Oligonucleotide compositions and methods thereof for EXON skipping

Abstract

Among other things, the present disclosure provides various technologies including chirally controlled oligonucleotide compositions and technologies for manufacturing and using such oligonucleotide compositions. In some embodiments, the present disclosure provides technologies useful for preventing or treating various conditions, disorders or diseases, e.g., Duchenne Muscular Dystrophy.

Description

Attorney Docket No.: 2010581-1542 OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF FOR EXON SKIPPING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application Nos.63 / 778,330, filed March 26, 2025, and 63 / 698,525, filed September 24, 2024, the entirety of each of which is incorporated herein by reference. BACKGROUND

[0002] Muscular dystrophy (MD) is a group of muscle conditions, diseases, or disorders that is reported to result in (increasing) weakening and breakdown of skeletal muscles over time. The conditions, diseases, or disorders differ in which muscles are primarily affected, the degree of weakness, when symptoms begin, and how quickly symptoms worsen. Many MD subjects eventually become unable to walk. In many cases muscular dystrophy is fatal. Some types are also associated with problems in other organs, including the central nervous system. In some embodiments, a muscular dystrophy is Duchenne (Duchenne’s) Muscular Dystrophy (DMD). In some embodiments, a muscular dystrophy is Becker (Becker’s) Muscular Dystrophy (BMD). SUMMARY

[0003] Among other things, the present disclosure provides technologies that can provide safe, and high and consistent dystrophin expression in DMD patients who amenable to exon 53 skipping. For example, in some embodiments, the present disclosure provides methods for treating DMD comprising administering or delivering WVE-N531 to a subject suffering therefrom according to a regimen as described herein, wherein the regimen demonstrates mean muscle-content adjusted, MHC-normalized dystrophin expression of about 9% (about 5.5% unadjusted) and about 89% subjects with muscle content- adjusted dystrophin levels of at least 5% in a population of ambulatory subjects. In some embodiments, provided technologies demonstrate evidence of improvement muscle health. In some embodiments, provided technologies demonstrate evidence of assessing stem cells. In some embodiments, provided technologies demonstrate evidence of myofiber regeneration. In some embodiments, provided technologies demonstrate best-in-class muscle delivery and / or tissue half-life, e.g., when compared to FDA-approved DMD exon 53 skipping therapeutics such as golodirsen and viltolarsen.

[0004] In some embodiments, provided technologies can provide various advantages, e.g., higher dystrophin expression, higher consistency, lower unit doses, lower total doses, less frequent administration (e.g., about every two weeks, about every four weeks, etc.), etc. (e.g., when compared to FDA-approved DMD exon 53 skipping therapeutics such as golodirsen and viltolarsen). For example, among other things, 1 of 176 13004462v1Attorney Docket No.: 2010581-1542 the present disclosure provides a method for treating DMD, comprising administering to a subject suffering therefrom WVE-N531 about every two weeks, wherein each dose of WVE-N531 is independently equivalent to about 10 mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for treating DMD, comprising administering to a subject suffering therefrom WVE- N531 about every four weeks, wherein each dose of WVE-N531 is independently equivalent to about 10 mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for treating DMD, comprising administering to a subject suffering therefrom WVE-N531 about monthly, wherein each dose of WVE-N531 is independently equivalent to about 10 mg / kg WVE-N531 free acid form. In some embodiments, each dose is administered about every 2 or 4 weeks or monthly. In some embodiments, each dose is administered about every 4 weeks or monthly. In some embodiments, once WVE-N531 is administered about every 4 weeks or about monthly, each dose thereafter is administered about every 4 weeks or about monthly.

[0005] In some embodiments, provided technologies are safe and well tolerated. For example, in one clinical trial readout, no SAEs and no oligonucleotide-class effects were observed after multiple doses (e.g., 10, 11, 12, 13, 14, 15 or more doses) of about 10 mg / kg (unless noted otherwise, dose is equivalent to WVE-N531 free acid form) WVE-N531 were administered about every two weeks.

[0006] In some embodiments, the present disclosure provides WVE-N531 dosing regimens that can deliver WVE-N531 to myocyte stem cells (e.g., marked by a biomarker such as Pax7), restore myocyte stem cell functions, increase myocyte stem cell differentiation, produce myogenic progenitor cells, increase muscle regeneration, and / or increase myofiber formation or repair.

[0007] In some embodiments, the present disclosure provides a method of treating muscular dystrophy, e.g., Duchenne muscular dystrophy (DMD), in a mammalian subject, e.g., a human subject, in need thereof, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping, the method comprising administering to the subject about 10 mg / kg of WVE-N531 about every two weeks (Q2W), e.g., once every 14 days, once every 15 days, or once every 16 days, wherein WVE-N531 is fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU* SfC, wherein: m represents 2’-OMe modified nucleoside; f represents 2’-F modified nucleoside; *S represents Sp phosphorothioate linkage; and n001R represents Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage.

[0008] In some embodiments, the present disclosure provides a method of treating muscular dystrophy, e.g., Duchenne muscular dystrophy (DMD), in a mammalian subject, e.g., a human subject, in need thereof, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping, 2 of 176 13004462v1Attorney Docket No.: 2010581-1542 the method comprising administering to the subject about 10 mg / kg of WVE-N531 about every four weeks (Q4W), e.g., once every 28, 29, 30, 31, or 32 days, wherein WVE-N531 is fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU* SfC, wherein: m represents 2’-OMe modified nucleoside; f represents 2’-F modified nucleoside; *S represents Sp phosphorothioate linkage; and n001R represents Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage.

[0009] In some embodiments, the present disclosure provides a method of treating muscular dystrophy, e.g., Duchenne muscular dystrophy (DMD), in a mammalian subject, e.g., a human subject, in need thereof, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping, the method comprising administering to the subject about 10 mg / kg of WVE-N531 about monthly, wherein WVE-N531 is fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU* SfC, wherein: m represents 2’-OMe modified nucleoside; f represents 2’-F modified nucleoside; *S represents Sp phosphorothioate linkage; and n001R represents Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage.

[0010] WVE-N531 is an oligonucleotide compound that has demonstrated exon skipping activity and DMD function restoration in vitro and in mouse models. Structurally, WVE-N531 belongs to a different type of oligonucleotide than golodirsen and viltolarsen. For example, both golodirsen and viltolarsen are phosphorodiamidate morpholino oligomers (PMOs), while WVE-N531 is a 2’-F and 2’-OMe modified oligonucleotide in which the internucleotidic linkages are independently phosphorothioate, n001 and natural phosphate linkages. Further, chiral linkage phosphorus centers are not stereodefined, and both golodirsen and viltolarsen are provided and administered as stereorandom compositions in which their chiral linkage phosphorus centers randomly exist in two of the potential configurations (Rp or Sp): the compositions are a random mixture of more than 33 million (225) and 1 million (220) diastereomers for golodirsen and viltolarsen, respectively. In contrast, chiral linkage phosphorus in WVE-N531 is each independently stereodefined: linkage phosphorus of each phosphorothioate linkage is Sp, and linkage phosphorus of each n001 linkage is Rp. As demonstrated herein, WVE-N531 compositions are chirally controlled compositions in which WVE-N531 or salts thereof are highly enriched over other diastereomers or salts thereof. Still further, to Applicant’s knowledge prior to the present disclosure oligonucleotides comprising phosphoryl guanidine linkages such as n001 have not been systematically administered to and 3 of 176 13004462v1Attorney Docket No.: 2010581-1542 assessed in human subjects for therapeutic uses. An oligonucleotide, suvodirsen, comprises certain chemistry moieties such as 2’-F, 2’-OMe, phosphorothioate linkages, etc. that are more similar to WVE- N531 than golodirsen and viltolarsen and was also manufactured and administered as a chirally controlled oligonucleotide composition, but it failed in human clinical trials at the assessed doses including about 4.5 mg / kg.

[0011] Among other things, the present disclosure provides technologies for treating muscular dystrophy such as DMD. In some embodiments, the present disclosure provides doses and dosage regimens that are sufficiently safe for clinical administration to subjects and are effective to provide DMD exon 53 skipping, production of truncated DMD polypeptide that can perform improved levels of one or more functions of wild-type DMD protein compared to absence of WVE-N531, DMD function restoration or improvement, and / or clinical benefits. In some embodiments, as demonstrated herein WVE-N531 can provide improved properties and / or activities, e.g., AUC, Cmax, tissue distribution, and / or plasma half life. compared to suvodirsen in primate subjects including human. Various results presented herein support that WVE-N531 can be safely and effectively utilized to treat conditions, disorders or diseases associated with DMD mutations that are amenable to exon 53 skipping including in human subjects.

[0012] In some embodiments, the present disclosure provides a method for treating muscular dystrophy, e.g., DMD, comprising administering to a subject suffering therefrom WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg (mg per kg of body weight) WVE-N531 free acid form, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping. In some embodiments, WVE-N531 is administered as a pharmaceutically acceptable salt form. In some embodiments, WVE-N531 is administered as a WVE-N531 hexadecasodium salt form. In some embodiments, WVE-N531 is administered as one or more forms including one or more pharmaceutically acceptable salt forms, and the total amount of all WVE-N531 forms is equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of the free acid form. In some embodiments, WVE-N531 is administered in a pharmaceutical composition.

[0013] In some embodiments, the present disclosure provides a method for treating muscular dystrophy, e.g., DMD, comprising administering to a subject suffering therefrom a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping. In some embodiments, a pharmaceutically acceptable carrier is or comprises a buffered solution. In some embodiments, it is a phosphate buffered solution. In 4 of 176 13004462v1Attorney Docket No.: 2010581-1542 some embodiments, WVE-N531 is dissolved in a solution and exist in one or more forms, wherein the total amount of all WVE-N531 forms is equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of the free acid form.

[0014] In some embodiments, the present disclosure provides a method for providing DMD exon 53 skipping in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for restoring DMD RNA reading frame in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for providing a truncated DMD polypeptide in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, a truncated (compared to wild- type DMD protein) DMD polypeptide is encoded by an exon53-skipped DMD mRNA. In some embodiments, such an mRNA may lack one or more additional exons in addition to exon 53. In some embodiments, such a truncated DMD polypeptide provides one or more functions, partially or fully, of a wild-type DMD protein. In some embodiments, the present disclosure provides a method for providing increased level of a DMD function in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for providing decreased muscle inflammation in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE- N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for providing decreased muscle necrosis in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 5 of 176 13004462v1Attorney Docket No.: 2010581-1542 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for providing decreased levels of one or more inflammatory biomarkers in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, one or more inflammatory biomarkers comprise one or more inflammatory cytokines. In some embodiments, one or more inflammatory biomarkers comprise monocyte chemoattractant protein-1 (MCP-1) and / or interleukin- 6 (IL-6). In some embodiments, one or more inflammatory biomarkers comprise one or more circulating inflammatory biomarkers. In some embodiments, the present disclosure provides a method for providing decreased levels of serum creatine kinase in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for providing decreased muscle fibrosis in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE- N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for reversing muscle fibrosis in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for improving muscle health in a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for increasing transition from regenerative to mature muscle in a subject, comprising administering to the subject WVE- N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, a subject has a mutation in the DMD gene that is amenable to exon 53 skipping. In some embodiments, a subject is suffering from or susceptible to a muscular dystrophy, e.g., DMD. In some 6 of 176 13004462v1Attorney Docket No.: 2010581-1542 embodiments, WVE-N531 is administered at doses each independently equivalent to about 10 mg / kg WVE- N531 free acid form and is administered about every 2 weeks. In some embodiments, WVE-N531 is administered at doses each independently equivalent to about 10 mg / kg WVE-N531 free acid form and is administered about every 4 weeks. In some embodiments, WVE-N531 is administered at doses each independently equivalent to about 10 mg / kg WVE-N531 free acid form and is administered about monthly.

[0015] In some embodiments, the present disclosure provides a method for slowing progression of muscular dystrophy, comprising administering to a subject suffering therefrom WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, slowing of progression comprises slowing of worsening of one or more functional assessments of a subject. In some embodiments, one or more functional assessments comprise time-to-rise (TTR). In some embodiments, a slowing of worsening of TTR comprises a decreased TTR as compared to a subject not administered WVE-N531. In some embodiments, one or more functional assessments comprise North Star Ambulatory Assessment (NSAA). In some embodiments, a slowing of worsening of NSAA comprises an increased NSAA total score as compared to a subject not administered WVE-N531. In some embodiments, the present disclosure provides a method for slowing increase in TTR of a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for slowing decrease in NSAA total score of a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for increasing grip strength of a subject, comprising administering to the subject WVE-N531, or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, grip strength is increased as compared to a baseline. In some embodiments, a baseline is grip strength prior to administering WVE-N531. In some embodiments, grip strength is increased in one or both hands.

[0016] In some embodiments, the present disclosure provides a method for sustaining dystrophin restoration in a subject, comprising administering to the subject WVE-N531 about monthly at a dose 7 of 176 13004462v1Attorney Docket No.: 2010581-1542 equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for sustaining dystrophin restoration in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for sustaining dystrophin restoration in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for sustaining dystrophin restoration in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for sustaining dystrophin restoration in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for sustaining dystrophin restoration in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping.

[0017] In some embodiments, the present disclosure provides a method for reversing muscle damage in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reversing muscle damage in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reversing muscle damage in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reversing muscle damage in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. 8 of 176 13004462v1Attorney Docket No.: 2010581-1542 In some embodiments, the present disclosure provides a method for reversing muscle damage in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reversing muscle damage in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reversion is or comprises a reversion relative to baseline. In some embodiments, a reversion is or comprises a reversion relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0018] In some embodiments, the present disclosure provides a method for reducing muscle fibrosis in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle fibrosis in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle fibrosis in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle fibrosis in a population of subjects, comprising independently administering to each subject WVE- N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle fibrosis in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle fibrosis in a population of subjects, comprising independently administering to each subject WVE- N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reduction is or comprises a reduction relative to baseline. In some embodiments, a reduction is or comprises a reduction relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0019] In some embodiments, the present disclosure provides a method for reversing muscle fibrosis 9 of 176 13004462v1Attorney Docket No.: 2010581-1542 in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reversing muscle fibrosis in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reversing muscle fibrosis in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reversing muscle fibrosis in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reversing muscle fibrosis in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reversing muscle fibrosis in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reversion is or comprises a reversion relative to baseline. In some embodiments, a reversion is or comprises a reversion relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0020] In some embodiments, the present disclosure provides a method for reducing muscle inflammation in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle inflammation in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE- N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle inflammation in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure 10 of 176 13004462v1Attorney Docket No.: 2010581-1542 provides a method for reducing muscle inflammation in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle inflammation in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle inflammation in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reduction is or comprises a reduction relative to baseline. In some embodiments, a reduction is or comprises a reduction relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0021] In some embodiments, the present disclosure provides a method for reducing level of a inflammatory biomarker in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of a inflammatory biomarker in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of a inflammatory biomarker in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of a inflammatory biomarker in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of a inflammatory biomarker in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of a inflammatory biomarker in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each 11 of 176 13004462v1Attorney Docket No.: 2010581-1542 subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reduction is or comprises a reduction relative to baseline. In some embodiments, a reduction is or comprises a reduction relative to an earlier time point of dosing (e.g., week 48 vs. week 24). In some embodiments, an inflammatory biomarker is a circulating inflammatory biomarker. In some embodiments, a circulating inflammatory biomarker is MCP-1. In some embodiments, a circulating inflammatory biomarker is IL-6.

[0022] In some embodiments, the present disclosure provides a method for reducing muscle necrosis in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle necrosis in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle necrosis in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle necrosis in a population of subjects, comprising independently administering to each subject WVE- N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle necrosis in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing muscle necrosis in a population of subjects, comprising independently administering to each subject WVE- N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reduction is or comprises a reduction relative to baseline. In some embodiments, a reduction is or comprises a reduction relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0023] In some embodiments, the present disclosure provides a method for reducing level of an inflammatory cytokine in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of an inflammatory cytokine in a population of subjects, comprising 12 of 176 13004462v1Attorney Docket No.: 2010581-1542 independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of an inflammatory cytokine in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of an inflammatory cytokine in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of an inflammatory cytokine in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of an inflammatory cytokine in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reduction is or comprises a reduction relative to baseline. In some embodiments, a reduction is or comprises a reduction relative to an earlier time point of dosing (e.g., week 48 vs. week 24). In some embodiments, an inflammatory cytokine is MCP-1. In some embodiments, an inflammatory cytokine is IL-6. In some embodiments, an inflammatory cytokine is TGF-β.

[0024] In some embodiments, the present disclosure provides a method for reducing level of creatine kinase in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of creatine kinase in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of creatine kinase in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of creatine kinase in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free 13 of 176 13004462v1Attorney Docket No.: 2010581-1542 acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of creatine kinase in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing level of creatine kinase in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reduction is or comprises a reduction relative to baseline. In some embodiments, a reduction is or comprises a reduction relative to an earlier time point of dosing (e.g., week 48 vs. week 24). In some embodiments, a reduction is relative to C-PATH natural history. In some embodiments, assessed level of creatine kinase is or comprises level of serum creatine kinase. In some embodiments, an improvement is or comprises an improvement on top of a stable corticosteroid regimen.

[0025] In some embodiments, the present disclosure provides a method for increasing or promoting transition from regenerative to mature muscle in a subject, comprising administering to the subject WVE- N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for increasing or promoting transition from regenerative to mature muscle in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for increasing or promoting transition from regenerative to mature muscle in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for increasing or promoting transition from regenerative to mature muscle in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for increasing or promoting transition from regenerative to mature muscle in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for 14 of 176 13004462v1Attorney Docket No.: 2010581-1542 increasing or promoting transition from regenerative to mature muscle in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, an increase is or comprises an increase relative to baseline. In some embodiments, an increase is or comprises an increase relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0026] In some embodiments, the present disclosure provides a method for increasing or promoting muscle maturation in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for increasing or promoting muscle maturation in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for increasing or promoting muscle maturation in a subject, comprising administering to the subject WVE- N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for increasing or promoting muscle maturation in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for increasing or promoting muscle maturation in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for increasing or promoting muscle maturation in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, an increase is or comprises an increase relative to baseline. In some embodiments, an increase is or comprises an increase relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0027] In some embodiments, the present disclosure provides a method for reducing density of myogenic stem cells in muscle in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering 15 of 176 13004462v1Attorney Docket No.: 2010581-1542 from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing density of myogenic stem cells in muscle in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing density of myogenic stem cells in muscle in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing density of myogenic stem cells in muscle in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing density of myogenic stem cells in muscle in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing density of myogenic stem cells in muscle in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reduction is or comprises a reduction relative to baseline. In some embodiments, a reduction is or comprises a reduction relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0028] In some embodiments, the present disclosure provides a method for reducing internalized nuclei in muscle in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing internalized nuclei in muscle in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing internalized nuclei in muscle in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing internalized nuclei in muscle in a population of subjects, 16 of 176 13004462v1Attorney Docket No.: 2010581-1542 comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing internalized nuclei in muscle in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing internalized nuclei in muscle in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reduction is or comprises a reduction relative to baseline. In some embodiments, a reduction is or comprises a reduction relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0029] In some embodiments, the present disclosure provides a method for reducing fibrotic muscle in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing fibrotic muscle in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing fibrotic muscle in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing fibrotic muscle in a population of subjects, comprising independently administering to each subject WVE- N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing fibrotic muscle in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for reducing fibrotic muscle in a population of subjects, comprising independently administering to each subject WVE- N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, a reduction is or comprises a reduction relative to baseline. In some embodiments, a 17 of 176 13004462v1Attorney Docket No.: 2010581-1542 reduction is or comprises a reduction relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0030] In some embodiments, the present disclosure provides a method for improving organization and uniformity of myofibers in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving organization and uniformity of myofibers in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving organization and uniformity of myofibers in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving organization and uniformity of myofibers in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving organization and uniformity of myofibers in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving organization and uniformity of myofibers in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, an improvement is or comprises improvement relative to baseline. In some embodiments, an improvement is or comprises improvement relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0031] In some embodiments, the present disclosure provides a method for improving muscle health in a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving muscle health in a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In 18 of 176 13004462v1Attorney Docket No.: 2010581-1542 some embodiments, the present disclosure provides a method for improving muscle health in a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving muscle health in a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving muscle health in a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving muscle health in a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, an improvement is or comprises improvement relative to baseline. In some embodiments, an improvement is or comprises improvement relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0032] In some embodiments, the present disclosure provides a method for improving a functional outcome of a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving a functional outcome of a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving a functional outcome of a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving a functional outcome of a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving a functional outcome of a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, 19 of 176 13004462v1Attorney Docket No.: 2010581-1542 wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving a functional outcome of a population of subjects, comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, an improvement is or comprises an improvement relative to baseline. In some embodiments, an improvement is or comprises improvement relative to an earlier time point of dosing (e.g., week 48 vs. week 24). In some embodiments, an improvement is or comprises an improvement relative to natural history.

[0033] In some embodiments, the present disclosure provides a method for improving time-to-rise (TTR) performance of a subject (e.g., relative to natural history), comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving TTR performance of a population of subjects (e.g., relative to natural history), comprising independently administering to each subject WVE- N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving TTR performance of a subject (e.g., relative to natural history), comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving TTR performance of a population of subjects (e.g., relative to natural history), comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving TTR performance of a subject (e.g., relative to natural history), comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving TTR performance of a population of subjects (e.g., relative to natural history), comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, an improvement is relative to natural history.

[0034] In some embodiments, the present disclosure provides a method for slowing disease progression of a subject (e.g., relative to natural history), comprising administering to the subject WVE- 20 of 176 13004462v1Attorney Docket No.: 2010581-1542 N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for slowing disease progression of a population of subjects (e.g., relative to natural history), comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for slowing disease progression of a subject (e.g., relative to natural history), comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for slowing disease progression of a population of subjects (e.g., relative to natural history), comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for slowing disease progression of a subject (e.g., relative to natural history), comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for slowing disease progression of a population of subjects (e.g., relative to natural history), comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, an improvement is relative to natural history. In some embodiments, slowing of disease progression is measured by TTR. In some embodiments, slowing of disease progression is measured by NSAA.

[0035] In some embodiments, the present disclosure provides a method for improving North Star Ambulatory Assessment (NSAA) performance of a subject (e.g., relative to natural history), comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving NSAA performance of a population of subjects (e.g., relative to natural history), comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE- N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving NSAA performance of a subject (e.g., relative to natural history), comprising administering to the subject WVE- 21 of 176 13004462v1Attorney Docket No.: 2010581-1542 N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving NSAA performance of a population of subjects (e.g., relative to natural history), comprising independently administering to each subject WVE- N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving NSAA performance of a subject (e.g., relative to natural history), comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving NSAA performance of a population of subjects (e.g., relative to natural history), comprising independently administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, an improvement is relative to natural history.

