assessment (NSAA) total score at week 144 compared to baseline. (Item 30) 30. The method of any one of items 1 to 29, wherein said treatment reduces loss of ambulation compared to baseline in said human subject as measured by a 6-minute walk test (6MWT). (Item 31) 31. The method of item 30, wherein locomotion is maintained compared to baseline. (Item 32) 32. The method of item 31, wherein locomotor activity is improved compared to baseline. (Item 33) 33. The method of any one of items 1 to 32, wherein the treatment reduces loss of lung function in the human subject compared to baseline, the loss being measured as % annualized decline in forced vital capacity (FVC). (Item 34) Item 34. The method of item 33, wherein lung function is maintained. (Item 35) 35. The method of any one of items 1 to 34, wherein the antisense oligomer is eteplirsen. (Item 36) 35. The method of any one of items 1 to 34, wherein the antisense oligomer is golodirsen. (Item 37) 35. The method of any one of items 1 to 34, wherein the antisense oligomer is casimersen. (Item 38) 1. A method of exon skipping in a human, comprising administering to a human subject having a mutation in the DMD gene that is suitable for exon skipping, an antisense oligomer, or a pharmaceutically acceptable salt thereof, to the human subject, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 80 mg / kg to about 300 mg / kg. (Item 39) 39. The method of claim 38, wherein the antisense oligomer induces skipping of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 target region of dystrophin pre-mRNA. (Item 40) The target region of the antisense oligonucleotide is designated as an annealing site, and the base sequence and the annealing site are as follows: [Table 10-1] [Table 10-2] or a pharmaceutically acceptable salt thereof, wherein each T and U in each of SEQ ID NOs: 1 to 51 is thymine or uracil. (Item 41) Item 41. The method of item 40, wherein each T in the base sequence is thymine. (Item 42) 42. The method of claim 40 or 41, wherein the annealing site is H51A(+66+95). (Item 43) 42. The method of claim 40 or 41, wherein the annealing site is H53A(+36+60) or H53A(+36+56). (Item 44) 42. The method of claim 40 or 41, wherein the annealing site is H45A(-03+19). (Item 45) The antisense oligomer has the formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: each Nu is a nucleobase that together form a targeting sequence; T' in formula (I) is [ka] is a moiety selected from; R 1 is a C1-C6 alkyl, and each Nu of 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of SEQ ID NOs: 1 to 51. (Item 46) Item 46. The method of item 45, wherein each T or U in the base sequence is thymine. (Item 47) The T moiety in the antisense oligomer of formula (I) is [ka] 47. The method according to item 45 or 46, wherein (Item 48) 48. The method of any one of items 38 to 47, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 50 mg / kg. (Item 49) 48. The method of any one of items 38 to 47, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. (Item 50) 48. The method of any one of items 38 to 47, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 150 mg / kg. (Item 51) 48. The method of any one of items 38 to 47, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. (Item 52) 48. The method of any one of items 38 to 47, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 250 mg / kg. (Item 53) 48. The method of any one of items 38 to 47, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. (Item 54) 54. The method of any one of items 38 to 53, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered once a week. (Item 55) 55. The method of any one of items 38 to 54, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered for up to about 24 weeks. (Item 56) 55. The method of any one of items 38 to 54, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered for up to about 48 weeks. (Item 57) 55. The method of any one of items 38 to 54, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered for up to about 144 weeks. (Item 58) 58. The method of any one of items 38 to 57, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is formulated for systemic administration. (Item 59) 59. The method of any one of items 38 to 58, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered intravenously. (Item 60) 60. The method of any one of items 38 to 59, wherein the human subject is male. (Item 61) Item 61. The method of item 60, wherein the human subject is 7 to 13 years old. (Item 62) 62. The method of any one of items 38 to 61, wherein said treatment increases the number of dystrophin-positive fibers in said human subject. (Item 63) 63. The method of any one of items 38 to 62, wherein the human subject achieves a higher North American Ambulatory Assessment (NSAA) total score at week 24 compared to baseline. (Item 64) 63. The method of any one of items 38 to 62, wherein the human subject achieves a higher North American Ambulatory Assessment (NSAA) total score at week 48 compared to baseline. (Item 65) 63. The method of any one of items 38 to 62, wherein the human subject achieves a higher North American Ambulatory Assessment (NSAA) total score at week 144 compared to baseline. (Item 66) 66. The method of any one of items 38 to 65, wherein the treatment reduces loss of ambulation compared to baseline in the human subject as measured by a 6-minute walk test (6MWT). (Item 67) 67. The method of item 66, wherein ambulatory activity is maintained compared to baseline. (Item 68) 68. The method of item 67, wherein locomotor activity is improved compared to baseline. (Item 69) 69. The method of any one of items 38 to 68, wherein the treatment reduces loss of lung function in the human subject compared to baseline, the loss being measured as % annualized decline in forced vital capacity (FVC). (Item 