[0036] In some embodiments, the present disclosure provides a method for improving hand grip strength of a subject, comprising administering to the subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving hand grip strength of a population of subjects, comprising independently administering to each subject WVE-N531 about monthly at a dose equivalent to about 10 mg / kg WVE- N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving hand grip strength of a subject, comprising administering to the subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving hand grip strength of a population of subjects, comprising independently administering to each subject WVE-N531 about every four weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving hand grip strength of a subject, comprising administering to the subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein the subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, the present disclosure provides a method for improving hand grip strength of a population of subjects, comprising independently 22 of 176 13004462v1Attorney Docket No.: 2010581-1542 administering to each subject WVE-N531 about every two weeks at a dose equivalent to about 10 mg / kg WVE-N531 free acid form, wherein each subject is suffering from or susceptible to DMD and is amenable to exon 53 skipping. In some embodiments, hand grip strength for both hands are improved. In some embodiments, an improvement is or comprises an improvement relative to baseline. In some embodiments, an improvement is or comprises improvement relative to an earlier time point of dosing (e.g., week 48 vs. week 24).

[0037] In some embodiments, an earlier time point of dosing is about 6 weeks on administration. In some embodiments, an earlier time point of dosing is about 24 weeks on administration. In some embodiments, an earlier time point of dosing is about 48 weeks on administration. In some embodiments, an earlier time point of dosing is after receiving about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 doses.

[0038] In some embodiments, two or more doses are administered, each independently of an amount as described herein, e.g., equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, about 2-10 doses are administered. In some embodiments, 10 or more doses are administered. In some embodiments, each dose is about the same.

[0039] In some embodiments, two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) doses are administered about weekly. In some embodiments, two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) doses are administered about every two weeks. In some embodiments, two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) doses are administered about every 3, 4, 5, 6, 7, 8, 9 or 10 weeks. In some embodiments, doses are administered about weekly. In some embodiments, doses are administered about every two weeks. In some embodiments, doses are administered about every three weeks. In some embodiments, doses are administered about every four weeks. In some embodiments, doses are administered about monthly. In some embodiments, doses are administered about every 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, doses are administered about every two weeks, and then about every four weeks. In some embodiments, doses are administered about every two weeks, and then about monthly. In some embodiments, three or more doses are administered about every two weeks. In some embodiments, doses are first administered with shorter intervals (e.g., about every two weeks) and then administered with longer intervals (e.g., about every 3, 4, 5, 6, 7, 8, 9 or 10 weeks). In some embodiments, two or more doses, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses, are administered about every two weeks, followed by one or more doses each independently administered about 3, 4, 5, 6, 7, 8, 9 or 10 weeks from its previous doses. In some embodiments, about 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses are administered about every two weeks, and one or more doses are administered about every 3, 4, 5, 6, 7, 8, 9 or 10 weeks, or about monthly. In some embodiments, doses are administered about every two weeks for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 23 of 176 13004462v1Attorney Docket No.: 2010581-1542 14, 15, or 16 or more weeks from first dose and then doses are administered about every 3, 4, 5, 6, 7, 8, 9 or 10 weeks. In some embodiments, doses are administered every two weeks for about 4, 8, 12, or 16 or more weeks from first dose and then doses are administered about every 4 weeks. In some embodiments, doses are administered every two weeks for about 4, 8, 12, or 16 or more weeks from first dose and then doses are administered about monthly. In some embodiments, doses are first administered about every two weeks, and then after about 4 weeks, 8 weeks, 12 weeks, or 16 weeks from first dose, doses are administered about every four weeks or about monthly. In some embodiments, each dose is independently about the same, e.g., about 10 mg / kg.

[0040] In some embodiments, WVE-N531 or a composition thereof is administered intravenously. In some embodiments, WVE-N531 or a composition thereof is administered intramuscularly. In some embodiments, a dose is administered as an about 20-60, e.g., about 20, 25, 30, 35, 40, 45, 50, 55 or 60, minutes intravenous infusion. In some embodiments, infusion is administered with about 1, 2, 3, or 4 hours. In some embodiments, if a dose is missed, it may be administered as soon as possible after the scheduled dose.

[0041] Among other things, the present disclosure provides technologies for preventing or treating DMD in a subject susceptible thereto or suffering therefrom, wherein the subject has a DMD mutation that is amenable to exon 53 skipping. In some embodiments, a subject is a pediatric subject. In some embodiments, a subject is boy subject. In some embodiments, a subject is no younger than 5 years old. In some embodiments, a subject is no older than 18 years old. In some embodiments, a subject meets one or more or all the inclusion criteria described in the Examples. In some embodiments, a subject is selected based on one or more or all the inclusion and exclusion criteria described in the Examples. In some embodiments, a subject is ambulatory. In some embodiments, a subject is a male, ambulatory and is about 5-11 years old. In some embodiments, a subject has not lost muscle. In some embodiments, a subject has not lost ambulation. In some embodiments, a method prevent DMD disease process cascade. In some embodiments, a method delays or prevent onset of DMD. In some embodiments, a subject has later stage DMD. In some embodiments, a subject is non-ambulatory. In some embodiments, a provided method improves cardiorespiratory function. In some embodiments, a subject is less than about 18 years of age. In some embodiments, a subject is less than about 12 years of age. In some embodiments, a subject is about 5-11 years of age. In some embodiments, a subject is more than about 11 years of age. In some embodiments, a subject is more than about 12 years of age. In some embodiments, a subject is more than about 15 years of age. In some embodiments, a subject is less than about 5 years of age (e.g., about 4, 3, 2, 1 year of age or younger). In some embodiments, a subject is about or less than about 4 years of age.

[0042] In some embodiments, WVE-N531 is administered at a dose that can achieve a clinically significant maximum plasma concentration. 24 of 176 13004462v1Attorney Docket No.: 2010581-1542

[0043] In some embodiments, a subject is administered a steroid. In some embodiments, a subject is administered a steroid at least about six months (or 24 weeks) prior to the first dose of WVE-N531 or a composition thereof. In some embodiments, a steroid is a corticosteroid. In some embodiments, a subject is administered a corticosteroid at least about six months (or 24 weeks) prior to the first dose of WVE-N531 or a composition thereof. In some embodiments, a subject is on a stable corticosteroid therapy regimen. In some embodiments, a corticosteroid is deflazacort. In some embodiments, a steroid is a glucocorticoid. In some embodiments, a subject is administered a glucocorticoid at least about six months (or 24 weeks) prior to the first dose of WVE-N531 or a composition thereof. In some embodiments, a subject is on a stable glucocorticoid therapy regimen. In some embodiments, a steroid is prednisone. In some embodiments, a subject is administered a steroid at least about one month prior to the first dose of WVE-N531 or a composition thereof.

[0044] In some embodiments, an oligonucleotide, e.g., WVE-N531, is utilized in combination with one or more other therapeutic agents. In some embodiments, a therapeutic agent is or comprises a steroid. In some embodiments, it is or comprises or veliflapon, difenpiramide, amopyroquine, cloricromen, alpelisib, benorilate, xipamide, fabomotizole and altizide, or a pharmaceutically acceptable salt thereof. See, e.g., WO 2023 / 156645. In some embodiments, an oligonucleotide and one or more other therapeutic agents are administered or delivered concurrently. In some embodiments, they are administered or delivered in the same composition. In some embodiments, an oligonucleotide is administered or delivered prior to or subsequently to one or more other therapeutic agents. In some embodiments, a subject is under the effects of an oligonucleotide when one or more other therapeutic agents are administered or delivered. In some embodiments, a subject is under the effects of one or more other therapeutic agents when an oligonucleotide is administered or delivered. In some embodiments, a subject is under the effects of an oligonucleotide and one or more other therapeutic agents at the same time.

[0045] In some embodiments, provided technologies (oligonucleotides, compositions, methods, doses, dosage regimens, etc.) provide one or more desired biological effects. In some embodiments, provided technologies provide exon 53 skipping. In some embodiments, provided technologies provide exon 53 skipping at clinically significant levels. In some embodiments, provided technologies increase levels of an internally truncated but functional DMD polypeptide, e.g., encoded by an exon 53-skipped DMD mRNA. In some embodiments, provided technologies provide one or more restored DMD functions, e.g., through truncated but functional DMD polypeptides. In some embodiments, provided technologies provide clinical benefits. In some embodiments, level of muscle inflammation is decreased. In some embodiments, level of muscle necrosis is decreased. In some embodiments, level of muscle fibrosis is decreased. In some embodiments, level of one or more inflammatory biomarkers, e.g., one or more inflammatory cytokines, e.g., MCP-1 and / or IL-6, is decreased. In some embodiments, disease progression is delayed, slowed or 25 of 176 13004462v1Attorney Docket No.: 2010581-1542 prevented. In some embodiments, disease progression is delayed, slowed or prevented as assessed by a 10 meter walk test. In some embodiments, provided technologies reduce loss of pulmonary function relative to baseline. In some embodiments, muscle weakness in a subject is delayed, slowed or prevented. In some embodiments, muscle mass loss in a subject is delayed, slowed or prevented. In some embodiments, a subject improves in a muscular dystrophin assessment. In some embodiments, a subject improves in a DMD assessment. In some embodiments, improvement is achieved in one or more functional assessments, including North Star Ambulatory Assessment (NSAA) 2.0, Performance of the Upper Limb (PUL) 2.0, four-stair climb, handheld myometry, and pulmonary function tests. In some embodiments, such assessments are performed in such an order. In some embodiments, an assessment is or comprise lower limb motor function by timed function tests (including 10-meter walk / run time, four-stair climb time, and time to rise from the floor). In some embodiments, an assessment is or comprises upper limb proximal strength assessed by handheld myometer. In some embodiments, an assessment is or comprises pulmonary function tests (peak flow rate [PFR], cough peak flow [CPF], and FVC). In some embodiments, improvements are achieved independently in one or more or all these assessments. In some embodiments, an assessment is one disclosed in the Examples. In some embodiments, an improvement is compared to a baseline. In some embodiments, an improvement is compared to prior to WVE-N531 administration. In some embodiments, an improvement is compared to predicted level, severity and / or function with WVE- N531 administration. In some embodiments, an improvement is compared to absence of WVE-N531 administration. In some embodiments, an improvement is compared to administration of a reference composition. In some embodiments, a reference composition is comparable to an administered WVE-N531 composition but does not contain WVE-N531 or contains less amount of WVE-N531.

[0046] In some embodiments, provided technologies reduce loss of ambulation relative to baseline.

[0047] In some embodiments, provided technologies provide increases from baseline in dystrophin levels of about 1% or more of normal levels after administration for a certain time period or after a certain number of doses. In some embodiments, it is about 1% or more. In some embodiments, it is about 2% or more. In some embodiments, it is about 3% or more. In some embodiments, it is about 4% or more. In some embodiments, it is about 5% or more. In some embodiments, it is about 6% or more. In some embodiments, it is about 7% or more. In some embodiments, it is about 8% or more. In some embodiments, it is about 9% or more. In some embodiments, it is about 10% or more. In some embodiments, it is about 11% or more. In some embodiments, it is about 12% or more. In some embodiments, it is about 13% or more. In some embodiments, it is about 14% or more. In some embodiments, it is about 15% or more. In some embodiments, it is about 16% or more. In some embodiments, it is about 17% or more. In some embodiments, it is about 18% or more. In some embodiments, it is about 19% or more. In some embodiments, it is about 20% or more. In some embodiments, a time period is about 10-60 weeks. In some 26 of 176 13004462v1Attorney Docket No.: 2010581-1542 embodiments, it is about 4 weeks. In some embodiments, it is about 8 weeks. In some embodiments, it is about 10 weeks. In some embodiments, it is about 12 weeks. In some embodiments, it is about 13 weeks. In some embodiments, it is about 14 weeks. In some embodiments, it is about 15 weeks. In some embodiments, it is about 16 weeks. In some embodiments, it is about 20 weeks. In some embodiments, it is about 24 weeks. In some embodiments, it is about 25 weeks. In some embodiments, it is about 28 weeks. In some embodiments, it is about 30 weeks. In some embodiments, it is about 32 weeks. In some embodiments, it is about 35 weeks. In some embodiments, it is about 36 weeks. In some embodiments, it is about 37 weeks. In some embodiments, it is about 38 weeks. In some embodiments, it is about 40 weeks. In some embodiments, it is about 45 weeks. In some embodiments, it is about 48 weeks. In some embodiments, it is about 49 weeks. In some embodiments, it is about 50 weeks. In some embodiments, it is about 72 weeks. In some embodiments, it is about 73 weeks. In some embodiments, it is about 74 weeks. In some embodiments, it is about 96 weeks. In some embodiments, it is about 97 weeks. In some embodiments, it is about 98 weeks or more. In some embodiments, it is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 months. In some embodiments, a certain number of doses is about 5 doses. In some embodiments, it is about 6 doses. In some embodiments, it is about 7 doses. In some embodiments, it is about 8 doses. In some embodiments, it is about 9 doses. In some embodiments, it is about 10 doses. In some embodiments, it is about 11 doses. In some embodiments, it is about 12 doses. In some embodiments, it is about 13 doses. In some embodiments, it is about 14 doses. In some embodiments, it is about 15 doses. In some embodiments, it is about 16 doses. In some embodiments, it is about 17 doses. In some embodiments, it is about 18 doses. In some embodiments, it is about 19 doses. In some embodiments, it is about 20 doses. In some embodiments, it is about 21 doses. In some embodiments, it is about 22 doses. In some embodiments, it is about 23 doses. In some embodiments, it is about 24 doses. In some embodiments, it is about 25 doses. In some embodiments, it is about 30 doses. In some embodiments, it is about 35 doses. In some embodiments, it is about 40 doses. In some embodiments, it is about 45 doses. In some embodiments, it is about 48 doses. In some embodiments, it is about 49 doses. In some embodiments, it is about 50 or more doses. In some embodiments, WVE-N531 is administered every two weeks. In some embodiments, WVE-N531 is administered about every two weeks. In some embodiments, WVE-N531 is administered about monthly.

[0048] In some embodiments, provided technologies do not lead to adverse events or low levels of adverse events, or no or low levels of severe and / or serious adverse events. In some embodiments, adverse events associated with provided technologies are tolerable or manageable.

[0049] Certain benefits and advantages of provided technologies are described in the Example(s).

[0050] In some embodiments, the present disclosure provides technologies for manufacturing WVE- N531. In some embodiments, the present disclosure provides technologies for manufacturing a WVE-N531 27 of 176 13004462v1Attorney Docket No.: 2010581-1542 drug substance. In some embodiments, the present disclosure provides technologies for manufacturing a WVE-N531 drug product. In some embodiments, the present disclosure provides technologies for characterizing WVE-N531 drug substance and / or product. In some embodiments, the present disclosure provides release specifications for characterizing WVE-N531 drug substance and / or product. In some embodiments, the present disclosure provides WVE-N531, compositions thereof, drug substances and / or products thereof manufactured by provided processes.

[0051] In some embodiment, delivery into satellite cells, and / or satellite cell renewal, activation and / or differentiation are utilized to assess DMD treatment efficacy. In some embodiments, delivery into satellite cells, and / or satellite cell renewal, activation and / or differentiation may be utilized to assess DMD treatment efficacy are utilized to monitor progress or efficacy of a treatment. In some embodiments, delivery into satellite cells, and / or satellite cell renewal, activation and / or differentiation are utilized to determine whether to administer an agent. In some embodiments, delivery into satellite cells, and / or satellite cell renewal, activation and / or differentiation are utilized to determine whether to continue administration of an agent (e.g., WVE-N531).

[0052] In some embodiments, reduction of serum creatine kinase level is utilized to assess a DMD treatment. In some embodiments, the present disclosure provides a method for treating DMD as described herein, comprising assessing serum creatine kinase level after one or more doses of WVE-N531 are administered or delivered. In some embodiments, reduction of serum creatine kinase is observed and administration of WVE-N531 is continued, e.g., according to a regimen as described herein. In some embodiments, reduction of serum aspartate aminotransferase level is utilized to assess a DMD treatment. In some embodiments, the present disclosure provides a method for treating DMD as described herein, comprising assessing serum aspartate aminotransferase level after one or more doses of WVE-N531 are administered or delivered. In some embodiments, reduction of serum aspartate aminotransferase is observed and administration of WVE-N531 is continued, e.g., according to a regimen as described herein.

[0053] In some embodiments, reversal of muscle damage, increases in muscle fiber organization, increases in uniformity of myofibers, and / or maturation of muscle tissue is utilized to assess a DMD treatment. In some embodiments, the present disclosure provides a method for treating DMD as described herein, comprising assessing muscle fiber organization, uniformity of myofibers, and / or maturation of muscle tissue after one or more doses of WVE-N531 are administered or delivered. In some embodiments, reduction of muscle inflammation is utilized to assess a DMD treatment. In some embodiments, the present disclosure provides a method for treating DMD as described herein, comprising assessing level of muscle inflammation after one or more doses of WVE-N531 are administered or delivered. In some embodiments, reduction of muscle necrosis is utilized to assess a DMD treatment. In some embodiments, the present disclosure provides a method for treating DMD as described herein, comprising assessing level of muscle 28 of 176 13004462v1Attorney Docket No.: 2010581-1542 necrosis after one or more doses of WVE-N531 are administered or delivered. In some embodiments, reduction of muscle fibrosis is utilized to assess a DMD treatment. In some embodiments, the present disclosure provides a method for treating DMD as described herein, comprising assessing level of muscle fibrosis after one or more doses of WVE-N531 are administered or delivered. In some embodiments, reduction of one or more inflammatory biomarkers, e.g., one or more inflammatory cytokines, e.g., MCP- 1 and / or IL-6, is utilized to assess a DMD treatment. In some embodiments, the present disclosure provides a method for treating DMD as described herein, comprising assessing level of one or more inflammatory biomarkers, e.g., one or more inflammatory cytokines, e.g., MCP-1 and / or IL-6, after one or more doses of WVE-N531 are administered or delivered. In some embodiments, improvement in TTR is utilized to assess a DMD treatment. In some embodiments, the present disclosure provides a method for treating DMD as described herein, comprising assessing TTR after one or more doses of WVE-N531 are administered or delivered. In some embodiments, improvement in NSAA total score is utilized to assess a DMD treatment. In some embodiments, the present disclosure provides a method for treating DMD as described herein, comprising assessing NSAA total score after one or more doses of WVE-N531 are administered or delivered. In some embodiments, improvement in grip strength is utilized to assess a DMD treatment. In some embodiments, the present disclosure provides a method for treating DMD as described herein, comprising assessing grip strength after one or more doses of WVE-N531 are administered or delivered. BRIEF DESCRIPTION OF THE DRAWING

[0054] Figure 1. Certain information of a clinical trial.

[0055] Figure 2. Provided technologies can provide high and consistent dystrophin expression. In an assessment, dystrophin expression of up to 14% with high consistency across participants was observed. Shown are percentage dystrophin expression across each patient after 24 weeks of treatment. Dystrophin levels adjusted and unadjusted for muscle content are shown. Dystrophin expression was quantified from two isoforms consistent with those observed in Becker patients who display milder disease. 89% of ambulatory participants achieve muscle content-adjusted dystrophin levels of at least 5%. * Excluded from prespecified mean analysis of ambulatory patients. Muscle content adjustment was done using the formula: MHC-normalized dystrophin / (total myofiber area / total area of biopsy section). Graph shows all patients (including non-ambulatory) with appropriate biopsy sample; dystrophin measured by Western Blot (AB15277).

[0056] Figure 3. WVE-N531 treatment led to substantial decreases in muscle-related biomarkers. (A): reduction of CK (Creatine Kinase). (B): reduction of AST (Aspartate Aminotransferase).

[0057] Figure 4. Certain information of a clinical trial. CK: creatine kinase; Q2W: every 2 weeks; SAE: serious adverse event. 29 of 176 13004462v1Attorney Docket No.: 2010581-1542

[0058] Figure 5. Provided technologies can provide high and consistent dystrophin expression. In an assessment, dystrophin expression was assessed after 24 weeks and after 48 weeks of treatment. Relative dystrophin levels after 24 weeks and after 48 weeks of treatment were scaled to range from 0 to 1 and plotted on the left graph. Pearson correlation (r) is presented inset on left graph. Average dystrophin levels (% of normal) between 24 weeks and 48 weeks of treatment for certain participants are shown in the right graph. Muscle content adjustment was done using the formula: MHC-normalized dystrophin / (total myofiber area / total area of biopsy section).

[0059] Figure 6. WVE-N531 treatment led to reversal of muscle damage. Hematoxylin and eosin (H&E) stained sections of muscle biopsies are presented at 20x magnification. (A): exemplary stained sections from participants after 24 weeks of treatment. (B): exemplary stained sections from same participants after 48 weeks of treatment.

[0060] Figure 7. WVE-N531 treatment led to decreased muscle inflammation (left graphs) and necrosis (right graphs). Severity scoring (x-axis) based on Schafer et al. Toxicol Pathol. 2018;46(3):256- 265. Severity scores: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked.

[0061] Figure 8. WVE-N531 treatment led to decreased levels of inflammatory cytokines. Reduction in MCP-1 levels shown in left graph. Reduction in IL-6 levels shown in right graph. Data are mean ± standard error (SE).

[0062] Figure 9. WVE-N531 treatment led to decreased levels of CK. (A): reduction of CK (n=11). (B): change in CK in WVE-N531-treated cohort (n=10; all ambulatory) versus DMD natural history cohort. *p<0.05; **p<0.01; ***p<0.001.

[0063] Figure 10. WVE-N531 treatment supported muscle maturation. (A): reduction of myogenic stem cell density. PAX7-positive cells / mm2quantified using Pax7 immunohistochemistry (IHC) with HALO. Data are mean ± SE (n=9). (B): reduction of internalized nuclei. Internalized nuclei quantified using 6 random fields in H&E stained sections. Data are mean ± SE (n=9 for week 24; n=8 for week 48). **p<0.01 by two-tailed Welch’s test.

[0064] Figure 11. WVE-N531 treatment decreased fibrotic muscle. Mean (± SE (n=7)) fibrotic muscle shown in left graph. Percent fibrotic muscle per individual shown in right graph. Fibrosis was stained with trichrome stain and analyzed with HALO. **p<0.01.

[0065] Figure 12. WVE-N531 treatment slowed disease progression as measured by time-to-rise (TTR). Data are mean ± SE. *p<0.05.

[0066] Figure 13. WVE-N531 treatment slowed disease progression as measured by North Star Ambulatory Assessment (NSAA). Data are mean ± SE.

[0067] Figure 14. WVE-N531 treatment increased grip strength. Grip strength was measured using handheld myometry. Data are mean ± SE (n=11). 30 of 176 13004462v1Attorney Docket No.: 2010581-1542 DEFINITIONS

[0068] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.

[0069] As used herein in the present disclosure, unless otherwise clear from context, (i) the term “a” or “an” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising”, “comprise”, “including” (whether used with “not limited to” or not), and “include” (whether used with “not limited to” or not) may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; (iv) the term “another” may be understood to mean at least an additional / second one or more; and (v) where ranges are provided, endpoints are included.

[0070] Unless otherwise specified, description of oligonucleotides and elements thereof (e.g., base sequence, sugar modifications, internucleotidic linkages, linkage phosphorus stereochemistry, etc.) is from 5’ to 3’. Unless otherwise specified, oligonucleotides described herein may be provided and / or utilized in various forms including salt forms, particularly pharmaceutically acceptable salt forms. As those skilled in the art will appreciate after reading the present disclosure, in some embodiments, oligonucleotides may be provided as salts such as sodium or potassium salts. As those skilled in the art will appreciate, in some embodiments, individual oligonucleotides within a composition may be considered to be of the same constitution and / or structure even though, within such composition (e.g., a liquid composition), particular such oligonucleotides might be in different form(s) including salt form(s) (and may be dissolved and the oligonucleotide chain may exist as an anion form when, e.g., in a liquid composition) at a particular moment in time. For example, those skilled in the art will appreciate that, at a given pH, individual internucleotidic linkages along an oligonucleotide chain may be in an acid (H) form, or in one of a plurality of possible salt forms (e.g., a sodium salt, or a salt of a different cation, depending on which ions might be present in the preparation or composition)), and will understand that, so long as their acid forms (e.g., replacing all cations, if any, with H) are of the same constitution and / or structure, such individual oligonucleotides may properly be considered to be of the same constitution and / or structure.

[0071] Approximately: As used herein, the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art. In some embodiments, the terms “approximately” or “about” in reference to a number are generally taken to include 31 of 176 13004462v1Attorney Docket No.: 2010581-1542 numbers that fall within a range of 5%, 10%, 15%, 20%, 25%, or 30%, in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context. In some embodiments, a range is ±5%. In some embodiments, a range is ±10%.

[0072] Dosing regimen: As used herein, a “dosing regimen” or “therapeutic regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.

[0073] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a controlled therapeutic effect when administered to a relevant population.

[0074] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, 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.

[0075] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

[0076] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and 32 of 176 13004462v1Attorney Docket No.: 2010581-1542 lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, a provided compound comprises more than one acid groups, for example, a provided oligonucleotide may comprise two or more acidic groups (e.g., in natural phosphate linkages and / or modified internucleotidic linkages). In some embodiments, a pharmaceutically acceptable salt, or generally a salt, of such a compound comprises two or more cations, which can be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or generally, a salt), each acidic group having sufficient acidity independently exists as its salt form (e.g., in an oligonucleotide comprising natural phosphate linkages and phosphorothioate internucleotidic linkages, each of the natural phosphate linkages and phosphorothioate internucleotidic linkages independently exists as its salt form). In some embodiments, a pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide. In some embodiments, a pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide, wherein each acidic linkage, e.g., each natural phosphate linkage and phosphorothioate internucleotidic linkage, exists as a sodium salt form (all sodium salt).

[0077] Subject: As used herein, the term “subject” or “test subject” refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. In some embodiments, a subject may be suffering from, and / or susceptible to a disease, disorder, and / or condition. In some embodiments, a subject is a human subject.

[0078] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0079] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0080] Susceptible to: An individual who is “susceptible to” a disease, disorder, and / or condition is one who has a higher risk of developing the disease, disorder, and / or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, 33 of 176 13004462v1Attorney Docket No.: 2010581-1542 disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0081] Systemic: The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” as used herein have their art-understood meaning referring to administration of a compound or composition such that it enters the recipient’s system.

[0082] Therapeutic agent: As used herein, the phrase “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0083] Therapeutically effective amount: In some embodiments, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. In some embodiments, a single dose is an infusion, which may take up to one or more hours.

[0084] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0085] Chirally controlled oligonucleotide composition: The terms “chirally controlled 34 of 176 13004462v1Attorney Docket No.: 2010581-1542 (stereocontrolled or stereodefined) oligonucleotide composition”, “chirally controlled (stereocontrolled or stereodefined) nucleic acid composition”, and the like, as used herein, refers to a composition that comprises a plurality of oligonucleotides (or nucleic acids, chirally controlled oligonucleotides or chirally controlled nucleic acids) which share 1) a common base sequence, 2) a common pattern of backbone linkages; 3) a common pattern of backbone chiral centers, and 4) a common pattern of backbone phosphorus modifications (oligonucleotides of a particular type), wherein the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotidic linkages (chirally controlled internucleotidic linkages, whose chiral linkage phosphorus is Rp or Sp, not a random Rp and Sp mixture as non-chirally controlled internucleotidic linkages). Level of the plurality of oligonucleotides (or nucleic acids) in a chirally controlled oligonucleotide composition is non-random (pre-determined, controlled). Chirally controlled oligonucleotide compositions are typically prepared through chirally controlled oligonucleotide preparation to stereoselectively form one or more chiral internucleotidic linkages (e.g., using chiral auxiliaries as exemplified in the present disclosure, compared to non-chirally controlled (stereorandom, non-stereoselective, racemic) oligonucleotide synthesis such as traditional phosphoramidite-based oligonucleotide synthesis using no chiral auxiliaries or chiral catalysts to purposefully control stereoselectivity). A chirally controlled oligonucleotide composition is enriched, relative to a substantially racemic preparation of oligonucleotides having the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications, for oligonucleotides of the plurality. In some embodiments, a chirally controlled oligonucleotide composition comprises a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications, wherein it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence, pattern of backbone linkages, and pattern of backbone phosphorus modifications, for oligonucleotides of the particular oligonucleotide type. As one having ordinary skill in the art readily appreciates, such enrichment can be characterized in that compared to a substantially racemic preparation, at each chirally controlled internucleotidic linkage, a higher level of the linkage phosphorus has the desired configuration. In some embodiments, each chirally controlled internucleotidic linkage independently has a diastereopurity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to its chiral linkage phosphorus. In some embodiments, each independently has a diastereopurity of at least 90%. In some embodiments, each independently has a diastereopurity of at least 95%. In some embodiments, each independently has a diastereopurity of at least 97%. In some embodiments, each independently has a diastereopurity of at least 98%. In some embodiments, oligonucleotides of a plurality have the same constitution. In some embodiments, oligonucleotides of a plurality have the same constitution and stereochemistry, and are structurally identical. 35 of 176 13004462v1Attorney Docket No.: 2010581-1542

[0086] In some embodiments, the plurality of oligonucleotides in a chirally controlled oligonucleotide composition share the same base sequence, the same, if any, nucleobase, sugar, and internucleotidic linkage modifications, and the same stereochemistry (Rp or Sp) independently at linkage phosphorus chiral centers of one or more chirally controlled internucleotidic linkages, though stereochemistry of certain linkage phosphorus chiral centers may differ. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80- 100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition are oligonucleotides of the plurality. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence are oligonucleotides of the plurality. In some embodiments, about 0.1%- 100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications are oligonucleotides of the plurality. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%- 100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition, or of all oligonucleotides in a composition that share a common base sequence (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share a common base sequence, a common patter of base modifications, a common pattern of sugar modifications, a common pattern of internucleotidic linkage types, and / or a common pattern of internucleotidic linkage modifications 36 of 176 13004462v1Attorney Docket No.: 2010581-1542 (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share the same constitution, are oligonucleotides of the plurality. In some embodiments, a percentage is at least (DP)NCI, wherein DP is a percentage selected from 85%-100%, and NCI is the number of chirally controlled internucleotidic linkage. In some embodiments, DP is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, DP is at least 85%. In some embodiments, DP is at least 90%. In some embodiments, DP is at least 95%. In some embodiments, DP is at least 96%. In some embodiments, DP is at least 97%. In some embodiments, DP is at least 98%. In some embodiments, DP is at least 99%. In some embodiments, DP reflects diastereopurity of linkage phosphorus chiral centers chirally controlled internucleotidic linkages. In some embodiments, diastereopurity of a linkage phosphorus chiral center of a internucleotidic linkage may be typically assessed using an appropriate dimer comprising such an internucleotidic linkage and the two nucleoside units being linked by the internucleotidic linkage. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotidic linkages. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90- 100%, 95-100%, 50%-90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of chiral internucleotidic linkages. In some embodiments, each chiral internucleotidic linkage is a chiral controlled internucleotidic linkage, and the composition is a completely chirally controlled oligonucleotide composition. In some embodiments, not all chiral internucleotidic linkages are chiral controlled internucleotidic linkages, and the composition is a partially chirally controlled oligonucleotide composition. In some embodiments, a chirally controlled oligonucleotide composition comprises predetermined levels of individual oligonucleotide or nucleic acid types. For instance, in some embodiments a chirally controlled oligonucleotide composition comprises one oligonucleotide type at a predetermined level (e.g., as described above). In some embodiments, a chirally controlled oligonucleotide composition comprises more than one oligonucleotide type, each independently at a predetermined level. In some embodiments, a chirally controlled oligonucleotide composition comprises multiple oligonucleotide types, each independently at a predetermined level. In some embodiments, a chirally controlled oligonucleotide composition is a composition of oligonucleotides of an oligonucleotide type, which composition comprises a predetermined level of a plurality of oligonucleotides of the oligonucleotide type. 37 of 176 13004462v1Attorney Docket No.: 2010581-1542 DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0087] Among other things, the present disclosure provides methods for treating muscular dystrophy, e.g., treatment of subjects having a mutation of the DMD gene that is amenable to exon 53 skipping, by administering an amount of WVE-N531 described herein to the subject. Dystrophin plays a vital role in muscle function, and various muscle-related diseases are characterized by mutated forms of this gene. In certain embodiments, methods described herein may be used for inducing exon skipping in transcripts, e.g., mRNA, of mutated forms of a human dystrophin gene, such as the mutated dystrophin gene forms found in muscular dystrophin, e.g., DMD and BMD. In some embodiments, provided technologies provide exon 53-skipped DMD mRNA. In some embodiments, present disclosure provide DMD polypeptides encoded by such DMD mRNA. In some embodiments, production of such DMD polypeptides restore, in some embodiments partially and in some embodiments fully, one or more DMD functions.

[0088] WVE-N531 can induce exon 53 skipping and dystrophin protein restoration. It is reported that mutations in the DMD gene that are amenable to exon 53 skipping occur in approximately 8% of subjects with DMD. As those skilled in the art appreciate, various DMD mutations amenable to exon 53 skipping have been reported, and various technologies are available to determine if a mutation is amenable to exon 53 skipping. In some embodiments, DMD mutations such as Δ3-52, Δ4-52, Δ5-52, Δ6-52, Δ9-52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17-52, Δ19-52, Δ21-52, Δ23-52, Δ24-52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32-52, Δ33-52, Δ34-52, Δ35-52, Δ36-52, Δ37-52, Δ38-52, Δ39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ45-52, Δ47-52, Δ48-52, Δ49-52, Δ50-52, Δ51-52, Δ52-52, Δ52, Δ54-58, Δ54-61, Δ54-63, Δ54-64, Δ54-66, Δ54-76, Δ54-77, etc. are amenable to exon 53 skipping. In some embodiments, a mutation is Δ45-52. In some embodiments, a mutation is Δ47-52. In some embodiments, a mutation is Δ48-52. In some embodiments, a mutation is Δ49-52. In some embodiments, a mutation is Δ50-52. In some embodiments, a mutation is Δ51-52. In some embodiments, a mutation is Δ52-52. In some embodiments, a mutation comprises Δ52. In some embodiments, a mutation is Δ52. In some embodiments, WVE-N531 mediates effective exon 53 skipping of DMD to restore the reading frame so that a shorter but at least partially functional dystrophin can be produced - a resulting dystrophin protein is not necessarily the “wild-type” form of dystrophin, but is a truncated, yet functional form of dystrophin and provides increased level of one or more dystrophin functions compared to absence of WVE-N531 / exon 53 skipping.

[0089] In some embodiments, WVE-N531 provides high muscle concentrations, e.g., at about or at least about 5 µg / g, 8 µg / g, 10 µg / g, 15 µg / g, 20 µg / g, 25 µg / g, 30 µg / g, 33 µg / g, 35 µg / g, 40 µg / g, 45 µg / g, 50 µg / g, 55 µg / g, 60 µg / g, 65 µg / g, 70 µg / g, 75 µg / g, 80 µg / g, 85 µg / g, 90 µg / g, etc., e.g., in various muscle. In some embodiments, such muscle concentrations are provided at a time point of about 6 weeks after an initial dose and / or after about 3 doses, wherein each dose is about 10 mg / kg WVE-N531. In some 38 of 176 13004462v1Attorney Docket No.: 2010581-1542 embodiments, WVE-N531 provides exon 53 skipping, e.g., at levels of about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, etc. In some embodiments, exon skipping is provided at a time point of about 6 weeks after an initial dose and / or after about 3 doses, wherein each dose is about 10 mg / kg WVE-N531 (which dose is significantly lower than golodirsen and / or viltolarsen).

[0090] In some embodiments, the present disclosure provides methods comprising using WVE-N531 to induce exon 53 skipping in DMD in muscle cells. In some embodiments, the present disclosure provides methods comprising using WVE-N531 to increase levels of functional dystrophin protein in muscle cells. It is useful in the prophylaxis and treatment of muscular dystrophy, especially those forms of muscular dystrophy that are amenable to exon 53 skipping. Among other things, methods described herein provide improved treatment options for subjects with muscular dystrophy and offer significant and practical advantages over alternate methods, e.g. methods using other oligonucleotide therapeutics.

[0091] In some embodiments, the present disclosure relates to improved methods for treating muscular dystrophy by inducing exon skipping in a subject. In some embodiments, exon 53 skipping in DMD in a subject induced by administering an effective amount of WVE-N531 as described herein. In some embodiments, the present disclosure relates to improved methods for treating muscular dystrophy, by restoring / increasing levels of dystrophin polypeptides and / or activities thereof (e.g., by restoring / increasing levels of functional dystrophin protein) in a subject.

[0092] In some embodiments, the present disclosure provides a method for treating DMD in a subject having a mutation in the DMD gene that is amenable to exon 53 skipping, comprising administering an effective dose amount of WVE-N531 (optionally as part of a composition, e.g., a pharmaceutical formulation or dosage form), e.g., equivalent to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg WVE-N531 free acid form, at a suitable frequency (e.g., weekly, biweekly, every three weeks, every four weeks, every five weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks or every 10 weeks).

[0093] In some embodiments, the present disclosure provides a method for slowing progression of DMD in a subject having a mutation in the DMD gene that is amenable to exon 53 skipping, comprising administering an effective dose amount of WVE-N531 (optionally as part of a composition, e.g., a pharmaceutical formulation or dosage form), e.g., equivalent to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg WVE-N531 free acid form, at a suitable frequency (e.g., weekly, biweekly, every three weeks, every four weeks, every five weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks or every 10 weeks).

[0094] In some embodiments, the present disclosure provides a method for treating DMD in a subject suffering therefrom, wherein the subject has a mutation in the DMD gene that is amenable to exon 53 skipping, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is 39 of 176 13004462v1Attorney Docket No.: 2010581-1542 independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

[0095] In some embodiments, the present disclosure provides a method for slowing progression of DMD in a subject suffering therefrom, wherein the subject has a mutation in the DMD gene that is amenable to exon 53 skipping, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

[0096] In some embodiments, the present disclosure provides a method for providing DMD exon skipping in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for increasing level of exon 53- skipped DMD transcripts in a subject, comprising administering to the subject one or more doses of WVE- N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10- 15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE- N531 free acid form. In some embodiments, the present disclosure provides a method for restoring DMD RNA reading frame in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for increasing level of a truncated DMD polypeptide in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for providing increased level of a DMD function in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, a truncated DMD polypeptide is encoded by an exon53- skipped DMD mRNA. In some embodiments, such an mRNA may lack one or more additional exons in addition to exon 53. In some embodiments, a subject has a mutation in the DMD gene that is amenable to exon 53 skipping. In some embodiments, a subject is suffering from or susceptible to a muscular dystrophy, e.g., DMD. In some embodiments, each dose of WVE-N531 is independently administered in a pharmaceutical composition independently comprising WVE-N531 and pharmaceutically acceptable 40 of 176 13004462v1Attorney Docket No.: 2010581-1542 carrier, wherein WVE-N531 in each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

[0097] In some embodiments, the present disclosure provides a method for providing decreased muscle inflammation in a subject, comprising administering to the subject one or more doses of WVE- N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10- 15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE- N531 free acid form. In some embodiments, the present disclosure provides a method for providing decreased muscle necrosis in a subject, comprising administering to the subject one or more doses of WVE- N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10- 15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE- N531 free acid form. In some embodiments, the present disclosure provides a method for providing decreased levels of one or more inflammatory biomarkers in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for providing decreased levels of serum creatine kinase in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for providing decreased muscle fibrosis in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for reversing muscle fibrosis in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for improving muscle health in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for increasing transition from regenerative to mature muscle in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is 41 of 176 13004462v1Attorney Docket No.: 2010581-1542 independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for slowing increase in TTR in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for slowing increase in NSAA total score in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for increasing grip strength in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, a subject has a mutation in the DMD gene that is amenable to exon 53 skipping. In some embodiments, a subject is suffering from or susceptible to a muscular dystrophy, e.g., DMD. In some embodiments, each dose of WVE-N531 is independently administered in a pharmaceutical composition independently comprising WVE-N531 and pharmaceutically acceptable carrier, wherein WVE-N531 in each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

[0098] WVE-N531 can be administered in various forms. For example, in many embodiments, WVE- N531 are administered as salt forms. In some embodiments, there is a single form in a composition. In some embodiments, there is a single form in a dose. In some embodiments, a form is a pharmaceutically acceptable salt form. In some embodiments, a form is WVE-N531 hexadecasodium salt. In some embodiments, there are two or more forms in a composition. In some embodiments, there are two or more forms in a dose. In some embodiments, each of the two or more forms is independently a pharmaceutically acceptable salt form. In some embodiments, one of them is WVE-N531 hexadecasodium salt. In some embodiments, a composition is a liquid composition comprising dissolved WVE-N531, e.g., in phosphate buffered saline. In some embodiments, there are one or more types of cations in a solution. In some embodiments, each is independently H+or a metal cation. In some embodiments, a metal cation is Na+. In some embodiments, a metal cation is K+. In some embodiments, each metal cation is independently Na+or K+.

[0099] In some embodiments, a dose is about 1-20 mg / kg. In some embodiments, a dose is about 1-5 mg / kg. In some embodiments, a dose is about 5-20 mg / kg. In some embodiments, a dose is about 5-10 42 of 176 13004462v1Attorney Docket No.: 2010581-1542 mg / kg. In some embodiments, a dose is about 10-20 mg / kg. In some embodiments, a dose is about 10-15 mg / kg. In some embodiments, a dose is about 15-20 mg / kg. In some embodiments, a dose is about 1 mg / kg. In some embodiments, a dose is about 1 mg / kg. In some embodiments, a dose is about 2 mg / kg. In some embodiments, a dose is about 3 mg / kg. In some embodiments, a dose is about 4 mg / kg. In some embodiments, a dose is about 5 mg / kg. In some embodiments, a dose is about 6 mg / kg. In some embodiments, a dose is about 7 mg / kg. In some embodiments, a dose is about 8 mg / kg. In some embodiments, a dose is about 9 mg / kg. In some embodiments, a dose is about 10 mg / kg. In some embodiments, a dose is about 11 mg / kg. In some embodiments, a dose is about 12 mg / kg. In some embodiments, a dose is about 13 mg / kg. In some embodiments, a dose is about 14 mg / kg. In some embodiments, a dose is about 15 mg / kg. In some embodiments, a dose is about 16 mg / kg. In some embodiments, a dose is about 17 mg / kg. In some embodiments, a dose is about 18 mg / kg. In some embodiments, a dose is about 19 mg / kg. In some embodiments, a dose is about 20 mg / kg.

[0100] In some embodiments, a dose is administered as a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is a WVE-N531 solution as described herein, e.g., in a buffer. In some embodiments, a pharmaceutically acceptable carrier is isotonic. In some embodiments, it is a WVE-N531 solution in phosphate buffered saline as described herein.

[0101] In some embodiments, two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 or more) doses are administered. In some embodiments, about 10 or more doses are administered. In some embodiments, about 20 or more doses are administered. In some embodiments, about 30 or more doses are administered. In some embodiments, about 40 or more doses are administered. In some embodiments, about 50 or more doses are administered. In some embodiments, about 60 or more doses are administered. In some embodiments, about 70 or more doses are administered. In some embodiments, about 80 or more doses are administered. In some embodiments, about 90 or more doses are administered. In some embodiments, about 100 or more doses are administered. In some embodiments, about 200 or more doses are administered. In some embodiments, about 500 or more doses are administered. In some embodiments, each does is independently about 1-20 mg / kg as described herein. In some embodiments, each dose is independently administered as a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier as described herein. In some embodiments, two or more doses are about the same mg / kg. In some embodiments, each dose is about the same mg / kg. In some embodiments, pharmaceutical compositions administered for two or more doses are about the same in terms of components and their relative amounts. In some embodiments, pharmaceutical compositions administered for all doses are about the same in terms of components and their relative amounts. In some 43 of 176 13004462v1Attorney Docket No.: 2010581-1542 embodiments, one or more earlier doses are independently lower than one or more later doses. In some embodiments, one or more earlier doses independently are higher than one or more later doses.

[0102] In some embodiments, doses are administered about weekly, or about every 2, 3, 4, 5, 6, 7, 8, 9, or 10, or about every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months. In some embodiments, WVE-N531 are administered about every two weeks wherein each dose is independently as described herein. In some embodiments, WVE-N531 are administered about every four weeks wherein each dose is independently as described herein. In some embodiments, WVE-N531 are administered about every four or more weeks wherein each dose is independently as described herein. In some embodiments, each dose interval (time between a dose and its preceding or subsequent dose) is about the same. In some embodiments, one or more earlier dose intervals are independently longer than one or more later ones. In some embodiments, one or more earlier dose intervals are independently shorter than one or more later ones.

[0103] In some embodiments, two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses are administered about every two weeks. In some embodiments, 3 or more doses are administered about every two weeks. In some embodiments, 4 or more doses are administered about every two weeks. In some embodiments, 5 or more doses are administered about every two weeks. In some embodiments, 6 or more doses are administered about every two weeks. In some embodiments, 7 or more doses are administered about every two weeks. In some embodiments, 8 or more doses are administered about every two weeks. In some embodiments, 9 or more doses are administered about every two weeks. In some embodiments, 10 or more doses are administered about every two weeks. In some embodiments, 11 or more doses are administered about every two weeks. In some embodiments, 3 or more doses are administered about every two weeks. In some embodiments, 12 or more doses are administered about every two weeks. In some embodiments, 13 or more doses are administered about every two weeks. In some embodiments, 14 or more doses are administered about every two weeks. In some embodiments, 15 or more doses are administered about every two weeks. In some embodiments, 16 or more doses are administered about every two weeks. In some embodiments, 17 or more doses are administered about every two weeks. In some embodiments, 18 or more doses are administered about every two weeks. In some embodiments, 19 or more doses are administered about every two weeks. In some embodiments, 20 or more doses are administered about every two weeks. In some embodiments, 21 or more doses are administered about every two weeks. In some embodiments, 22 or more doses are administered about every two weeks. In some embodiments, 23 or more doses are administered about every two weeks. In some embodiments, 24 or more doses are administered about every two weeks. In some embodiments, 25 or more doses are administered about every two weeks. In some embodiments, 26 or more doses are administered about every two weeks. In some embodiments, 27 or more doses are administered about every two weeks. In some embodiments, 28 or more doses are 44 of 176 13004462v1Attorney Docket No.: 2010581-1542 administered about every two weeks. In some embodiments, 29 or more doses are administered about every two weeks. In some embodiments, 30 or more doses are administered about every two weeks. In some embodiments, 20 or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses are administered about every two weeks and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) are administered about every four weeks. In some embodiments, 20 or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses are administered about every two weeks and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) are administered about monthly.

[0104] In some embodiments, “about” is + / − 1% (99% to 101% of a value). In some embodiments, it is + / − 2%. In some embodiments, it is + / − 3%. In some embodiments, it is + / − 4%. In some embodiments, it is + / − 5%. In some embodiments, it is + / − 6%. In some embodiments, it is + / − 7%. In some embodiments, it is + / − 8%. In some embodiments, it is + / − 9%. In some embodiments, it is + / − 10%.