70) 70. The method of item 69, wherein lung function is maintained. (Item 71) 71. The method of any one of items 38 to 70, wherein the antisense oligomer is eteplirsen. (Item 72) 71. The method of any one of items 38 to 70, wherein the antisense oligomer is golodirsen. (Item 73) 71. The method of any one of items 38 to 70, wherein the antisense oligomer is casimersen. (Item 74) 1. A method for treating Duchenne muscular dystrophy (DMD) in a human subject having a mutation in the DMD gene that is amenable to exon skipping, the method comprising administering to the human subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer is administered at a dose of about 80 mg / kg to about 300 mg / kg. (Item 75) 75. The method of item 74, wherein the antisense oligomer induces skipping of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 target region of dystrophin pre-mRNA. (Item 76) The target region of the antisense oligonucleotide is designated as an annealing site, and the base sequence and the annealing site are as follows: [Table 11-1] [Table 11-2] or a pharmaceutically acceptable salt thereof, wherein each T and U in each of SEQ ID NOs: 1 to 51 is thymine or uracil. (Item 77) Item 77. The method of item 76, wherein each T and U in the base sequence is thymine. (Item 78) 78. The method of claim 76 or 77, wherein the annealing site is H51A(+66+95). (Item 79) 78. The method of claim 76 or 77, wherein the annealing site is H53A(+36+60) or H53A(+36+56). (Item 80) 78. The method of claim 76 or 77, wherein the annealing site is H45A(-03+19). (Item 81) The antisense oligomer has the formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: each Nu is a nucleobase that together form a targeting sequence; T' in formula (I) is [ka] is a moiety selected from; R 1 is a C1-C6 alkyl, and each Nu of 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of SEQ ID NOs: 1 to 51. (Item 82) 82. The method of item 81, wherein each T in the base sequence is thymine. (Item 83) The T moiety in the antisense oligomer of formula (I) is [ka] 83. The method according to item 81 or 82, wherein (Item 84) 84. The method of any one of items 74 to 83, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 50 mg / kg. (Item 85) 84. The method of any one of items 74 to 83, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg / kg. (Item 86) 84. The method of any one of items 74 to 83, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 150 mg / kg. (Item 87) 84. The method of any one of items 74 to 83, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg / kg. (Item 88) 84. The method of any one of items 74 to 83, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 250 mg / kg. (Item 89) 84. The method of any one of items 74 to 83, wherein the antisense oligomer, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg / kg. (Item 90) 89. The method of any one of items 74 to 89, wherein the pharmaceutical composition is administered once a week. (Item 91) 91. The method of any one of items 74 to 90, wherein the pharmaceutical composition is administered for up to about 24 weeks. (Item 92) 91. The method of any one of items 74 to 90, wherein the pharmaceutical composition is administered for up to about 48 weeks. (Item 93) 91. The method of any one of items 74 to 90, wherein the pharmaceutical composition is administered for up to about 144 weeks. (Item 94) 94. The method according to any one of items 74 to 93, wherein the pharmaceutical composition is administered systemically. (Item 95) 95. The method according to any one of items 74 to 94, wherein the pharmaceutical composition is administered intravenously. (Item 96) 96. The method of any one of items 74 to 95, wherein the pharmaceutical composition comprises phosphate buffered saline. (Item 97) 97. The method of any one of items 74 to 96, wherein the human subject is male. (Item 98) Item 98. The method of item 97, wherein the human subject is 7 to 13 years old. (Item 99) 99. The method of any one of items 74 to 98, wherein said treating increases the number of dystrophin-positive fibers in said human subject. (Item 100) 99. The method of any one of items 74 to 99, wherein the human subject achieves a higher North American Ambulatory Assessment (NSAA) total score at week 24 compared to baseline. (Item 101) 99. The method of any one of items 74 to 99, wherein the human subject achieves a higher North American Ambulatory Assessment (NSAA) total score at week 48 compared to baseline. (Item 102) 99. The method of any one of paragraphs 74 to 99, wherein the human subject achieves a higher North American Ambulatory Assessment (NSAA) total score at week 144 compared to baseline. (Item 103) 103. The method of any one of paragraphs 74 to 102, wherein the treatment reduces loss of ambulation compared to baseline in the human subject as measured by a 6-minute walk test (6MWT). (Item 104) 104. The method of claim 103, wherein locomotion is maintained compared to baseline. (Item 105) 104. The method of claim 103, wherein locomotor activity is improved compared to baseline. (Item 106) 106. The method of any one of items 74 to 105, wherein the treatment reduces loss of lung function in the human subject compared to baseline, the loss being measured as % annualized decline in forced vital capacity (FVC). (Item 107) 107. The method of claim 106, wherein lung function is maintained. (Item 108) 108. The method of any one of items 74 to 107, wherein the antisense oligomer is eteplirsen. (Item 109) 108. The method of any one of items 74 to 107, wherein the antisense oligomer is golodirsen. (Item 110) 108. The method of any one of items 74 to 107, wherein the antisense oligomer is casimersen.

Claims

[Claim 1] The invention described in the specification.

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