[0105] Unless specified otherwise, amounts, concentrations, doses, etc., of WVE-N531 are of the free acid form.

[0106] Amounts, concentrations, doses, etc. are typically purity corrected. In some embodiments, purity is measured by IP-RP-UPLC as described herein based on % area.

[0107] Vyondys 53TM(golodirsen) is an oligonucleotide of the phosphorodiamidate morpholino oligomer (PMO) subclass intended to increase dystrophin production via exon-skipping. Golodirsen was approved in 2019 in the US for the treatment of patients who have a confirmed mutation of the DMD gene that is amenable to exon 53 skipping. Approval was based on a mean change from baseline in dystrophin levels of 0.92% (standard deviation [SD] 1.01) of normal levels following 48 weeks of treatment at 30 mg / kg / week. Viltepso™ (viltolarsen), another PMO subclass exon skipping ASO, was approved in the US in 2020 for the treatment of DMD patients amenable to exon 53 skipping. The approval was also based on an observed increase in dystrophin production with a mean increase of 5.3% (SD 4.5) of normal levels measured following 25 weeks of treatment at 80 mg / kg / week. Both golodirsen and viltolarsen were approved via the US accelerated approval mechanism on the basis of changes in the surrogate endpoint of dystrophin production, and the clinical benefit of either drug has not yet been demonstrated in patients with DMD at approval. Continued approval in the US is contingent upon verification of a clinical benefit in confirmatory trials. At the time of the present invention neither drug is approved in the United Kingdom or European Union (EU).

[0108] Among other things, provided technologies provide various advantages compared to golodirsen and / or viltolarsen, e.g., lower single doses, lower total doses, less frequent administration, improved exon 53 skipping in DMD, higher level of functional DMD polypeptides, improved clinical results, etc. Dystrophin 45 of 176 13004462v1Attorney Docket No.: 2010581-1542

[0109] In some embodiments, the dystrophin (DMD) gene or a product thereof, or a variant or portion thereof, may be referred to as DMD, BMD, CMD3B, DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270, DXS272, MRX85, or dystrophin; External IDs: OMIM: 300377 MGI: 94909; HomoloGene: 20856; GeneCards: DMD; In Human: Entrez: 1756; Ensembl: ENSG00000198947; UniProt: P11532; RefSeq (mRNA): NM_000109; NM_004006; NM_004007; NM_004009; NM_004010; RefSeq (protein): NP_000100; NP_003997; NP_004000; NP_004001; NP_004002; Location (UCSC): Chr X: 31.1 – 33.34 Mb; In Mouse: Entrez: 13405; Ensembl: ENSMUSG00000045103; UniProt: P11531; RefSeq (mRNA): NM_007868; NM_001314034; NM_001314035; NM_001314036; NM_001314037; RefSeq (protein): NP_001300963; NP_001300964; NP_001300965; NP_001300966; NP_001300967; Location (UCSC): Chr X: 82.95 – 85.21 Mb.

[0110] Dystrophin is reported to be found in muscle cells and is crucial in strengthening and protecting muscle fibers. Normal dystrophin protein is reported to be part of a protein complex called the dystrophin-glycoprotein complex, which according to various reports provides structural stability to skeletal muscle and protects the muscle from injury during contraction and relaxation. In addition, dystrophin is reported to be essential for cell survival via a transmembrane signaling function and modulation of vasomotor response to physical activity.

[0111] The DMD gene reportedly contains 79 exons distributed over 2.3 million bp of genetic real estate on the X chromosome; however, only approximately 14,000 bp (<1%) is reported to be used for translation into protein (coding sequence). It is reported that about 99.5% of the genetic sequence, the intronic sequences, is spliced out of the 2.3 million bp initial heteronuclear RNA transcript to provide a mature 14,000 bp mRNA that includes all key information for dystrophin protein production. In some embodiments, subjects with DMD have mutation(s) in the DMD gene that prevent the appropriate construction of the wild-type DMD mRNA and / or the production of the wild-type dystrophin protein, and subjects with DMD often show marked dystrophin deficiency in their muscle.

[0112] In some embodiments, a dystrophin transcript, e.g., mRNA, or protein encompasses those related to or produced from alternative splicing. For example, at least sixteen alternative transcripts of the dystrophin gene were reported following an analysis of splicing patterns of the DMD gene in skeletal muscle, brain and heart tissues.

[0113] It is reported that dystrophin has several isoforms. In some embodiments, dystrophin refers to a specific isoform. At least three full-length dystrophin isoforms have been reported, each controlled by a tissue-specific promoter. The muscle isoform is reportedly mainly expressed in skeletal muscle but also in smooth and cardiac muscles, the brain dystrophin is reportedly specific for cortical neurons but can also be detected in heart and cerebellar neurons, while the Purkinje-cell type reportedly accounts for nearly all cerebellar dystrophin. Alternative splicing reportedly provides a means for dystrophin diversification: the 46 of 176 13004462v1Attorney Docket No.: 2010581-1542 3’ region of the gene reportedly undergoes alternative splicing resulting in tissue-specific transcripts in brain neurons, cardiac Purkinje fibers, and smooth muscle cells while 12 patterns of alternative splicing have been reported in the 5’ region of the gene in skeletal muscle.

[0114] In some embodiments, a dystrophin mRNA, gene or protein is a revertant version.

[0115] Various mutations in the DMD gene can and / or were reported to cause muscular dystrophy. Mutations in the form of large deletions (1 or more exons) are reported to account for approximately two- thirds of all DMD gene mutations; the remaining mutations are reported to be due to duplications and small deletions, insertions, point mutations, or splicing mutations. According to certain reports, deletions are typically clustered in a hotspot region between exons 45 and 55 and prevent translation of dystrophin. Absent or defective dystrophin protein, resulting from DMD gene mutations, is reported to disrupt the dystrophin-glycoprotein complex, leading to increased muscle membrane fragility, chronic muscle damage, inflammation, replacement of muscle fibers with fat and fibrotic tissue, and then loss of muscle function.

[0116] It is reported that in healthy muscle cells, dystrophin can act to stabilize fibers during contractions, and DMD-related dystrophin reduction in muscle cells can lead to myofiber injury and progressive muscle degeneration. For example, absence of dystrophin in muscle stem cells may impair cell division and myogenesis, and inhibit self-renewal, leading to a reduction of viable stem pools and halting of subsequent muscle repair. Among other things, technologies can deliver WVE-N531 into muscle stem cells and provide dystrophin expression therein. In some embodiments, provided technologies can restore dystrophin in muscle stem cells which can enable them to function properly, including allowing quiescent cells to “wake up,” differentiate, and initiate repair of muscle fibers. Muscular Dystrophy

[0117] Muscular dystrophy (MD) is any of a group of muscle conditions, diseases, or disorders that results in (increasing) weakening and breakdown of skeletal muscles over time. The conditions, diseases, or disorders differ in which muscles are primarily affected, the degree of weakness, when symptoms begin, and how quickly symptoms worsen. Many MD subjects eventually become unable to walk. In many cases muscular dystrophy is fatal. Some types are also associated with problems in other organs, including the central nervous system. In some embodiments, a muscular dystrophy is Duchenne (Duchenne’s) Muscular Dystrophy (DMD). In some embodiments, a muscular dystrophy Becker (Becker’s) Muscular Dystrophy (BMD).

[0118] In some embodiments, a symptom of Duchenne Muscular Dystrophy is muscle weakness associated with muscle wasting, with the voluntary muscles being first affected, especially those of the hips, pelvic area, thighs, shoulders, and calves. Muscle weakness can also occur later, in the arms, neck, and other areas. Calves are often enlarged. Symptoms usually appear before age six and may appear in early 47 of 176 13004462v1Attorney Docket No.: 2010581-1542 infancy. Other physical symptoms are: awkward manner of walking, stepping, or running (in some cases, subjects tend to walk on their forefeet, because of an increased calf muscle tone), frequent falls, fatigue, difficulty with motor skills (e.g., running, hopping, jumping), lumbar hyperlordosis, possibly leading to shortening of the hip-flexor muscles, unusual overall posture and / or manner of walking, stepping, or running, muscle contractures of Achilles tendon and hamstrings impair functionality, progressive difficulty walking, muscle fiber deformities, pseudohypertrophy (enlarging) of tongue and calf muscles, higher risk of neurobehavioral disorders (e.g., ADHD), learning disorders (e.g., dyslexia), and non-progressive weaknesses in specific cognitive skills (e.g., short-term verbal memory), which are believed to be the result of absent or dysfunctional dystrophin in the brain, eventual loss of ability to walk (usually by the age of 12), skeletal deformities (including scoliosis in some cases), and trouble getting up from lying or sitting position. In some embodiments, provided technologies delay onset of, slow progression of, reduce severity of, and / or prevent one or more symptoms.

[0119] In some embodiments, Becker muscular dystrophy (BMD) is caused by mutations that give rise to shortened but in-frame transcripts resulting in the production of truncated but partially functional protein(s). Such partially functional protein(s) were reported to retain the critical amino terminal, cysteine rich and C-terminal domains but usually lack elements of the central rod domains which were reported to be of less functional significance. England et al.1990 Nature, 343, 180–182.

[0120] In some embodiments, BMD phenotypes range from mild DMD to virtually asymptomatic, depending on the precise mutation and the level of dystrophin produced. Yin et al.2008 Hum. Mol. Genet. 17: 3909-3918.

[0121] In some embodiments, dystrophy subjects with out-of-frame mutations are generally diagnosed with the more severe Duchenne Muscular Dystrophy, and dystrophy subjects with in-frame mutations are generally diagnosed with the less severe Becker Muscular Dystrophy. Exon Skipping as a Treatment for Muscular Dystrophy

[0122] Exon skipping can induce cellular machinery to skip over one or more targeted exons and restore the reading frame resulting in the production of internally truncated but functional dystrophin polypeptides. In various exon-skipping approaches, oligonucleotides can bind to target complementary sequences in the messenger ribonucleic acids (mRNAs), which engage with the splicing machineries to exclude exons from final transcripts. In subjects with DMD, this approach may slow disease progression by converting severe DMD symptoms to the milder symptoms such as those seen in patients with Becker muscular dystrophy (BMD), in which patients are ambulatory longer and have a longer life expectancy.

[0123] Various DMD genotypes are amenable to exon skipping including exon 53 skipping. Exon skipping can restore DMD mRNA reading frame, resulting in the production of a shorter but functional 48 of 176 13004462v1Attorney Docket No.: 2010581-1542 dystrophin protein. Exon 53 skipping is applicable, but not limited to, patients with deletions in the DMD gene of exons 43–52, 45–52, 47–52, 48–52, 49–52, 50–52, 51–52, or exon 52 alone. According to some reports, approximately 8% to 10% of patients with DMD may be treated by exon 53 skipping. Thirty-six month longitudinal natural history data in a cohort of patients with deletions amenable to skip exons 44, 45, 51, or 53 showed that patients amenable to exon 53 skipping in particular appear to have earlier onset of decline and are at a higher risk of dramatic functional loss (e.g., loss of ambulation) compared to other subgroups.

[0124] In some embodiments, exon skipping restores or maintains a proper reading frame, and / or creating an artificially internally truncated DMD protein which provides at least partially improved or fully restored biological activity. In some embodiments, an internally truncated DMD protein produced from a dystrophin transcript with a skipped exon(s) is more functional than a terminally truncated DMD protein e.g., produced from a dystrophin transcript with an out-of-frame deletion.

[0125] In some embodiments, an internally truncated DMD protein produced from a dystrophin transcript with a skipped exon(s) is more resistant to nonsense-mediated decay, which can degrade a terminally truncated DMD protein, e.g., produced from a dystrophin transcript with an out-of-frame deletion.

[0126] In some embodiments, a DMD subject is analyzed for DMD genotype and is confirmed to have a mutation of the DMD gene that is amenable to exon 53 skipping prior to administration of WVE-N531. In some embodiments, a mutation of the DMD gene that is amenable to exon 53 skipping is or comprises one or more nucleobase changes (e.g., replaced by a different nucleobase), insertions, and / or deletions, etc., wherein exon 53 skipping can produce a DMD polypeptide that can provide improved functions compared to without exon 53 skipping.

[0127] Prior to the present disclosure, Emflaza®(deflazacort) is a corticosteroid approved in the US for the treatment of DMD. Emflaza was shown to improve muscle strength (mean change 0.15 points as measured by the modified Medical Research Council sale) following a 12-week treatment period at a dose of 0.9 mg / kg / day. In a long-term placebo-controlled study in children with DMD aged 6 to 12 years, there was no significant effect of deflazacort on average muscle strength score at 2 years. While Emflaza is not approved for the treatment of DMD in the European Union (EU), corticosteroids such as deflazacort are commonly used off-label in the EU in patients with DMD to slow the decline in muscle strength, as well as to prolong ambulation and respiratory function.However, the chronic use of corticosteroids has been associated with serious side effects such as immunosuppression and increased risk of infection, changes in endocrine function, hypertension, cataracts, bone demineralization, gastrointestinal perforation, and growth retardation in children. Further, corticosteroids do not correct the underlying genetic defect in DMD.

[0128] Vyondys 53TM(golodirsen) is an oligonucleotide of the phosphorodiamidate morpholino 49 of 176 13004462v1Attorney Docket No.: 2010581-1542 oligomer (PMO) subclass intended to increase dystrophin production via exon 53 skipping. Golodirsen was approved in 2019 in the US for the treatment of patients who have a confirmed mutation of the DMD gene that is amenable to exon 53 skipping. Approval was based on a mean change from baseline in dystrophin levels of 0.92% (standard deviation [SD] 1.01) of normal levels following 48 weeks of treatment at 30 mg / kg / week. ViltepsoTM(viltolarsen), another PMO subclass exon skipping oligonucleotide, was approved in the US and Japan in 2020 for the treatment of patients with DMD who have a mutation amenable to exon 53 skipping. The approval was also based on an observed increase in dystrophin production with a mean increase of 5.3% (SD 4.5) of normal levels measured following 25 weeks of treatment at 80 mg / kg / week. Both golodirsen and viltolarsen were approved via the US accelerated approval mechanism on the basis of an increase in the surrogate endpoint of dystrophin production and neither drug has been shown to provide clinical benefit in patients with DMD. Continued approval in the US is contingent upon verification of a clinical benefit in confirmatory trials. Neither drug is approved in the United Kingdom (UK) or EU prior to the present disclosure.

[0129] Similarly, some patients may have a mutation that makes them eligible for treatment with both an exon 53-skipping oligonucleotide and Translarna™ (ataluren) or Exondys 51®(eteplirsen). Ataluren is a small molecule intended to enhance ribosomal read-through of nonsense mutations that is not approved for use in the United States (US). It received conditional marketing approval in the EU for the treatment of DMD resulting from a nonsense mutation in the dystrophin gene in ambulatory patients aged 5 years or older. Clinical efficacy of ataluren has not been established in non-ambulatory patients and additional evidence is required in order to support its continued approval in the EU. It is anticipated that ataluren’s use would be limited to a small percentage of the DMD population in consideration of the low prevalence of nonsense mutations and its limited indication for ambulatory patients. Eteplirsen is an oligonucleotide of the PMO subclass intended to increase dystrophin production via exon skipping. Eteplirsen was approved in the US for the treatment of patients who have a confirmed mutation of the DMD gene that is amenable to exon 51 skipping. Approval was based on detection of an average dystrophin protein level of 0.93% of the dystrophin level in normal muscle as well as a median increase in truncated dystrophin expression of 0.1% in the only study where an estimate of dystrophin expression in response to eteplirsen treatment was possible. The drug is not approved in the EU prior to the present disclosure.

[0130] The plasma Cmaxand area under the plasma concentration-time curve (AUC) of golodirsen increases proportionally with dose and half-life is approximately 3 hours with no plasma accumulation observed. Based on a Phase 1 study of viltolarsen, the urinary excretion rate indicated removal of 70% to 80% of the total administered drug within 24 hours, indicating a short half-life (<24 hours). The kidney function of all patients was in the normal range. Eteplirsen is rapidly cleared from the plasma with the majority of drug elimination occurring within 24 hours. Twenty-four hours after the end of the infusion, 50 of 176 13004462v1Attorney Docket No.: 2010581-1542 mean concentrations of eteplirsen were 0.07% of the maximum (peak) concentration of drug in plasma (Cmax). Renal clearance of eteplirsen accounts for over 60% of the administered dose with a terminal half- life (t½) of 3 to 4 hours. Accumulation of eteplirsen during once-weekly dosing has not been observed.

[0131] Among other things, the present disclosure provides technologies for treating DMD, inducing DMD exon 53 skipping, increasing levels of exon 53-skipped DMD transcripts, producing or increasing levels of truncated DMD polypeptides from exon 53 skipping, increasing levels of and / or restoring one or more DMD functions, etc., comprising administering to a subject WVE-N531 or a composition thereof in accordance with the present disclosure, wherein the subject has a mutated DMD amenable to exon 53 skipping. In some embodiments, provided technologies provide various advantages over one or more existing approved drugs, for example, improved safety, tolerability, clearance, accumulation, exon skipping, clinical effects, and / or one or more other pharmacokinetic or pharmacodynamic aspects.

[0132] In some embodiments, the present disclosure provides technologies for DMD exon 53 skipping which can restore DMD reading frame. In some embodiments, provided technologies produce various functional dystrophin isoforms. For example, in some embodiments, provided technologies can provide dystrophin isoforms observed in BMD. In some embodiments, provided technologies can delay loss of function, e.g., loss of ambulation. In some embodiments, provided technologies can consistently produce 5% or more dystrophin of normal in a population of patients. See, e.g., Feraudy, Y., et al. 2020 Annals Neurology 89, 280. WVE-N531

[0133] WVE-N531 may be described as (as those skilled in the art appreciate, 5’ to 3’ unless specified otherwise): fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU* SfC m: 2’-OMe modified nucleoside; f: 2’-F modified nucleoside; *S: Sp phosphorothioate linkage; and n001R: Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage.

[0134] WVE-N531 may be named by accepted oligonucleotide nomenclature: (Sp)-2’-fluoro-2’-deoxy-P-thio-cytidylyl-(O3'→O5')-(Sp)-2’-fluoro-2’-deoxy-P-thio-uridylyl-(O3'→O5')- (Rp)-2’-fluoro-2’-deoxy-P-((1,3-dimethylimidazolidin-2-ylidenyl)amino)-cytidylyl-(O3'→O5')-(Sp)-2’- fluoro-2’-deoxy-P-thio-cytidylyl-(O3'→O5')-(Sp)-2’-fluoro-2’-deoxy-P-thio-guanylyl-(O3'→O5')-(Rp)-2’- fluoro-2’-deoxy-P-((1,3-2-ylidenyl)amino-guanylyl-(O3'→O5')-(Sp)-2’-fluoro-2’- deoxy-P-thio-uridylyl-(O3'→O5')-(Sp)-2’-fluoro-2’-deoxy-P-thio-uridylyl-(O3'→O5')-2’-O- 51 of 176 13004462v1Attorney Docket No.: 2010581-1542 methylcytidylyl-(O3'→O5')-(Sp)-2’-fluoro-2’-deoxy-P-thio-uridylyl-(O3'→O5')-(Sp)-2’-O-methyl-P-thio- guanylyl-(O3'→O5')-(Sp)-2’-fluoro-2’-deoxy-P-thio-adenylyl-(O3'→O5')-2’-O-methyladenylyl-(O3'→O5')- (Sp)-2’-fluoro-2’-deoxy-P-thio-guanylyl-(O3'→O5')-(Sp)-2’-fluoro-2’-deoxy-P-thio-guanylyl-(O3'→O5')- (Sp)-2’-fluoro-2’-deoxy-P-thio-uridylyl-(O3'→O5')-(Rp)-2’-fluoro-2’-deoxy-P-((1,3- dimethylimidazolidin-2-ylidenyl)amino)-guanylyl-(O3'→O5')-(Sp)-2’-fluoro-2’-deoxy-P-thio-uridylyl- (O3'→O5')-(Sp)-2’-fluoro-2’-deoxy-P-thio-uridylyl-(O3'→O5')-2’-fluoro-2’-deoxycytidine.

[0135] In some embodiments, WVE-N531 is provided in a composition, e.g., a pharmaceutical composition, and / or administered as one or more pharmaceutically acceptable salts. In some embodiments, the present disclosure provides pharmaceutically acceptable salts of WVE-N531. In some embodiments, a pharmaceutically acceptable salt is a sodium salt. In some embodiments, the present disclosure provides WVE-N531 hexadecasodium salt. In some embodiments, a WVE-N531 drug substance is WVE-N531 hexadecasodium salt.

[0136] Molecular formulae and molecular weights of WVE-N531 and its hexadecasodium salt is described below: Molecular Formula (free acid form): C206H250F17N78O109P19S14 Molecular Weight (free acid form): 6923.02 g / mol Molecular Formula (hexadecasodium salt form): C206H234F17N78O109P19S14Na16 Molecular Weight (hexadecasodium salt form): 7274.73 g / mol

[0137] WVE-N531 has 17 stereodefined internucleotide linkages, 14 of which are identified as Sp phosphorothioate linkages, and three as Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkages. Its internucleotide linkages can be illustrated as: 5’-SSRSSRSSOSSSOSSSRSS-3’, where ‘S’, ‘R’, and ‘O’ represent Sp phosphorothioate linkage, Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage, and phosphate linkages, respectively. A N-(1,3-dimethylimidazolidin-2- ylidenyl) phosphoramidate linkage may be referred to as a n001 linkage.

[0138] The structure of WVE-N531 may be depicted as Formula I: 52 of 176 13004462v1Attorney Docket No.: 2010581-154253 of 176 13004462v1Attorney Docket No.: 2010581-1542in each of which there are five nucleoside units. As shown in these Formulae, each linkage in WVE-N531 is independently a Sp phosphorothioate linkage, a Rp n001 linkage or a natural phosphate linkage, and in a WVE-N531 salt, acidic hydrogen atoms of the phosphorothioate and phosphate linkages may be 54 of 176 13004462v1Attorney Docket No.: 2010581-1542 independently replaced with cations, e.g., in the hexadecasodium salt, each replaced with sodium.

[0141] Water solution of WVE-N531 hexadecasodium salt is typically a clear, colorless to pale yellow solution, and can have a concentration of about 150 mg / mL or more as determined by UV. In some embodiments, pH of WVE-N531 hexadecasodium salt in purified water ranges from 6.0-8.0.

[0142] Extinction coefficient of WVE-N531 is 186,486 M-1cm-1(determined in water) at 260 nm. Unless noted otherwise, when UV is utilized to measure concentration / amount of WVE-N531, 260 nm is utilized and this number is utilized in calculation. Using a molecular weight of 6918.72 g / mol (free acid form), this equates to an absorptivity factor of 27.0 OD / mg.

[0143] In some embodiments, WVE-N531 is supplied as an isotonic solution for dilution for IV infusion. In some embodiments, the total volume of infusion is 100-500 mL based on body weight of a subject. In some embodiments, infusion is administered over approximately a period of 1 hour. In some embodiments, infusion is extended to about 3 hours, e.g., to enhance tolerability. In some embodiments, WVE-N531 diluted infusion solution contains no preservatives and is administered without delay or within four hours of dilution. In some embodiments, 0.45% sodium chloride injection is utilized for dilution. In some embodiments, 0.9% sodium chloride injection is utilized for dilution.

[0144] In some embodiments, WVE-N531 is provided and administered as chirally controlled composition in which WVE-N531 is enriched over other diastereomers.

[0145] Among other things, the present disclosure provides WVE-N531 of high purity. In some embodiments, WVE-N531 has a level of purity of about 70%-90%, e.g., about 70%-85%, about 70%-80%, about 75%-85%, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, a level of purity is determined by IP-RP- UPLC described herein. In some embodiments, it is about 70% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 71% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 72% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 73% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 74% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 75% as determined by IP- RP-UPLC described herein. In some embodiments, it is about 76% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 77% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 78% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 79% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 80% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 81% or more as determined by IP-RP-UPLC described herein. In some embodiments, it is about 82% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 83% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 84% as determined by IP-RP-UPLC described herein. 55 of 176 13004462v1Attorney Docket No.: 2010581-1542 In some embodiments, it is about 85% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 86% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 87% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 88% as determined by IP-RP-UPLC described herein. In some embodiments, it is about 89% as determined by IP- RP-UPLC described herein. In some embodiments, it is about 90% or more as determined by IP-RP-UPLC described herein. In some embodiments, an IP-RP-UPLC utilize the Set A parameters. In some embodiments, purity is % area at a given wavelength, e.g., around 260 nm.

[0146] In some embodiments, level of impurities is about 10%-30% or lower. In some embodiments, it is about 10%-30%. In some embodiments, it is about 15%-30%. In some embodiments, it is about 20%- 30%. In some embodiments, it is about 30% or lower. In some embodiments, it is about 29% or lower. In some embodiments, it is about 28% or lower. In some embodiments, it is about 27% or lower. In some embodiments, it is about 26% or lower. In some embodiments, it is about 25% or lower. In some embodiments, it is about 24% or lower. In some embodiments, it is about 23% or lower. In someembodiments, it is about 22% or lower. In some embodiments, it is about 21% or lower. In some embodiments, it is about 20% or lower. In some embodiments, it is about 19% or lower. In some embodiments, it is about 18% or lower. In some embodiments, it is about 17% or lower. In some embodiments, it is about 16% or lower. In some embodiments, it is about 15% or lower. In some embodiments, it is about 14% or lower. In some embodiments, it is about 13% or lower. In some embodiments, it is about 12% or lower. In some embodiments, it is about 11% or lower. In some embodiments, it is about 10% or lower. In some embodiments, level of impurity is assessed using IP-RP- UPLC. In some embodiments, an IP-RP-UPLC utilize the Set A parameters. In some embodiments, purity is % area at a given wavelength, e.g., around 260 nm.

[0147] In some embodiments, the present disclosure provides various technologies for characterizing WVE-N531 preparations. In some embodiments, the present disclosure provides technologies for assessing purity of WVE-N531 or compositions thereof.

[0148] In some embodiments, the present disclosure provides technologies for manufacturing WVE- N531 or compositions thereof. In some embodiments, the present disclosure provides technologies for releasing WVE-N531 preparations. Manufacturing

[0149] In some embodiments, the present disclosure provides technologies for manufacturing WVE- N531 or a sat thereof, e.g., WVE-N531 hexadecasodium salt. In some embodiments, the present disclosure provides technologies for manufacturing a WVE-N531 drug substance. In some embodiments, the present disclosure provides technologies for manufacturing a pharmaceutical composition comprising WVE-N531 56 of 176 13004462v1Attorney Docket No.: 2010581-1542 or a pharmaceutically acceptable salt form thereof and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides WVE-N531 drug products. Certain useful technologies for manufacturing WVE-N531 are described in WO 2023 / 168014 and are incorporated herein by reference.

[0150] In some embodiments, WVE-N531 or a salt thereof, e.g., WVE-N531 hexadecasodium salt, is chemically manufactured in compliance with Good Manufacturing Practice (cGMP) regulations.

[0151] In some embodiments, a provided technology comprises a multi-step process that includes solid-phase oligonucleotide synthesis, cleavage of the crude protected oligonucleotide from the solid support, removal of protecting groups (deprotection), preparative anion exchange (AEX) chromatographic purification, concentration and / or desalting.

[0152] In some embodiments, stereochemistry of WVE-N531 or salts thereof is established through control of starting materials for synthesis as well as synthetic processes. For example, in some embodiments, provided technologies comprise use of phosphoramidites prepared from the chiral auxiliaries (L)-DPSE and (L)-PSM during coupling steps to ensure that the intended stereodefined Sp phosphorothioate and Rp n001 linkages respectively, are obtained.

[0153] A useful manufacturing process for WVE-N531 hexadecasodium salt as a drug substance and certain results are described below as an example. A Useful Manufacturing Process

[0154] In some embodiments, a manufacturing process consists of synthesizing the oligonucleotide, containing a combination of three kinds of internucleotide linkage (phosphodiester, stereodefined phosphorothioate, and stereodefined n001), on solid support using phosphoramidite chemistry, as shown in the synthesis cycles below. Each cycle to introduce a stereodefined phosphorothioate or stereodefined n001 consists of 5’-detritylation, coupling, capping of exposed chiral auxiliary secondary amine (Capping-1), thiolation or imidation respectively, and capping of unreacted 5’-hydroxyl groups (Capping-2). Each cycle to introduce a phosphodiester consists of detritylation, coupling, oxidation, and Capping-2. Each coupling reaction is carried out by activation of an appropriate phosphoramidite and reaction with the free 5’- hydroxyl group of a support-immobilized protected nucleotide or oligonucleotide. After the appropriate number of cycles and final detritylation, the (L)-PSM chiral auxiliary and cyanoethyl phosphate protecting groups are removed from a crude oligonucleotide by on-column treatment with diethylamine (DEA) in acetonitrile (ACN).The (L)-DPSE chiral auxiliary is then removed by treatment with NEt3·HF (triethylamine hydrogen fluoride) solution. The crude oligonucleotide is then cleaved from the solid support and removed from the column by ammonia treatment. The base protecting groups are removed with continued ammonia treatment in an appropriately sized reaction vessel. The resulting crude oligonucleotide is purified using preparative anion exchange (AEX) high-performance liquid chromatography (HPLC) followed by concentration and desalting to yield WVE-N531 or a salt thereof, 57 of 176 13004462v1Attorney Docket No.: 2010581-1542 e.g., WVE-N531 hexadecasodium salt, in some embodiments, as drug substance. A useful cycle for phosphorothioate assembly DMTrOOB3 B1 Y158 of 176 13004462v1Attorney Docket No.: 2010581-1542 A useful cycle for n001 assembly59 of 176 13004462v1Attorney Docket No.: 2010581-1542 A useful cycle for phosphate assembly60 of 176 13004462v1Attorney Docket No.: 2010581-1542 X YnR Bn S F (L)-DPSE-Ac ABzStage 1: Synthesis

[0156] Synthesis of the oligonucleotide is carried out on Controlled Pore Glass (CPG) solid support functionalized with 5’-ODMTr-2’-F-dC(N4-Ac), on an automated oligonucleotide synthesizer. All reactions take place on the solid support packed in a column.

[0157] 1. Detritylation

[0158] Tosupport is subjected to acid-catalyzed removal of the DMTr protecting group from the 5’-hydroxyl by treatment with 3% dichloroacetic acid (DCA) in toluene. Complete DMTr removal is ensured by inline UV monitoring based on a watch command in the synthesis program. The DMTr removal is performed in the same way at the beginning of each synthesis cycle and after the final cycle. In every case, following detritylation, the support-bound material is washed with acetonitrile in preparation for the next step of the synthesis.

[0159] 2. Coupling 61 of 176 13004462v1Attorney Docket No.: 2010581-1542

[0160] the 5’-hydroxyl group of the support-bound oligonucleotide with an excess of a solution of the protected phosphoramidite, in the presence of an activator, either 5-(ethylthio)-1H-tetrazole (ETT) or 1-cyanomethyl imidazolium triflate salt (CMIMT) dissolved in acetonitrile. The phosphoramidites are dissolved in acetonitrile or an 80:20 v / v acetonitrile : isobutyronitrile mixture. The phosphoramidite required in each coupling step is determined by the oligonucleotide sequence. For introduction of phosphate linkage, phosphoramidites and ETT are used. For introduction of a stereodefined phosphorothioate or n001 linkage, (L)-DPSE or (L)- PSM derived phosphoramidite, and CMIMT are used. In either case, the phosphoramidite / activator solutions are mixed inline, pushed onto the synthesis column, then circulated through the column for appropriate amount of time. Subsequently, excess reagents are removed by flushing the synthesis column with acetonitrile.

[0161] 3. Capping-1 62 of 176 13004462v1Attorney Docket No.: 2010581-1542

[0162] Capping 1 can protect a secondary amine whichring opening on an auxiliary group during a coupling step rendering it unreactive for the remainder of the synthesis. The secondary amine is capped by flowing capping reagent B ([Cap B]: acetic anhydride / 2,6- lutidine / acetonitrile (20:30:50, v:v:v) through the synthesis column. Excess reagents are flushed from the synthesis column with acetonitrile.

[0163] 4. Oxidation, Thiolation or Imidation

[0164] A newly created P(III) phosphite triester linkage is treated in one of three ways: 1. Oxidation by treatment with iodine in pyridine : water (90:10,v:v), resulting in generation of a P(V) phosphodiester linkage. 2.in the generation of a stereodefined P(V) phosphorothioate triester linkage. 3.in acetonitrile resulting in the generation of a stereodefined P(V) phosphorimidate triester linkage. 63 of 176 13004462v1Attorney Docket No.: 2010581-1542

[0165] column withacetonitrile.

[0166] 5. Capping-2

[0167] Typically, a coupling reaction proceeds in very high yield but it is not quantitative. A small proportion of the 5’-hydroxy groups, available in any given cycle can fail to couple with an activated phosphoramidite. To prevent reaction during subsequent cycles, these sites are blocked by capping with a mixture of reagents (1:1, v:v) (Capping Reagent A [Cap A]: N-methylimidazole / acetonitrile (20:80,v:v) and Cap B). As a result, 5’-O-acetylated (‘capped’) support-bound oligonucleotide sequences are formed. Excess capping reagents are washed from the column with acetonitrile.

[0168] Oligonucleotide Assembly and Final Step

[0169] Independent iteration of the three cycles shown above, using appropriate protected phosphoramidites assemble an entire protected sequence with a DMTr protecting group present at the 5’- terminal position. After addition of the last nucleotide in the sequence, the 5’-terminal DMTr group is removed during a final detritylation step. Stage 2: Cleavage and Deprotection

[0170] Removal of phosphate protecting groups, and (L)-PSM and (L)-DPSE chiral auxiliaries from stereodefined phosphorimidate triester and phosphorothioate triester, cleavage of the crude oligonucleotide from the solid support, and removal of exocyclic nucleobase protecting groups is performed in a three-step 64 of 176 13004462v1Attorney Docket No.: 2010581-1542 process.

[0171] Step 1: (L)-PSM Chiral Auxiliary and Cyanoethyl Removal with Diethylamine Treatment incan remove groups and (L)-PSM chiral auxiliary from the phosphorimidate triester to generate phosphodiester and phosphoramidate diester linkages, respectively.

[0173] Step 2: (L)-DPSE Chiral Auxiliary Removal with Fluoride Treatmentsolution of NEt3·3HF in a mixture of dimethyl sulfoxide (DMSO), NEt3 and H2O to effect removal of the chiral auxiliary. This process converts a stereodefined phosphorothioate triester to a stereodefined phosphorothioate diester as shown above.

[0175] Step 3: Cleavage and Global Nucleobase Deprotection 65 of 176 13004462v1Attorney Docket No.: 2010581-154266 of 176 13004462v1Attorney Docket No.: 2010581-1542hydroxide in an appropriately sized pressure-rated vessel. This reaction can provide global deprotection of cyanoethyl phosphate groups (may have been deprotected in an earlier step) and exocyclic amino groups (e.g., removal of protecting groups such as acetyl, benzoyl, isobutyryl, etc.). In Formula P, each M+is independently a cation. In some embodiments, each M+is H+(such a compound the “Formula P Acid”). 67 of 176 13004462v1Attorney Docket No.: 2010581-1542 In some embodiments, each M+is independently a cation. In some embodiments, a crude oligonucleotide is a salt of the Formula P Acid. In some embodiments, a crude oligonucleotide composition comprise a salt of the Formula P Acid. In some embodiments, a crude oligonucleotide composition comprise two or more salts of the Formula P Acid.

[0177] Typically, after deprotection one or more WVE-N531 salts are provided. In some embodiments, a salt is an ammonium salt. In some embodiments, a deprotection product composition comprises one or more WVE-N531 salts. In some embodiments, a deprotection product composition comprises a WVE-N531 ammonium salt. Stage 3: Purification by Anion Exchange Chromatography

[0178] Purification of the crude oligonucleotide solution is accomplished by AEX-HPLC. A solution of crude oligonucleotide is loaded onto the purification column packed with TSK-GEL Super Q-5PW media. A purification run is performed using sodium hydroxide buffered eluents. A sodium chloride gradient is used to elute the oligonucleotide from the column. The purification is carried out at ambient temperature. The elution profile is monitored by ultraviolet (UV) spectrophotometry. Fractions are collected and mock pools are evaluated by IP-RP-UPLC. The pool containing oligonucleotide at or above a target purity level is subjected to the next step in the process. Stage 4: Concentration and Desalting (Final UF / DF)

[0179] The selected fraction pool is then concentrated and diafiltered against purified water to remove the purification buffer by tangential flow filtration (TFF) using regenerated cellulose membrane cassettes. The pool is concentrated, and pH adjusted to 6.5 -7.5 with hydrochloric acid or sodium hydroxide. The concentrated oligonucleotide is diafiltered against purified water and concentrated to a target of 1200 – 1400 OD / mL prior to collecting the desalted oligonucleotide. The system is flushed with purified water to maximize yield combining the concentrated desalted oligonucleotide with the rinses to a target final desalt concentration of 878 ± 68 OD / mL (32.5 ± 2.5 mg / mL). Stage 5: Filtration and Packaging

[0180] The oligonucleotide solution is filtered through a 0.2-micron filter before filling into drug substance storage containers. The drug substance is packaged in sterile high-density polyethylene (HDPE) bottles, each of which is labeled and sealed in a Mylar foil pouch and stored at -20°C. Starting Materials

[0181] Certain useful starting materials are listed below: Material (Grade) Step68 of 176 13004462v1Attorney Docket No.: 2010581-1542 Capping reagent B: Synthesis (Capping 1 and 2) Acetic anhydride: 2,6-Lutidine: Acetonitrile (20:30:50) PAbbreviations: CoA = Certificate of Analysis; NF = National Formulary; CIP = clean in place; USP = United States Pharmacopoeia; ACS = American Chemical Society 69 of 176 13004462v1Attorney Docket No.: 2010581-1542 Material Molecular Formula (Average MW)70 of 176 13004462v1Attorney Docket No.: 2010581-1542 Material Molecular Formula (Average MW)71 of 176 13004462v1Attorney Docket No.: 2010581-1542 Material Molecular Formula (Average MW)72 of 176 13004462v1Attorney Docket No.: 2010581-1542 Material Molecular Formula (Average MW), port and 2-azido-1,3-dimethylimidazolinium hexafluorophosphate. The reactive exocyclic groups on nucleobases are appropriately protected to render them unreactive during oligonucleotide synthesis, and the 5’-hydroxy functionality is protected as a 4,4’-dimethoxytrityl ether (DMTr). Phosphoramidites have purity levels of about 90%, about 95% or in some cases about 98% or more (RP-HPLC at about 260 nm (area %) and / or31P NMR by integration), about 97% or more P(III) purity (by31P NMR integration) and water content of less than 0.2% (w / w). Controlled Pore Glass -5’-ODMTr-2’-F-dC (N4-Ac) succinate solid support is about 120-200 mesh in particle size (analytical sieving), about 540- 660 Å in pore diameter (mercury intrusion), about 0.20 – 0.24 g / cc in density (tap density), ≥ about 0.8 cc / g in pore volume (mercury intrusion), ≥ about 75 m2 / g in surface area Hg intrusion (mercury intrusion), and 70 – 80 µmol / g assessed by DMT ligand assay (spectrophotometric at 498 nm). 2-Azido-1,3-dimethylimidazolinium hexafluorophosphate has a purity of about 98.0% or more (HPLC) and nitrogen content of about 23.70 – 24.80 % (elemental analysis).

[0183] Stereochemistry can be established through control of the starting materials for synthesis as well as the synthetic process. Stereodefined phosphoramidites are prepared from appropriately protected nucleosides and a chiral auxiliary, e.g., (L)-DPSE or (L)-PSM. The chemical purity of the stereodefined phosphoramidite can be determined by31P NMR. Configuration can be determined by a combination of31P NMR,1H NMR, and13C NMR. As the stereochemical configuration of the protected nucleoside and chiral auxiliaries are fixed, there are 2 possible diastereomers (trans and cis) that can result from the coupling reaction, of which the trans form is predominant, with the cis form being present as a minor impurity.

[0184] During the manufacture process, various in-process controls are applied. Upon completion of the synthesis and the cleavage and deprotection stages, the presence of WVE-N531 drug substance in the resulting crude oligonucleotide is identified by LC / MS and its purity quantified by IP-RP-UPLC. During 73 of 176 13004462v1Attorney Docket No.: 2010581-1542 purification, individual fractions as well as selected mock pools are evaluated for purity and impurities by IP-RP-UPLC. Once selected fractions are pooled and concentrated, desalting of the resulting solution is controlled via in-process measurements of conductivity, pH and concentration. In some embodiments, a drug substance or a drug product is manufactured meeting one or more or all of the following criteria. In some embodiments, a drug substance, e.g., WVE-N531 or a salt form thereof, has a certain level of purity as described herein. In some embodiments, WVE-N531 hexadecasodium salt has a certain level of purity as described herein. In some embodiments, WVE-N531 or a salt thereof has a certain level of purity in a composition, e.g., a pharmaceutical composition. In some embodiments, WVE-N531 or a salt thereof has a certain level of purity in a drug product. In some embodiments, WVE-N531 or a salt thereof is WVE- N531 hexadecasodium salt. In some embodiments, WVE-N531 has a level of purity of about 70%-90%, about 70%-85%,about 70%-80%, about 75%-85%, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or more as determined by IP- RP-UPLC described herein. Characterization

[0185] Among other things, the present disclosure provides technologies for assessing WVE-N531 or compositions thereof.

[0186] Products can be characterized by various technologies. For example, WVE-N531 preparations may be characterized by mass spectrometry. For one preparation, electrospray ionization mass spectrometry (ESI-MS) analysis was performed and the experimentally determined value of 6722 Da is consistent with the theoretical average mass (free acid form) of WVE-N531 drug substance (6723 Da). In some embodiments, the sequence of WVE-N531 or a salt thereof was confirmed by ESI-MS / MS sequencing. For example, in one assessment, at least one sequence-relevant fragment ion was observed for 19 nucleobases in the sequence within 5 ppm error of each calculated (expected) mass. Since the monoisotopic mass of the full-length oligonucleotide was also verified in the ESI-MS / MS analysis, then the identities and locations of all 20 nucleobases have been demonstrated and the sequence was confirmed.

[0187] Stereochemical identity comprises the combination of the absolute stereochemical configuration of each chiral linkage phosphorus in an oligonucleotide (Rp or Sp) (for WVE-N531 or a salt thereof, each linkage phosphorus in a phosphorothioate or phosphoramidate group). This combination can give rise to a single diastereomer which is unique among 2ndiastereomers (n = number of chiral linkage phosphorus). In the case of WVE-N531 or a salt thereof, this combination can be illustrated with 5’- SSRSSRSSOSSSOSSSRSS-3’, wherein S, P, and O represent Sp phosphorothioate, Rp N-(1,3- dimethylimidazolidin-2-ylidenyl) phosphoramidate, and phosphate linkages, respectively. WVE-N531 or a salt thereof also comprises 2’-fluoro (F) and 2’- O-Methyl (OMe) modifications.

[0188] Establishment of stereochemical identity comprises control of starting materials for synthesis. 74 of 176 13004462v1Attorney Docket No.: 2010581-1542 Synthetic coupling of phosphoramidites prepared from (L)-DPSE or (L)-PSM gives rise to phosphorothioate linkages of Sp chirality and phosphoramidate linkages of Rp chirality respectively, and those prepared from (D)-DPSE or (D)-PSM gives rise to phosphorothioate linkages of Rp chirality and phosphoramidate linkages of Sp chirality. Consistent use of stereodefined phosphoramidites for each coupling reaction allows for control of stereochemical identity of each phosphorothioate or phosphoramidate linkage.

[0189] Stereochemical identity can be confirmed using various technologies in accordance with the present disclosure. For example, it can be confirmed by measurement using several different techniques, which in combination, and by comparison with appropriate standards, provide accurate information about, and confirmation of, this attribute. Useful analytical techniques include NMR (1H,19F,31P, multidimensional, etc.) and enzymatic digestion. In some instances, NMR are performed in phosphate buffer (e.g., pH 7.0). Results including1H,19F and31P NMR and enzymatic digestion of various preparations are consistent with product structures. In some embodiments, NMR is referenced to water based on DSS standard. Observed data from certain experiments are described below.31P NMR data for a preparation of WVE-N531 drug substance (hexadecasodium salt). Acquired and recorded at 161.98 MHz. Chemical Shift (ppm) Integral Possible Assignment e)75 of 176 13004462v1Attorney Docket No.: 2010581-1542 -3.16 1 PN19recorded at 348K. Chemical Shift (ppm) Integral -198.82 1

[0190] In some embodiments, a reference standard, e.g., one characterized by NMR, enzymatic digestion, etc. is used to determine stereochemical identity by UPLC as part of batch release. In some embodiments, the provided technology provides reference standards.

[0191] FTIR spectrum of solid WVE-N531 hexadecasodium salt as a drug substance was recorded. The major absorbance bands at 1643 cm-1and 1682 cm-1are consistent with amide C=O, C=N and C=C stretch vibrations. Broad peaks in the 3200 –3300 cm-1region are also consistent with oligonucleotide functional groups.

[0192] Counter ions can be analyzed in accordance with the present disclosure. For example, a sodium content value of 5.1% has been determined by ICP-OES for a preparation of a WVE-N531 drug substance lot, in agreement with the theoretical sodium content value of 5.1% (w / w) (hexadecasodium salt). 76 of 176 13004462v1Attorney Docket No.: 2010581-1542

[0193] A prepared solution of WVE-N531 drug substance is a clear (no visible material and essentially free of particulate matter), colorless solution. The pH of WVE-N531 drug substance in purified water was found to be about 7.3.

[0194] The molar extinction coefficient of WVE-N531 drug substance was experimentally determined in water to be 186486 M-1cm-1using a lot. Using a molecular weight of 6918.72 g / mol (free acid form), this equates to an absorptivity factor of 27.0 OD / mg.

[0195] In various embodiments, impurities are controlled at low levels as described herein. For example, in some embodiments, impurity level is lower than about 30% as described herein.

[0196] WVE-N531 drug substance preparations (including associated impurities) were assessed in GLP in vitro genotoxicity studies and in an in vivo micronucleus study. No genotoxicity was observed.

[0197] Various preparations of WVE-N531 drug substance have met specified limits for residual solvents, including acetonitrile, toluene and pyridine (ICH Class 2), and elemental impurities, including various elements in ICH Class 1, 2A, 3, etc.

[0198] The fidelity of transfer of chiral information (diastereoselectivity) from each stereodefined phosphoramidite to its stereodefined Sp phosphorothioate or Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate (PN) linkage in WVE-N531 or a salt thereof during oligonucleotide synthesis is very high. The overall stereochemical purity of the oligonucleotide can be represented by a product of the diastereoselectivities of incorporation of each of the 17 stereodefined internucleotide linkages.

[0199] In some embodiments, stereopurity is assessed using dimer modeling. The 14 dimer units that represent the composition of the stereodefined phosphorothioate or PN internucleotide linkages within WVE-N531 drug substance (the fGn001RfU, fG*SfG and fU*SfC dimers are each represented twice) were independently synthesized and assessed for stereochemical purity. The stereochemical purity of WVE- N531 or a salt thereof can be estimated as the product of the measured diastereoselectivities of the 17 individual dimers. The synthesis conditions used to produce each dimer were identical to those applied to the WVE-N531 drug substance. Compared to full length WVE-N531 drug substance, these model dimer units are more easily analyzed by standard analytical methods such as IP-RP-UPLC, and both Rp and Sp diastereoisomers are readily separated and quantified by this technique. IP-RP-UPLC data for the model dimers are presented below. Diastereoselectivity Dimer77 of 176 13004462v1Attorney Docket No.: 2010581-1542 fCn001RfC 0.69 99.31 fGn001RfU 0.38 99.62y , with stereochemical purity values of ≥98% at each location in the sequence. The overall stereochemical purity, based on the product of the stereochemical purity of each individual constituent dimers, is 85% (84.9% = product of the 17 diastereoselectivity fidelity values above).

[0201] In some embodiments, the present disclosure provides WVE-N531 with high stereopurity, e.g., as assessed using dimer modeling described herein. In some embodiments, a stereopurity is about 80% or more. In some embodiments, a stereopurity is about 81% or more. In some embodiments, a stereopurity is about 82% or more. In some embodiments, a stereopurity is about 83% or more. In some embodiments, a stereopurity is about 84% or more. In some embodiments, a stereopurity is about 85% or more. In some embodiments, a stereopurity is about 86% or more. In some embodiments, a stereopurity is about 87% or more. In some embodiments, a stereopurity is about 88% or more. In some embodiments, a stereopurity is about 89% or more. In some embodiments, a stereopurity is about 90% or more. In some embodiments, a stereopurity is about 80%-90%. In some embodiments, a stereopurity is about 83%-87%.

[0202] Release specifications of WVE-N531 drug substance may include one or more specifications described herein, e.g., appearance (e.g., visual), sequence identity (e.g., by MS / MS), molecular weight, purity (e.g., area % by IP-RP-UPLC), impurities (e.g., area % by IP-RP-UPLC), sodium content (e.g., by ICP-OES, etc.), concentration (free acid form) (e.g., by UV spectrophotometry), pH (e.g., of solution in purified water), residual solvents (e.g., by gas chromatography), elemental impurities (e.g., by ICP-MS), stereochemical identity (e.g., by UPLC), bacterial endotoxins (e.g., by USP <85>, Ph. Eur 2.6.14), 78 of 176 13004462v1Attorney Docket No.: 2010581-1542 bioburden (total microbial aerobic count, total yeast and bolds count, etc. by USP <61) and / or Ph. Eur 2.6.12), etc.

[0203] Useful parameters for identity (molecular weight) by LC MS are described below: Parameter Value Column Waters Acquity BEH C18 Column, 1.7 µm, 2.1 x 50 mm, or equivalentIn some embodiments, system suitability is confirmed when the molecular weight (deconvoluted mass) for full length product (FLP) for 3 initial injections of the analytical reference material should be 6923±3 Da for WVE-N531; in addition, blank chromatogram has no interfering peaks other than the solvent front and gradient shift.

[0204] The identity of WVE-N531 or a salt thereof can be determined by liquid chromatography mass spectrometry (LC-MS). Samples are prepared in water and injected on a Waters Acquity BEH C18 column. Analysis employs a gradient of mobile phase A (hexafluoroisopropanol [HFIP] and triethylamine [TEA] in 79 of 176 13004462v1Attorney Docket No.: 2010581-1542 water) and mobile phase B (acetonitrile). A summary of method parameters is provided below as an example. Parameter Value Column Flow Injection ) 3)

[0205] In some embodiments, the present disclosure provides technologies for assessing WVE-N531 purity and / or impurities. In some embodiments, purity and impurities of WVE-N531 can be determined by ion-pair reversed-phase UPLC (IP-RP-UPLC), e.g., using a Waters BEH C18 Column. A useful procedure is described below as an example (Protocol A). The separation is accomplished using a gradient of mobile phase A (hexafluoroisopropanol [HFIP] and diisopropylethylamine [DIPEA] in water) and mobile phase B (30% acetonitrile in water). A useful set of parameters are provided below (Set A) and have been utilized to determine levels of purity / impurity for various preparations described herein. Various impurities including diastereomer impurities can be separated using this method (of the 17 WVE-N531 diastereomers 80 of 176 13004462v1Attorney Docket No.: 2010581-1542 each of which only differs from WVE-N531 at a single linkage phosphorus (DS1-DS17), 6 diastereomers are completely resolved and 9 are partially resolved). In some embodiments, the present disclosure provides a method for characterizing WVE-N531 or a salt thereof, or a composition comprising WVE-N531 or a salt thereof, comprising HPLC or UPLC analysis comprising one or more or all of the parameters (e.g., column, one or more solvents in Mobile Phase A, one or more solvents in Mobile Phase B, a mobile phase comprising one or more or all solvents in Mobile Phase A, a mobile phase comprising one or more or all solvents in Mobile Phase B, Mobile Phase A, Mobile phase B, flow rate, run time, gradient, etc.). Parameter Value Column Waters BEH Oligonucleotide C18 Column 1.7 µm 2.1 x 150 mm or equivalentDiastereomer Description S S S S81 of 176 13004462v1Attorney Docket No.: 2010581-1542 fC*SfU*SfCn001RfC*SfG*RfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*S DS5 fGn001RfU*SfU*SfC S S S S S *S S S R S S S

[0206] In some embodiments, all individual impurities ≥0.10 area%, as a function of relative retention time to the main peak, as well as total of all impurities ≥0.10 area% are included in analysis. In some embodiments, WVE-N531 identity is confirmed by comparing relative retention time with system suitability standard.

[0207] In some embodiments, the present disclosure provide technologies for confirming stereochemical identity of WVE-N531 or a diastereomer thereof with respect to one or more linkage phosphorus. In some embodiments, stereochemical identity of WVE-N531 is determined by IP-RP-UPLC 82 of 176 13004462v1Attorney Docket No.: 2010581-1542 using a Waters BEH C18 Column. A useful procedure is described below as an example (Protocol B). The separation is accomplished using a gradient of mobile phase A (hexafluoroisopropanol [HFIP] and DIPEA in water) and mobile phase B (30% acetonitrile in water). A summary of method parameters (Set B) is provided below. In some embodiments, the present disclosure provides a method for characterizing WVE- N531 or a salt thereof, or a composition comprising WVE-N531 or a salt thereof, comprising HPLC or UPLC analysis comprising one or more or all of the parameters (e.g., column, one or more solvents in Mobile Phase A, one or more solvents in Mobile Phase B, a mobile phase comprising one or more or all solvents in Mobile Phase A, a mobile phase comprising one or more or all solvents in Mobile Phase B, Mobile Phase A, Mobile phase B, flow rate, run time, gradient, etc.). Parameter Value Column Waters BEH Oligonucleotide C18 Column 17 µm 21 x 150 mm or equivalent

[0208] Among other things, the present disclosure provides WVE-N531 of high purity as described herein. In some embodiments, the present disclosure provides WVE-N531 of high stereochemical purity, e.g., as determined utilizing IP-RP-UPLC with parameters of Set A and / or Set B. In some embodiments, 83 of 176 13004462v1Attorney Docket No.: 2010581-1542 the present disclosure provides compositions comprising one or more (“the first group”) of WVE-N531 and DS1 to DS17, none of one or more (“the second group) of WVE-N531 and DS1 to DS17, and optionally reduced levels of one or more (“the third group”) of WVE-N531 and DS1 to DS17, wherein each reduced level is independently lower than (e.g., about or no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of; in some embodiments, about or no more than about 50% of) one or more or each of the levels of the first group members. In some embodiments, the present disclosure provides a composition comprising one of WVE-N531 and DS1 to DS17, wherein the composition is free of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of one of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of two of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of three of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of four of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of five of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of six of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of seven of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of eight of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of nine of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of 10 of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of 11 of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of 12 of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of 13 of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of 14 of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of 15 of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of 16 of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition is free of 17 of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition comprises WVE-N531. In some embodiments, a composition comprises DS1. In some embodiments, a composition comprises DS2. In some embodiments, a composition comprises DS3. In some embodiments, a composition comprises DS4. In some embodiments, a composition comprises DS5. In some embodiments, a composition comprises DS6. In some embodiments, a composition comprises DS7. In some embodiments, a composition comprises DS8. In some embodiments, a composition comprises DS9. In some embodiments, a composition comprises DS10. In some embodiments, a composition comprises DS11. In some embodiments, a composition comprises DS12. In some embodiments, a composition comprises DS13. In some embodiments, a composition comprises DS14. In some embodiments, a composition comprises DS15. In some embodiments, a composition 84 of 176 13004462v1Attorney Docket No.: 2010581-1542 comprises DS16. In some embodiments, a composition comprises DS17. In some embodiments, at least WVE-N531 is absent from a composition. In some embodiments, at least DS1 is absent from a composition. In some embodiments, at least DS2 is absent from a composition. In some embodiments, at least DS3 is absent from a composition. In some embodiments, at least DS4 is absent from a composition. In some embodiments, at least DS5 is absent from a composition. In some embodiments, at least DS6 is absent from a composition. In some embodiments, at least DS7 is absent from a composition. In some embodiments, at least DS8 is absent from a composition. In some embodiments, at least DS9 is absent from a composition. In some embodiments, at least DS10 is absent from a composition. In some embodiments, at least DS11 is absent from a composition. In some embodiments, at least DS12 is absent from a composition. In some embodiments, at least DS13 is absent from a composition. In some embodiments, at least DS14 is absent from a composition. In some embodiments, at least DS15 is absent from a composition. In some embodiments, at least DS16 is absent from a composition. In some embodiments, at least DS17 is absent from a composition. In some embodiments, the present disclosure provides technologies for preparing such compositions, e.g., IP-RP-UPLC technologies described herein (e.g., those that can fully or partially resolve diastereomers). For example, in some embodiments, when a peak for one of WVE-N531 and DS1 to DS17 is fully resolved from one or more peaks of one or more of WVE-N531 and DS1 to DS17, the peak represents a composition comprising the one of WVE-N531 and DS1 to DS17 that is free of the one or more of WVE-N531 and DS1 to DS17. In some embodiments, presence, absence and / or levels are assessed using provided technologies, e.g., IP-RP-UPLC as described herein.

[0209] In some embodiments, the present disclosure provides a composition comprising WVE-N531, wherein the composition is free of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) of DS1 to DS17. In some embodiments, a composition is free of one of DS1 to DS17. In some embodiments, a composition is free of two of DS1 to DS17. In some embodiments, a composition is free of three of DS1 to DS17. In some embodiments, a composition is free of four of DS1 to DS17. In some embodiments, a composition is free of five of DS1 to DS17. In some embodiments, a composition is free of six of DS1 to DS17. In some embodiments, a composition is free of seven of DS1 to DS17. In some embodiments, a composition is free of eight of DS1 to DS17. In some embodiments, a composition is free of nine of DS1 to DS17. In some embodiments, a composition is free of 10 of DS1 to DS17. In some embodiments, a composition is free of 11 of DS1 to DS17. In some embodiments, a composition is free of 12 of DS1 to DS17. In some embodiments, a composition is free of 13 of DS1 to DS17. In some embodiments, a composition is free of 14 of DS1 to DS17. In some embodiments, a composition is free of 15 of DS1 to DS17. In some embodiments, a composition is free of 16 of DS1 to DS17. In some embodiments, a composition is free of 17 of DS1 to DS17. In some embodiments, at least DS1 is absent 85 of 176 13004462v1Attorney Docket No.: 2010581-1542 from a composition. In some embodiments, at least DS2 is absent from a composition. In some embodiments, at least DS3 is absent from a composition. In some embodiments, at least DS4 is absent from a composition. In some embodiments, at least DS5 is absent from a composition. In some embodiments, at least DS6 is absent from a composition. In some embodiments, at least DS7 is absent from a composition. In some embodiments, at least DS8 is absent from a composition. In some embodiments, at least DS9 is absent from a composition. In some embodiments, at least DS10 is absent from a composition. In some embodiments, at least DS11 is absent from a composition. In some embodiments, at least DS12 is absent from a composition. In some embodiments, at least DS13 is absent from a composition. In some embodiments, at least DS14 is absent from a composition. In some embodiments, at least DS15 is absent from a composition. In some embodiments, at least DS16 is absent from a composition. In some embodiments, at least DS17 is absent from a composition.

[0210] Similar to WVE-N531, each of DS1 to DS17 may independently be in various forms. In some embodiments, in a composition it is in one form; in some embodiments, it is in two or more forms. In some embodiments, a form is a salt form. In some embodiments, a form is a pharmaceutically acceptable salt form. In some embodiments, each form is independently a salt form. In some embodiments, each form is independently a pharmaceutically acceptable salt form. In some embodiments, a form is hexadecasodium salt. In some embodiments, in a liquid composition, e.g., an optionally buffered solution, WVE-N531 and / or D1 to DS17 are dissolved and may dissociate to yield cations and anions.

[0211] The liquid WVE-N531 drug substance is packaged in sterile high density polyethylene (HDPE) bottles with polypropylene screw closures, labeled and sealed in a Mylar foil pouches which provide a gas / moisture barrier with high levels of abrasion and puncture resistance.

[0212] Various batches of WVE-N531 hexadecasodium salt have been manufactured as drug substance, in some instances, at about 20 mmol scale (e.g., in one instance, 2 x 23 mmol scale). Certain preparations was utilized in nonclinical studies including GLP toxicology studies. Certain preparations were manufactured for clinical studies. Manufactured products are stable: No significant changes in stability indicating attributes have been observed for GMP drug substance of multiple lots after 24 months of storage at the long-term storage condition of -20ºC ± 5ºC and after 14 days of storage at an accelerated condition of 5ºC ± 3ºC. Drug Product

[0213] In some embodiments, the present disclosure provides a pharmaceutical composition comprising or delivering WVE-N531 or a pharmaceutically acceptable salt form thereof and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides a pharmaceutical composition comprising WVE-N531 hexadecasodium salt and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides a WVE-N531 drug product. In 86 of 176 13004462v1Attorney Docket No.: 2010581-1542 some embodiments, a WVE-N531 drug product comprises a WVE-N531 drug substance, e.g., WVE-N531 hexadecasodium salt manufactured using a process described above.

[0214] In some embodiments, a pharmaceutically acceptable carrier is or comprises a buffered solution. In some embodiments, a pharmaceutically acceptable carrier is or comprises a phosphate buffered solution. In some embodiments, a pharmaceutically acceptable carrier is or comprises an isotonic phosphate buffered solution. In some embodiments, a WVE-N531 drug product is or comprises aWVE- N531 drug substance in an isotonic phosphate buffered solution. In some embodiments, a pharmaceutical composition or a drug product that closely matches physiological conditions in blood with respect to pH and tonicity. In some embodiments, it is for intravenous (IV) administration.

[0215] Certain WVE-N531 drug products and useful manufacturing technologies are described below as examples.

[0216] In some embodiments, a WVE-N531 pharmaceutical composition, e.g., a drug product is packaged in a 10 mL USP Type 1 clear glass vial capped with a FluroTec®coated chlorobutyl rubber stopper and an aluminum overseal with a white flip-off cap. In some embodiments, each drug product vial contains WVE-N531 drug substance equivalent to 36 mg WVE-N531 free acid form (6 mg / mL; nominal content 36 mg, total extractable volume 6 mL). In some embodiments, the components of a WVE-N531 drug product comprise or are WVE-N531 drug substance, potassium phosphate monobasic, sodium phosphate dibasic, and sodium chloride in water for injection (WFI). Sodium hydroxide and / or hydrochloric acid may also be used to adjust the pH of the formulated solution into the required range.

[0217] An example composition of WVE-N531 drug product is presented below: 87 of 176 13004462v1Attorney Docket No.: 2010581-1542 Total Amount Concentration Component Quality Standard Function Per Vial (mg / mL) eereva ons: = n e a es armacope a, = a ona ormuary, . ur. = uropean Pharmacopoeia, JP = Japanese Pharmacopoeia, BP = British Pharmacopoeia; qs = quantity sufficient, N / A = Not Applicable.

[0218] In some embodiments, a WVE-N531 drug product is a colorless solution with no visible material and essentially free of particulates. In some embodiments, pH of a 6 mg / mL solution in phosphate buffered saline solution is 7.0 – 7.8 with osmolality in the range of 280 – 320 mOsm / kg. In some embodiments, density of WVE-N531 drug product is 1.0 g / mL.

[0219] A WVE-N531 drug product lot was manufactured at a concentration of 36 mg / vial (free acid form). The 36 mg strength is achieved by filling 6.35 mL of a 6 mg / mL formulation which included a 0.35 mL overfill above the nominal volume of 6 mL. All material contact components were sterilized prior to use. The container closure system for WVE-N531 drug product comprises or consists of a 10 mL USP Type 1 clear glass vial sealed with a FluroTec®-coated rubber stopper and an aluminum overseal with a white flip-off cap. This container closure can protect the sterilized product from microbiological contamination.

[0220] A WVE-N531 drug product is typically a sterile solution. In some embodiments, a WVE-N531 drug product can be diluted; in some embodiments, with 0.9% sodium chloride or 0.45% sodium chloride as a sterile, preservative-free solution prior to intravenous (IV) administration. A drug product lot may be 88 of 176 13004462v1Attorney Docket No.: 2010581-1542 made by pooling multiple drug substance lots.

[0221] In some embodiments, a drug product has a purity as described herein, e.g., those described for WVE-N531 or a salt thereof (e.g., hexadecasodium salt salt), a WVE-N531 drug substance, etc. In some embodiments, purity is determined assessed using an IP-RP-UPLC method described herein for purity.

[0222] A flow diagram for a WVE-N531 drug product manufacturing process is provided in Figure 1 as an example.

[0223] WVE-N531 drug substance containers can be thawed at 2-8°C for approximately 48 hours. While drug substance thawing occurs, phosphate buffered saline (PBS) solutions at 1x and 2x strengths are prepared where the 1x solution contains 10 mM sodium / potassium phosphate and 142 mM of sodium chloride at pH 7.4 ± 0.2 and 285-315 mOsm / kg osmolality. Contents of the thawed drug substance from each container are pooled into the formulation vessel and mixed adequately.

[0224] If a single lot of drug substance is used for formulation, the release purity result is used as the purity correction factor. If multiple lots of drug substance are to be pooled, then a purity correction factor that is based on the quantity of active ingredient from each drug substance lot is determined.

[0225] In some embodiments, water for injection (WFI) is used to rinse each drug substance container and the rinsate is added to next drug substance container. The rinsate from final drug substance container is added to the formulation vessel. The total amount of drug substance is weighed and an equal amount of 2x PBS solution is added to the formulation vessel. An in-process sample is taken to measure the concentration by ultraviolet (UV) spectrophotometry and density.

[0226] Based on the results of WVE-N531 drug substance concentration and solution mass, the required volume of 1x PBS to accomplish WVE-N531 drug substance concentration of 6.0 mg / mL (WVE- N531 free acid form) is calculated. The required amount of 1x PBS is then added directly to the formulation vessel, and the contents of the vessel are mixed to ensure solution homogeneity.

[0227] In some embodiments, final in-process samples are collected from the formulation vessel for concentration by UV spectrophotometry, density, osmolality and pH. In some embodiments, WVE-N531 concentration in a drug product is about 6 mg / mL. In some embodiments, it is about 5.5-6.5 mg / mL (free acid form, purity corrected). The final formulation undergoes clarification filtration.

[0228] In some embodiments, an in-process pre-filtration bioburden sample is collected prior to sterile filtration. In some embodiments, the final formulation is sterile filtered through two 0.2 µm filters in series prior to filling. In some embodiments, sterile filters are checked for filter integrity by bubble point method prior to and post filtration.

[0229] Based upon the final measured value of density, the target fill weight is determined (to reach target fill volume of 6.35 mL / vial) along with alert limits and action limits. Filling occurs with periodic fill weight checks. The vials are filled, stoppered and sealed aseptically in a Grade A environment. Finished 89 of 176 13004462v1Attorney Docket No.: 2010581-1542 vials are visually inspected prior to release testing, bulk packaging, and / or stability studies.

[0230] In some embodiments, process controls and in-process control tests and acceptance limits applied during the manufacture of WVE-N531 drug product include WVE-N531 concentration, pH, osmolality, bioburden, filter integrity, fill weight, appearance, etc.

[0231] In some embodiments, manufacture of WVE-N531 drug product comprises in-process concentration measurements via ultraviolet (UV) spectrophotometry. In some embodiments, an in-process concentration measurement is made upon the initial dilution of WVE-N531 drug substance with equal amounts of 2x phosphate buffered saline (PBS). The result is used to determine the amount of 1x PBS to be added for the final concentration which is measured prior to sterile filtration and filling.

[0232] Release specifications of WVE-N531 drug product may include one or more specifications described herein, e.g., appearance (e.g., visual), identity (e.g., by retention time of IP-RP-UPLC), purity (e.g., area % by IP-RP-UPLC), impurities (e.g., area % by IP-RP-UPLC), % label claim (e.g., by UV; e.g., + / −10%), pH (e.g., USP <791> and / or Ph. Eur.2.2.3), osmolality (e.g., USP <785> and / or Ph. Eur.2.2.35), bacterial endotoxin (e.g., USP <85> and / or Ph. Eur.2.6.14), sterility (e.g., USP <71> and / or Ph. Eur.2.6.1), uniformity of dose (e.g., USP <905> and / or Ph. Eur. 2.9.40), particulate matter (e.g., USP <788> and / or Ph. Eur. 2.9.19), container content (e.g., USP <697>), container closure integrity testing (e.g., USP <1207>), etc. In some embodiments, IP-RP-UPLC technologies utilizing one or more or all Set A parameters are utilized for assessing identity (retention time), purity and / or impurities. In some embodiments, purity level is about 70%-85% as described herein. In some embodiments, it is about 70% or more. In some embodiments, it is about 71% or more. In some embodiments, it is about 72% or more. In some embodiments, it is about 73% or more. In some embodiments, it is about 74% or more. In some embodiments, it is about 75% or more. In some embodiments, it is about 76% or more. In some embodiments, it is about 77% or more. In some embodiments, it is about 78% or more. In some embodiments, it is about 79% or more. In some embodiments, it is about 80% or more. In some embodiments, it is about 81% or more. In some embodiments, it is about 82% or more. In some embodiments, it is about 83% or more. In some embodiments, it is about 84% or more. In some embodiments, it is about 85% or more. In some embodiments, it is measured by IP-RP-UPLC area % at 260 nm using Set A parameters.

[0233] A WVE-N531 drug product may comprise similar impurities as a WVE-N531 drug substance at levels as described herein.

[0234] In some embodiments, a container closure system for WVE-N531 drug product comprises or consists of USP Type 1 glass vials (10 mL capacity, 20 mm opening) with stoppers molded from FluroTec®coated elastomeric formula 4432 / 50 gray with aluminum overseals, and white flip-off caps.

[0235] In some embodiments, a storage condition for WVE-N531 drug product is -20 ± 5ºC. In some 90 of 176 13004462v1Attorney Docket No.: 2010581-1542 embodiments, a condition of 5°C ± 3°C, e.g., for relatively short-term storage.

[0236] The present disclosure incorporates various disclosures by reference. In the case of any conflict, the present disclosure will control.

[0237] Among other things, the present disclosure provides the following example Embodiments: 1. A method for treating muscular dystrophy, comprising administering to a subject suffering therefrom WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping. 2. A method for treating muscular dystrophy in a subject suffering therefrom, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, and wherein the subject has a mutation in the DMD gene that is amenable to exon 53 skipping. 3. A method, comprising administering to a subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 4. A method, comprising administering to a subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 5. A method for providing DMD exon 53 skipping in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15- 20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 6. A method for providing DMD exon 53 skipping in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 7. A method for restoring DMD RNA reading frame in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15- 20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 8. A method for restoring DMD RNA reading frame in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 91 of 176 13004462v1Attorney Docket No.: 2010581-1542 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 9. A method for providing a DMD polypeptide in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein. 10. A method for providing a DMD polypeptide in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein. 11. The method of any one of Embodiments 9-10, wherein the DMD polypeptide is encoded by an exon 53-skipped DMD mRNA. 12. The method of any one of Embodiments 9-11, wherein the DMD polypeptide provides one or more functions of a wild-type DMD protein. 13. A method for providing increased level of a DMD function in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 14. A method for providing increased level of a DMD function in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1- 20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 15. A method for providing decreased muscle inflammation in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 16. A method for providing decreased muscle inflammation in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 17. A method for providing decreased muscle necrosis in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15- 20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 92 of 176 13004462v1Attorney Docket No.: 2010581-1542 18. A method for providing decreased muscle necrosis in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 19. A method for providing decreased levels of one or more inflammatory biomarkers in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 20. A method for providing decreased levels of one or more inflammatory biomarkers in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 21. The method of Embodiment 19 or 20, wherein one or more inflammatory biomarkers comprise one or more inflammatory cytokines. 22. The method of any one of Embodiments 19-21, wherein one or more inflammatory biomarkers comprise monocyte chemoattractant protein-1 (MCP-1) and / or interleukin-6 (IL-6). 23. The method of any one of Embodiments 19-22, wherein one or more inflammatory biomarkers comprise one or more circulating inflammatory biomarkers. 24. A method for providing decreased levels of serum creatine kinase in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 25. A method for providing decreased levels of serum creatine kinase in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 26. A method for providing decreased muscle fibrosis in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15- 20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 27. A method for providing decreased muscle fibrosis in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 93 of 176 13004462v1Attorney Docket No.: 2010581-1542 28. A method for reversing muscle fibrosis in a subject, comprising administering to the subject WVE- N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 29. A method for reversing muscle fibrosis in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 30. A method for improving muscle health in a subject, comprising administering to the subject WVE- N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 31. A method for improving muscle health in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 32. A method for increasing transition from regenerative to mature muscle in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 33. A method for increasing transition from regenerative to mature muscle in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 34. A method for slowing progression of muscular dystrophy, comprising administering to a subject suffering therefrom WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10- 15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE- N531 free acid form. 35. A method for slowing progression of muscular dystrophy, comprising administering to a subject suffering therefrom one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 36. The method of Embodiment 34 or 35, wherein slowing of progression comprises slowing of worsening of one or more functional assessments of the subject. 37. The method of Embodiment 36, wherein the one or more functional assessments comprise time-to- rise (TTR). 94 of 176 13004462v1Attorney Docket No.: 2010581-1542 38. The method of Embodiment 37, wherein a slowing of worsening of TTR comprises a decreased TTR as compared to a subject not administered one or more doses of WVE-N531. 39. The method of any one of Embodiments 36-38, wherein the one or more functional assessments comprise North Star Ambulatory Assessment (NSAA). 40. The method of Embodiment 39, wherein a slowing of worsening of NSAA comprises an increased NSAA total score as compared to a subject not administered one or more doses of WVE-N531. 41. A method for slowing increase in time-to-rise (TTR) of a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15- 20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 42. A method, comprising administering to a subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein when TTR is observed, an improvement is achieved relative to natural history. 43. A method for slowing increase in time-to-rise (TTR) of a subject, comprising administering to a subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 44. A method, comprising administering one or more doses of WVE-N531 to a subject, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, and wherein when TTR is observed, an improvement is achieved relative to natural history. 45. A method for slowing decrease in North Star Ambulatory Assessment (NSAA) total score of a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 46. A method for slowing decrease in North Star Ambulatory Assessment (NSAA) total score of a subject, comprising administering one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 47. A method for increasing grip strength of a subject, comprising administering to the subject WVE- N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 48. A method for increasing grip strength of a subject, comprising administering one or more doses of 95 of 176 13004462v1Attorney Docket No.: 2010581-1542 WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 49. The method of Embodiment 47 or 48, wherein grip strength is increased as compared to a baseline. 50. The method of Embodiment 49, wherein the baseline is grip strength prior to administering one or more doses of WVE-N531. 51. The method of any one of Embodiments 3-50, wherein the subject is suffering from a muscular dystrophy. 52. The method of any one of Embodiments 3-51, wherein the subject is suffering from DMD. 53. The method of any one of Embodiments 3-52, wherein the subject has a mutation in the DMD gene that is amenable to exon 53 skipping. 54. The method of any one of the preceding Embodiments, wherein WVE-N531 is administered in one or more forms. 55. The method of any one of the preceding Embodiments, wherein WVE-N531 is administered in one or more pharmaceutically acceptable salt forms. 56. The method of any one of Embodiments 54-55, wherein one form is WVE-N531 hexadecasodium salt. 57. The method of any one of Embodiments 54-56, wherein each dose of WVE-N531 is independently administered in a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier. 58. A method for treating muscular dystrophy, comprising administering to a subject suffering therefrom a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping. 59. A method, comprising administering to a subject a pharmaceutical composition comprising WVE- N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 60. A method for providing DMD exon 53 skipping in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 61. A method for restoring DMD RNA reading frame, comprising administering to the subject a 96 of 176 13004462v1Attorney Docket No.: 2010581-1542 pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 62. A method for providing a DMD polypeptide in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein. 63. The method of Embodiment 62, wherein the DMD polypeptide is encoded by an exon 53-skipped DMD mRNA. 64. The method of any one of Embodiments 62-63, wherein the DMD polypeptide provides one or more functions of a wild-type DMD protein. 65. A method for providing increased level of a DMD function in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 66. A method for providing decreased muscle inflammation in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 67. A method for providing decreased muscle necrosis in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 68. A method for providing decreased levels of one or more inflammatory biomarkers in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 69. The method of Embodiment 68, wherein the one or more inflammatory biomarkers comprise one or more inflammatory cytokines. 70. The method of Embodiment 68 or 69, wherein one or more inflammatory biomarkers comprise monocyte chemoattractant protein-1 (MCP-1) and / or interleukin-6 (IL-6). 71. The method of any one of Embodiments 68-70, wherein one or more inflammatory biomarkers 97 of 176 13004462v1Attorney Docket No.: 2010581-1542 comprise one or more circulating inflammatory biomarkers. 72. A method for providing decreased levels of serum creatine kinase in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 73. A method for providing decreased muscle fibrosis in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 74. A method for reversing muscle fibrosis in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 75. A method for improving muscle health in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 76. A method for increasing transition from regenerative to mature muscle in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 77. A method for slowing progression of muscular dystrophy, comprising administering to a subject suffering therefrom a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 78. The method of Embodiment 77, wherein slowing of progression comprises slowing of worsening of one or more functional assessments of the subject. 79. The method of Embodiment 78, wherein one or more functional assessments comprise time-to-rise (TTR). 80. The method of Embodiment 79, wherein a slowing of worsening of TTR comprises a decreased TTR as compared to a subject not administered one or more doses of WVE-N531. 98 of 176 13004462v1Attorney Docket No.: 2010581-1542 81. The method of any one of Embodiments 78-80, wherein one or more functional assessments comprise North Star Ambulatory Assessment (NSAA). 82. The method of Embodiment 81, wherein a slowing of worsening of NSAA comprises an increased NSAA total score as compared to a subject not administered one or more doses of WVE-N531. 83. A method for slowing increase in time-to-rise (TTR) of a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 84. A method, comprising administering to a subject a pharmaceutical composition comprising WVE- N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein when TTR is assessed, an improvement is achieved relative to natural history. 85. A method for slowing decrease in North Star Ambulatory Assessment (NSAA) total score of a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 86. A method for increasing grip strength of a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 87. The method of Embodiment 86, wherein grip strength is increased as compared to a baseline. 88. The method of Embodiment 87, wherein the baseline is grip strength prior to administering one or more doses of WVE-N531. 89. The method of any one of Embodiments 59-88, wherein the subject is suffering from a muscular dystrophy. 90. The method of any one of Embodiments 59-89, wherein the subject is suffering from DMD. 91. The method of any one of Embodiments 59-90, wherein the subject has a mutation in the DMD gene that is amenable to exon 53 skipping. 92. The method of any one of the preceding Embodiments, wherein WVE-N531 in a dose is equivalent to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg WVE-N531 free acid form. 93. The method of any one of the preceding Embodiments, wherein WVE-N531 in a dose is equivalent 99 of 176 13004462v1Attorney Docket No.: 2010581-1542 to about 10 mg / kg WVE-N531 free acid form. 94. The method of any one of Embodiments 92-93, wherein about is ±1%. 95. The method of any one of Embodiments 92-93, wherein about is ±2%. 96. The method of any one of Embodiments 92-93, wherein about is ±3%. 97. The method of any one of Embodiments 92-93, wherein about is ±4%. 98. The method of any one of Embodiments 92-93, wherein about is ±5%. 99. The method of any one of Embodiments 92-93, wherein about is ±6%. 100. The method of any one of Embodiments 92-93, wherein about is ±7%. 101. The method of any one of Embodiments 92-93, wherein about is ±8%. 102. The method of any one of Embodiments 92-93, wherein about is ±9%. 103. The method of any one of Embodiments 92-93, wherein about is ±10%. 104. The method of any one of the preceding Embodiments, wherein two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) doses are administered. 105. The method of any one of the preceding Embodiments, wherein about 10 or more doses are administered. 106. The method of any one of the preceding Embodiments, wherein about 12 or more doses are administered. 107. The method of any one of the preceding Embodiments, wherein about 13 or more doses are administered. 108. The method of any one of the preceding Embodiments, wherein about 24 or more doses are administered. 109. The method of any one of the preceding Embodiments, wherein about 25 or more doses are administered. 110. The method of any one of Embodiments 104-109, wherein each dose is independently administered in a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier. 111. The method of any one of Embodiments 104-110, wherein each dose has about the same amount of WVE-N531. 112. The method of any one of Embodiments 104-111, wherein WVE-N531 in each dose is equivalent to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg WVE-N531 free acid form. 113. The method of any one of Embodiments 104-111, wherein WVE-N531 in each dose is equivalent to about 10 mg / kg WVE-N531 free acid form. 114. The method of any one of Embodiments 111-113, wherein about is ±1%. 100 of 176 13004462v1Attorney Docket No.: 2010581-1542 115. The method of any one of Embodiments 111-113, wherein about is ±2%. 116. The method of any one of Embodiments 111-113, wherein about is ±3%. 117. The method of any one of Embodiments 111-113, wherein about is ±4%. 118. The method of any one of Embodiments 111-113, wherein about is ±5%. 119. The method of any one of Embodiments 111-113, wherein about is ±6%. 120. The method of any one of Embodiments 111-113, wherein about is ±7%. 121. The method of any one of Embodiments 111-113, wherein about is ±8%. 122. The method of any one of Embodiments 111-113, wherein about is ±9%. 123. The method of any one of Embodiments 111-113, wherein about is ±10%. 124. The method of any one of the preceding Embodiments, wherein WVE-N531 exists in the pharmaceutical composition as one or more pharmaceutically acceptable salt forms. 125. The method of Embodiment 124, wherein a pharmaceutically acceptable salt form is hexadecasodium salt. 126. The method of any one of the preceding Embodiments, wherein a pharmaceutical composition is a liquid composition comprising dissolved WVE-N531. 127. The method of any one of the preceding Embodiments, wherein a pharmaceutically acceptable carrier is or comprises a phosphate buffered solution. 128. The method of any one of the preceding Embodiments, wherein the components in a pharmaceutical composition are WVE-N531, potassium phosphate monobasic, sodium phosphate dibasic, sodium chloride and water, and hydrochloric acid and / or sodium hydroxide for pH adjustment. 129. The method of any one of the preceding Embodiments, wherein a pharmaceutical composition is isotonic. 130. The method of any one of the preceding Embodiments, wherein a pharmaceutical composition has a pH of about 7-8. 131. The method of any one of the preceding Embodiments, wherein a pharmaceutical composition has a pH of about 7.3. 132. The method of Embodiment 130, wherein a pharmaceutical composition has a pH of about 7.4. 133. The method of any one of the preceding Embodiments, wherein two or more doses are administered about every four weeks. 134. The method of any one of the preceding Embodiments, wherein two or more doses are administered about monthly. 135. The method of any one of the preceding Embodiments, wherein two or more consecutive doses are administered about weekly, or about every 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or about every 1, 2, 3, 4, 5, 6, or more months. 101 of 176 13004462v1Attorney Docket No.: 2010581-1542 136. The method of any one of the preceding Embodiments, wherein 10 or more consecutive doses are administered about weekly, or about every 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or about every 1, 2, 3, 4, 5, 6, or more months. 137. The method of any one of the preceding Embodiments, wherein all doses are administered about weekly, or about every 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or about every 1, 2, 3, 4, 5, 6, or more months. 138. The method of any one of the preceding Embodiments, wherein two or more consecutive doses are administered about every two weeks. 139. The method of any one of the preceding Embodiments, wherein three consecutive doses are administered about every two weeks. 140. The method of any one of the preceding Embodiments, wherein three or more consecutive doses are administered about every two weeks. 141. The method of any one of the preceding Embodiments, wherein 10 or more consecutive doses are administered about every two weeks. 142. The method of any one of the preceding Embodiments, wherein all doses are administered about every two weeks. 143. The method of any one of Embodiments 1-137, wherein two or more consecutive doses are administered about every four weeks. 144. The method of any one of Embodiments 1-137, wherein 10 or more consecutive doses are administered about every four weeks. 145. The method of any one of Embodiments 1-137, wherein all doses are administered about every four weeks. 146. The method of any one of Embodiments 1-137, wherein two or more consecutive doses are administered about monthly. 147. The method of any one of Embodiments 1-137, wherein 10 or more consecutive doses are administered about monthly. 148. The method of any one of Embodiments 1-137, wherein all doses are administered about monthly. 149. The method of any one of Embodiments 135-148, wherein each of the consecutive doses is independently equivalent to about 10 mg / kg WVE-N531 free acid form. 150. The method of any one of Embodiments 1-137, wherein one or more doses are administered about every two weeks for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks from first dose and then doses are administered about every 3, 4, 5, 6, 7, 8, 9 or 10 weeks. 151. The method of any one of Embodiments 1-137, wherein doses are first administered about every two weeks, and then after about 4 weeks, 8 weeks, 12 weeks, or 16 weeks from first dose, doses are administered about every four weeks. 102 of 176 13004462v1Attorney Docket No.: 2010581-1542 152. The method of any one of Embodiments 1-137, wherein doses are first administered about every two weeks, and then after about 4 weeks, 8 weeks, 12 weeks, or 16 weeks from first dose, doses are administered about monthly. 153. The method of any one of Embodiments 1-137, wherein two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every two weeks, and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every four weeks. 154. The method of any one of Embodiments 1-137, wherein two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every two weeks, and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about monthly. 155. The method of any one of Embodiments 153-154, wherein the two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses administered about every two weeks are administered before two or more doses administered about every four weeks. 156. The method of any one of Embodiments 153-154, wherein the two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses administered about every two weeks are administered before two or more doses administered about monthly. 157. The method of any one of Embodiments 1-137, wherein 20 or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses are administered about every two weeks, and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every four weeks. 158. The method of any one of Embodiments 1-137, wherein 20 or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses are administered about every two weeks, and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about monthly. 159. The method of any one of Embodiments 157-158, wherein the 20 or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses administered about every two weeks are administered before two or more doses administered about every four weeks. 160. The method of any one of Embodiments 157-158, wherein the 20 or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses administered about every two weeks are administered before two or more doses administered about monthly. 161. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 70%-85%. 162. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 70%-90%. 163. The method of any one of the preceding Embodiments, wherein the composition has a purity of 103 of 176 13004462v1Attorney Docket No.: 2010581-1542 about 70%-80%. 164. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 70% or more. 165. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 71% or more. 166. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 72% or more. 167. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 73% or more. 168. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 74% or more. 169. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 75% or more. 170. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 76% or more. 171. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 77% or more. 172. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 78% or more. 173. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 79% or more. 174. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 80% or more. 175. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 81% or more. 176. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 82% or more. 177. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 83% or more. 178. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 84% or more. 179. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 85% or more. 180. The method of any one of the preceding Embodiments, wherein the composition has a purity of 104 of 176 13004462v1Attorney Docket No.: 2010581-1542 about 86% or more. 181. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 87% or more. 182. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 88% or more. 183. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 89% or more. 184. The method of any one of the preceding Embodiments, wherein the composition has a purity of about 90% or more. 185. The method of any one of Embodiments 161-184, wherein the purity is measured by IP-RP-UPLC using area % at 260 nm. 186. The method of any one of Embodiments 161-185, wherein the purity is measured by IP-RP-UPLC using area % at 260 nm and the Set A parameters. 187. The method of any one of Embodiments 161-185, wherein the purity is measured by an IP-RP- UPLC method for purity as described herein. 188. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about 10%-30%. 189. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about 15%-30%. 190. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about 20%-30%. 191. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 30%. 192. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 29%. 193. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 28%. 194. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 27%. 195. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 26%. 196. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 25%. 197. The method of any one of the preceding Embodiments, wherein impurities in the composition are 105 of 176 13004462v1Attorney Docket No.: 2010581-1542 no more than about or about 24%. 198. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 23%. 199. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 22%. 200. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 21%. 201. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 20%. 202. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 19%. 203. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 18%. 204. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 17%. 205. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 16%. 206. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 15%. 207. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 14%. 208. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 13%. 209. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 12%. 210. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 11%. 211. The method of any one of the preceding Embodiments, wherein impurities in the composition are no more than about or about 10%. 212. The method of any one of Embodiments 188-211, wherein the impurities are measured by IP-RP- UPLC using area % at 260 nm. 213. The method of any one of Embodiments 188-212, wherein the impurities are measured by IP-RP- UPLC using area % at 260 nm and the Set A parameters. 214. The method of any one of Embodiments 188-213, wherein the impurities are measured by an IP- 106 of 176 13004462v1Attorney Docket No.: 2010581-1542 RP-UPLC method for purity as described herein. 215. The method of any one of the preceding Embodiments, wherein stereochemical purity of WVE- N531 is about 80% or more. 216. The method of any one of the preceding Embodiments, wherein stereochemical purity of WVE- N531 is about 85% or more. 217. The method of any one of the preceding Embodiments, wherein stereochemical purity of WVE- N531 is about 80%-90%. 218. The method of any one of the preceding Embodiments, wherein stereochemical purity is assessed by dimer modeling. 219. The method of any one of the preceding Embodiments, wherein the amount of WVE-N531 is measured by UV at 260 nm and 27 OD / mg. 220. The method of any one of the preceding Embodiments, wherein a WVE-N531 drug substance is manufactured by a process described herein. 221. The method of any one of the preceding Embodiments, wherein a WVE-N531 drug substance is characterized by one or more method described herein. 222. The method of any one of the preceding Embodiments, wherein stereochemical identity of WVE- N531 is confirmed by IP-RP-UPLC. 223. The method of any one of the preceding Embodiments, wherein stereochemical identity of WVE- N531 is confirmed by IP-RP-UPLC according to Set B parameters. 224. The method of any one of the preceding Embodiments, wherein stereochemical identity of WVE- N531 is confirmed by an IP-RP-UPLC method for stereochemical identity as described herein. 225. The method of any one of the preceding Embodiments, wherein a WVE-N531 drug substance is released by one or more method described herein. 226. The method of any one of the preceding Embodiments, wherein a WVE-N531 drug substance is stored by one or more method described herein. 227. The method of any one of Embodiments 220-226, wherein the WVE-N531 drug substance is hexadecasodium salt. 228. The method of any one of the preceding Embodiments, wherein a WVE-N531 drug product is manufactured by a process described herein. 229. The method of any one of the preceding Embodiments, wherein a WVE-N531 drug product is characterized by one or more method described herein. 230. The method of any one of the preceding Embodiments, wherein a WVE-N531 drug product is released by one or more method described herein. 231. The method of any one of the preceding Embodiments, wherein a WVE-N531 drug product is 107 of 176 13004462v1Attorney Docket No.: 2010581-1542 stored by one or more method described herein. 232. The method of any one of the preceding Embodiments, wherein a pharmaceutical composition is manufactured by a process described herein. 233. The method of any one of the preceding Embodiments, wherein a pharmaceutical composition is characterized by one or more method described herein. 234. The method of any one of the preceding Embodiments, wherein a pharmaceutical composition is released by one or more method described herein. 235. The method of any one of the preceding Embodiments, wherein a pharmaceutical composition is stored by one or more method described herein. 236. The method of any one of the preceding Embodiments, wherein WVE-N531 is administered intravenously. 237. The method of any one of the preceding Embodiments, wherein a DMD mutation is Δ3-52, Δ4-52, Δ5-52, Δ6-52, Δ9-52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17-52, Δ19-52, Δ21-52, Δ23- 52, Δ24-52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32-52, Δ33-52, Δ34-52, Δ35- 52, Δ36-52, Δ37-52, Δ38-52, Δ39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ45-52, Δ47-52, Δ48-52, Δ49- 52, Δ50-52, Δ51-52, Δ52-52, Δ52, Δ54-58, Δ54-61, Δ54-63, Δ54-64, Δ54-66, Δ54-76, or Δ54-77. 238. The method of Embodiment 237, wherein the DMD mutation is Δ3-52, Δ4-52, Δ5-52, Δ6-52, Δ9- 52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17-52, Δ19-52, Δ21-52, Δ23-52, Δ24-52, Δ25- 52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32-52, Δ33-52, Δ34-52, Δ35-52, Δ36-52, Δ37- 52, Δ38-52, Δ39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ45-52, Δ47-52, Δ48-52, Δ49-52, Δ50-52, Δ51- 52, Δ52-52, or Δ52. 239. The method of Embodiment 237, wherein the DMD mutation is Δ45-52, Δ48-52, Δ49-52, Δ50-52, or Δ52-52. 240. The method of any one of the preceding Embodiments, wherein the mutation comprises Δ52. 241. The method of any one of the preceding Embodiments, wherein the mutation is Δ52. 242. The method of Embodiment 240, wherein the mutation is Δ45-52. 243. The method of Embodiment 240, wherein the mutation is Δ47-52. 244. The method of Embodiment 240, wherein the mutation is Δ48-52. 245. The method of Embodiment 240, wherein the mutation is Δ49-52. 246. The method of Embodiment 240, wherein the mutation is Δ50-52. 247. The method of Embodiment 240, wherein the mutation is Δ51-52. 248. The method of Embodiment 240, wherein the mutation is Δ52-52. 249. The method of any one of the preceding Embodiments, wherein the muscular dystrophin is DMD. 250. The method of any one of the preceding Embodiments, wherein exon 53 is skipped in DMD mRNA. 108 of 176 13004462v1Attorney Docket No.: 2010581-1542 251. The method of any one of the preceding Embodiments, wherein level of exon 53-skipped DMD mRNA is increased. 252. The method of any one of the preceding Embodiments, wherein about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more of DMD mRNA is exon 53-skipped DMD mRNA after administration for a certain time period or after a certain number of doses. 253. The method of any one of the preceding Embodiments, wherein a truncated DMD polypeptide is produced compared to a wild-type DMD protein. 254. The method of any one of the preceding Embodiments, wherein level of a truncated DMD polypeptide is increased. 255. The method of any one of Embodiments 253-254, wherein the truncated DMD polypeptide performs one or more functions of a wild-type DMD protein. 256. The method of any one of the preceding Embodiments, wherein the method provide increases from baseline in dystrophin levels of about 1% or more of normal levels after administration for a certain time period or after a certain number of doses. 257. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 2% or more of normal levels. 258. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 5% or more of normal levels.259. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 5.3% or more of normal levels. 260. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 6% or more of normal levels. 261. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 7% or more of normal levels. 262. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 8% or more of normal levels. 263. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 9% or more of normal levels. 264. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 10% or more of normal levels.265. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 11% or more of normal levels. 266. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 12% or more of normal levels. 109 of 176 13004462v1Attorney Docket No.: 2010581-1542 267. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 13% or more of normal levels. 268. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 14% or more of normal levels. 269. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 15% or more of normal levels. 270. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 16% or more of normal levels. 271. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 17% or more of normal levels. 272. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 18% or more of normal levels. 273. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 19% or more of normal levels. 274. The method of Embodiment 256, wherein the increases from baseline in dystrophin levels is of about 20% or more of normal levels. 275. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 12 weeks from first dose. 276. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 13 weeks from first dose. 277. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 14 weeks from first dose. 278. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 24 weeks from first dose. 279. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 25 weeks from first dose. 280. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 26 weeks from first dose. 281. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 36 weeks from first dose. 282. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 37 weeks from first dose. 283. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 38 weeks from first dose. 110 of 176 13004462v1Attorney Docket No.: 2010581-1542 284. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 48 weeks from first dose. 285. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 49 weeks from first dose. 286. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 50 weeks from first dose. 287. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 72 weeks from first dose. 288. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 73 weeks from first dose. 289. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 74 weeks from first dose. 290. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 96 weeks from first dose. 291. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 97 weeks from first dose. 292. The method of any one of Embodiments 256-274, wherein the increase is measured after administration for about 98 weeks from first dose. 293. The method of any one of Embodiments 256-292, wherein the increase is measured after 6 doses. 294. The method of any one of Embodiments 256-292, wherein the increase is measured after 7 doses. 295. The method of any one of Embodiments 256-292, wherein the increase is measured after 8 doses. 296. The method of any one of Embodiments 256-292, wherein the increase is measured after 12 doses. 297. The method of any one of Embodiments 256-292, wherein the increase is measured after 13 doses. 298. The method of any one of Embodiments 256-292, wherein the increase is measured after 14 doses. 299. The method of any one of Embodiments 256-292, wherein the increase is measured after 24 doses. 300. The method of any one of Embodiments 256-292, wherein the increase is measured after 25 doses. 301. The method of any one of Embodiments 256-292, wherein the increase is measured after 30 doses. 302. The method of any one of Embodiments 256-292, wherein the increase is measured after 35 doses. 303. The method of any one of Embodiments 256-292, wherein the increase is measured after 40 doses. 304. The method of any one of Embodiments 256-292, wherein the increase is measured after 45 doses. 305. The method of any one of Embodiments 256-292, wherein the increase is measured after 48 doses. 306. The method of any one of the preceding Embodiments, wherein a DMD function is increased. 307. The method of any one of the preceding Embodiments, wherein a DMD function is restored. 308. The method of any one of the preceding Embodiments, wherein level of muscle inflammation in 111 of 176 13004462v1Attorney Docket No.: 2010581-1542 the subject is decreased. 309. The method of Embodiment 308, wherein level of muscle inflammation is assessed using a biopsy sample from the subject. 310. The method of Embodiment 308 or 309, wherein level of muscle inflammation is assessed as described in Figure 7. 311. The method of any one of Embodiments 308-310, wherein level of muscle inflammation is assessed after administration of 24 doses of WVE-N531 and the level of muscle inflammation is decreased as compared to after administration of 12 doses of WVE-N531. 312. The method of any one of Embodiments 308-311, wherein level of muscle inflammation is assessed after administration for about 48 weeks from first dose and the level of muscle inflammation is decreased as compared to after administration for about 24 weeks from first dose. 313. The method of any one of the preceding Embodiments, wherein level of muscle necrosis in the subject is decreased. 314. The method of Embodiment 313, wherein level of muscle necrosis is assessed using a biopsy sample from the subject. 315. The method of Embodiment 313 or 314, wherein level of muscle necrosis is assessed as described in Figure 7. 316. The method of any one of Embodiments 313-315, wherein level of muscle necrosis is assessed after administration of 24 doses of WVE-N531 and the level of muscle necrosis is decreased as compared to after administration of 12 doses of WVE-N531. 317. The method of any one of Embodiments 313-316, wherein level of muscle necrosis is assessed after administration for about 48 weeks from first dose and the level of muscle necrosis is decreased as compared to after administration for about 24 weeks from first dose. 318. The method of any one of the preceding Embodiments, wherein level of muscle fibrosis in the subject is decreased. 319. The method of Embodiment 318, wherein level of muscle fibrosis is assessed using a biopsy sample from the subject. 320. The method of Embodiment 318 or 319, wherein level of muscle fibrosis is assessed using trichrome staining of a biopsy sample from the subject. 321. The method of any one of Embodiments 318-320, wherein level of muscle fibrosis is assessed after administration of 24 doses of WVE-N531 and the level of muscle fibrosis is decreased as compared to after administration of 12 doses of WVE-N531. 322. The method of any one of Embodiments 318-321, wherein level of muscle fibrosis is assessed after administration for about 48 weeks from first dose and the level of muscle fibrosis is decreased as compared 112 of 176 13004462v1Attorney Docket No.: 2010581-1542 to after administration for about 24 weeks from first dose. 323. The method of any one of the preceding Embodiments, wherein muscle fibrosis in the subject is reversed. 324. The method of any one of the preceding Embodiments, wherein level of one or more inflammatory biomarkers in the subject is decreased. 325. The method of Embodiment 324, wherein one or more inflammatory biomarkers comprise one or more inflammatory cytokines. 326. The method of Embodiment 324 or 325, wherein one or more inflammatory biomarkers comprise monocyte chemoattractant protein-1 (MCP-1) and / or interleukin-6 (IL-6). 327. The method of any one of Embodiments 324-326, wherein one or more inflammatory biomarkers comprise one or more circulating inflammatory biomarkers. 328. The method of any one of Embodiments 324-327, wherein level of one or more inflammatory biomarkers is assessed using a serum sample from the subject. 329. The method of any one of Embodiments 324-328, wherein levels of one or more inflammatory biomarkers is assessed after administration of 12 doses of WVE-N531 and the level of one or more inflammatory biomarkers is decreased as compared to baseline. 330. The method of any one of Embodiments 324-328, wherein levels of one or more inflammatory biomarkers is assessed after administration of 24 doses of WVE-N531 and the level of one or more inflammatory biomarkers is decreased as compared to baseline. 331. The method of any one of Embodiments 324-328, wherein levels of one or more inflammatory biomarkers is assessed after administration of 24 doses of WVE-N531 and the level of one or more inflammatory biomarkers is decreased as compared to after administration of 12 doses of WVE-N531. 332. The method of any one of Embodiments 324-331, wherein level of one or more inflammatory biomarkers is assessed after administration for about 24 weeks from first dose and the level of one or more inflammatory biomarkers is decreased as compared to baseline. 333. The method of any one of Embodiments 324-331, wherein level of one or more inflammatory biomarkers is assessed after administration for about 48 weeks from first dose and the level of one or more inflammatory biomarkers is decreased as compared to baseline. 334. The method of any one of Embodiments 324-331, wherein level of one or more inflammatory biomarkers is assessed after administration for about 48 weeks from first dose and the level of one or more inflammatory biomarkers is decreased as compared to after administration for about 24 weeks from first dose. 335. The method of any one of the preceding Embodiments, wherein loss of ambulation in the subject is reduced. 113 of 176 13004462v1Attorney Docket No.: 2010581-1542 336. The method of any one of the preceding Embodiments, wherein disease progression in the subject is delayed or slowed. 337. The method of any one of the preceding Embodiments, wherein muscle health in the subject is improved. 338. The method of any one of the preceding Embodiments, wherein transition from regenerative muscle to mature muscle in the subject is increased. 339. The method of any one of the preceding Embodiments, wherein muscle weakness in the subject is delayed or slowed. 340. The method of any one of the preceding Embodiments, wherein loss of muscle mass in the subject is delayed or slowed. 341. The method of any one of the preceding Embodiments, wherein loss of pulmonary function in the subject is delayed or slowed. 342. The method of any one of the preceding Embodiments, wherein the subject improves in a muscular dystrophy assessment. 343. The method of any one of the preceding Embodiments, wherein the subject improves in a 10 meter walk test. 344. The method of any one of the preceding Embodiments, wherein the subject improves in one or more functional assessments. 345. The method of any one of the preceding Embodiments, wherein the subject improves in North Star Ambulatory Assessment (NSAA) 2.0. 346. The method of any one of the preceding Embodiments, wherein the subject improves in Performance of the Upper Limb (PUL) 2.0. 347. The method of any one of the preceding Embodiments, wherein the subject improves in one or more lower limb motor function by timed function tests. 348. The method of any one of the preceding Embodiments, wherein the subject improves in four-stair climb. 349. The method of any one of the preceding Embodiments, wherein the subject improves in time to rise from the floor. 350. The method of any one of the preceding Embodiments, wherein the subject improves in upper limb proximal strength. 351. The method of any one of the preceding Embodiments, wherein the subject improves in handheld myometry. 352. The method of any one of the preceding Embodiments, wherein the subject improves in grip strength. 114 of 176 13004462v1Attorney Docket No.: 2010581-1542 353. The method of any one of the preceding Embodiments, wherein the subject improves in grip strength in both hands. 354. The method of Embodiment 352 or 353, wherein the improvement in grip strength is from baseline. 355. The method of any one of Embodiments 352-354, wherein grip strength is assessed using handheld myometry. 356. The method of any one of Embodiments 352-355, wherein grip strength is assessed after administration of 24 doses of WVE-N531 and the improvement in grip strength is as compared to after administration of 12 doses of WVE-N531. 357. The method of any one of the preceding Embodiments, wherein the subject improves in one or more pulmonary function tests. 358. The method of any one of the preceding Embodiments, wherein the subject improves in one or more of peak flow rate [PFR], cough peak flow [CPF], and FVC. 359. The method of any one of the preceding Embodiments, wherein the increase or improvement is assessed in a population of subjects. 360. The method of any one of the preceding Embodiments, comprising administering WVE-N531 to a subject according to a regimen, wherein the regimen comprises 12 or more doses of WVE-N531 administered about every two weeks, wherein each dose is independently equivalent to about 10 mg / kg WVE-N531 free acid form. 361. The method of any one of the preceding Embodiments, wherein the first dose of the 12 or more doses is the first dose of the regimen. 362. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects mean muscle content-adjusted dystrophin expression of about 9% of dystrophin level in healthy subjects (normal). 363. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects mean muscle content-adjusted myosin heavy chain (MHC)-normalized dystrophin expression of about 9% of dystrophin level in healthy subjects (normal). 364. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects mean myosin heavy chain (MHC)-normalized dystrophin expression of about 5.5% of dystrophin level in healthy subjects (normal). 365. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects muscle content-adjusted MHC-normalized dystrophin levels independently of at least 5% of normal in about 89% of the ambulatory subjects. 366. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects muscle-content adjusted MHC-normalized dystrophin levels of 115 of 176 13004462v1Attorney Docket No.: 2010581-1542 independently at least 5% of normal in 8 out of 9 subjects, wherein there are only 9 subjects in the population who are ambulatory and whose biopsy samples are assessed for dystrophin level. 367. The method of any one of the preceding Embodiments, wherein dystrophin expression was quantified from two isoforms consistent with those observed in Becker patients who display milder disease. 368. The method of any one of the preceding Embodiments, wherein dystrophin expression was quantified by western blot. 369. The method of any one of the preceding Embodiments, wherein dystrophin expression was quantified by western blot using AB15277. 370. The method of any one of the preceding Embodiments, wherein muscle-content adjustment is by total myofiber area / total area of biopsy section. 371. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve results described in Figure 2. 372. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve results described in Example 1. 373. The method of any one of the preceding Embodiments, wherein dystrophin expression comprises expression of two or more functional dystrophin isoforms. 374. The method of any one of the preceding Embodiments, wherein dystrophin expression comprises expression of one or more dystrophin isoforms observed in BMD subjects. 375. The method of any one of the preceding Embodiments, wherein dystrophin localization to sarcolemma membrane is demonstrated. 376. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects mean exon 53 skipping of about 57%. 377. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects mean muscle concentration about 41 ug / g. 378. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects muscle tissue half-life about 61 days. 379. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects detection in myocyte nuclei in all subjects. 380. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects detection in myogenic stem cells in the majority of participants. 381. The method of Embodiment 380, wherein myogenic stem cells are identified by PAX7 immunohistochemistry. 382. The method of any one of Embodiments 379-380, wherein WVE-N531 are detected by in-situ hybridization. 116 of 176 13004462v1Attorney Docket No.: 2010581-1542 383. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve evidence of myocyte regeneration. 384. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve evidence of improvements in muscle health. 385. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve an increase in the mean percentage of myocytes with internalized nuclei. 386. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects an improvement in myofiber size and / or diameter. 387. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects an improvement in myofiber size and / or diameter in one or more subjects of the population. 388. The method of any one of the preceding Embodiments, wherein no serious adverse effects are observed. 389. The method of any one of the preceding Embodiments, wherein no discontinuation due to adverse effects are observed. 390. The method of any one of Embodiments 360-389, wherein the assessment is performed about 2 weeks after the 12thdose, wherein each dose is independently equivalent to about 10 mg / kg WVE-N531 free acid form and administered about every 2 weeks. 391. The method of any one of the preceding Embodiments, comprising administering WVE-N531 to a subject according to a regimen, wherein the regimen comprises 24 or more doses of WVE-N531 administered about every two weeks, wherein each dose is independently equivalent to about 10 mg / kg WVE-N531 free acid form. 392. The method of any one of the preceding Embodiments, wherein the first dose of the 24 or more doses is the first dose of the regimen. 393. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects mean muscle content-adjusted dystrophin expression of about 7.8% of dystrophin level in healthy subjects (normal). 394. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects mean muscle content-adjusted myosin heavy chain (MHC)-normalized dystrophin expression of about 7.8% of dystrophin level in healthy subjects (normal). 395. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects mean muscle content-adjusted dystrophin expression of about 6.4% of dystrophin level in healthy subjects (normal). 396. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to 117 of 176 13004462v1Attorney Docket No.: 2010581-1542 achieve in a population of subjects mean muscle content-adjusted myosin heavy chain (MHC)-normalized dystrophin expression of about 6.4% of dystrophin level in healthy subjects (normal). 397. The method of any one of the preceding Embodiments, wherein the regimen is demonstrated to achieve in a population of subjects muscle content-adjusted MHC-no...

Claims

Attorney Docket No.: 2010581-1542 CLAIMS 1. A method for providing decreased muscle fibrosis in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15- 20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for providing decreased muscle fibrosis in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

2. A method for reversing muscle fibrosis in a subject, comprising administering to the subject WVE- N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for reversing muscle fibrosis in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

3. A method for providing decreased muscle inflammation in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for providing decreased muscle inflammation in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

4. A method for providing decreased muscle necrosis in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15- 20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for providing decreased muscle necrosis in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

5. A method for improving muscle health in a subject, comprising administering to the subject WVE- 153 of 176 13004462v1Attorney Docket No.: 2010581-1542 N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for improving muscle health in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

6. A method for increasing transition from regenerative to mature muscle in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for increasing transition from regenerative to mature muscle in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

7. A method for providing decreased levels of one or more inflammatory biomarkers in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for providing decreased levels of one or more inflammatory biomarkers in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

8. A method for providing decreased levels of serum creatine kinase in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for providing decreased levels of serum creatine kinase in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

9. A method for slowing progression of muscular dystrophy, comprising administering to a subject suffering therefrom WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10- 15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE- N531 free acid form; or 154 of 176 13004462v1Attorney Docket No.: 2010581-1542 a method for slowing progression of muscular dystrophy, comprising administering to a subject suffering therefrom one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

10. A method for slowing increase in time-to-rise (TTR) of a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15- 20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for slowing increase in time-to-rise (TTR) of a subject, comprising administering to a subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method, comprising administering to a subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein when TTR is observed, an improvement is achieved relative to natural history; or a method, comprising administering one or more doses of WVE-N531 to a subject, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, and wherein when TTR is observed, an improvement is achieved relative to natural history.

11. A method for slowing decrease in North Star Ambulatory Assessment (NSAA) total score of a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for slowing decrease in North Star Ambulatory Assessment (NSAA) total score of a subject, comprising administering one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

12. A method for increasing grip strength of a subject, comprising administering to the subject WVE- N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for increasing grip strength of a subject, comprising administering one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 155 of 176 13004462v1Attorney Docket No.: 2010581-1542 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

13. A method for treating muscular dystrophy, comprising administering to a subject suffering therefrom WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping; or a method for treating muscular dystrophy in a subject suffering therefrom, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, and wherein the subject has a mutation in the DMD gene that is amenable to exon 53 skipping; or a method, comprising administering to a subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method, comprising administering to a subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for providing DMD exon 53 skipping in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for providing DMD exon 53 skipping in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for restoring DMD RNA reading frame in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15- 20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for restoring DMD RNA reading frame in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or 156 of 176 13004462v1Attorney Docket No.: 2010581-1542 a method for providing a DMD polypeptide in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein; or a method for providing a DMD polypeptide in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein; or a method for providing increased level of a DMD function in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for providing increased level of a DMD function in a subject, comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1- 20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

14. A method for providing decreased muscle fibrosis in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

15. A method for reversing muscle fibrosis in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

16. A method for providing decreased muscle inflammation in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

17. A method for providing decreased muscle necrosis in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

18. A method for improving muscle health in a subject, comprising administering to the subject a 157 of 176 13004462v1Attorney Docket No.: 2010581-1542 pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

19. A method for increasing transition from regenerative to mature muscle in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

20. A method for providing decreased levels of one or more inflammatory biomarkers in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

21. A method for providing decreased levels of serum creatine kinase in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

22. A method for slowing progression of muscular dystrophy, comprising administering to a subject suffering therefrom a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

23. A method for slowing increase in time-to-rise (TTR) of a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method, comprising administering to a subject a pharmaceutical composition comprising WVE- N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein when TTR is assessed, an improvement is achieved relative to natural history.

24. A method for slowing decrease in North Star Ambulatory Assessment (NSAA) total score of a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and 158 of 176 13004462v1Attorney Docket No.: 2010581-1542 a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

25. A method for increasing grip strength of a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

26. A method for treating muscular dystrophy, comprising administering to a subject suffering therefrom a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping; or a method, comprising administering to a subject a pharmaceutical composition comprising WVE- N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for providing DMD exon 53 skipping in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for restoring DMD RNA reading frame, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or a method for providing a DMD polypeptide in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein; or a method for providing increased level of a DMD function in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

27. The method of claim 7 or 20, wherein: 159 of 176 13004462v1Attorney Docket No.: 2010581-1542 (i) one or more inflammatory biomarkers comprise one or more inflammatory cytokines; (ii) one or more inflammatory biomarkers comprise monocyte chemoattractant protein-1 (MCP-1) and / or interleukin-6 (IL-6); and / or (iii) one or more inflammatory biomarkers comprise one or more circulating inflammatory biomarkers.

28. The method of claim 9 or 22, wherein slowing of progression comprises slowing of worsening of one or more functional assessments of the subject, optionally wherein: (i) one or more functional assessments comprise time-to-rise (TTR), optionally wherein a slowing of worsening of TTR comprises a decreased TTR as compared to a subject not administered one or more doses of WVE-N531; and / or (ii) one or more functional assessments comprise North Star Ambulatory Assessment (NSAA), optionally wherein a slowing of worsening of NSAA comprises an increased NSAA total score as compared to a subject not administered one or more doses of WVE-N531.

29. The method of claim 12 or 25, wherein grip strength is increased as compared to a baseline, optionally wherein the baseline is grip strength prior to administering one or more doses of WVE-N531.

30. The method of claim 13 or 26, wherein the DMD polypeptide is encoded by an exon 53-skipped DMD mRNA and / or wherein the DMD polypeptide provides one or more functions of a wild-type DMD protein.

31. The method of any one of the preceding claims, wherein: (i) the subject is suffering from a muscular dystrophy; (ii) the subject is suffering from DMD; and / or (iii) the subject has a mutation in the DMD gene that is amenable to exon 53 skipping.

32. The method of any one of the preceding claims, wherein WVE-N531 is administered in one or more forms and / or WVE-N531 is administered in one or more pharmaceutically acceptable salt forms; optionally wherein: (i) one form is WVE-N531 hexadecasodium salt; and / or (ii) each dose of WVE-N531 is independently administered in a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier.

33. The method of any one of the preceding claims, wherein WVE-N531 in a dose is equivalent to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg WVE-N531 free acid form; and / or wherein WVE-N531 in a dose is equivalent to about 10 mg / kg WVE-N531 free acid form.

34. The method of any one of the preceding claims, wherein two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) doses are administered; and / or wherein about 10, 12, 13, 24, or 25 or more doses are administered; optionally wherein: 160 of 176 13004462v1Attorney Docket No.: 2010581-1542 (i) each dose is independently administered in a pharmaceutical composition comprising WVE- N531 and a pharmaceutically acceptable carrier; (ii) each dose has about the same amount of WVE-N531; (iii) WVE-N531 in each dose is equivalent to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg WVE-N531 free acid form; and / or (iv) WVE-N531 in each dose is equivalent to about 10 mg / kg WVE-N531 free acid form.

35. The method of any one of the preceding claims, wherein: (i) WVE-N531 exists in the pharmaceutical composition as one or more pharmaceutically acceptable salt forms, optionally wherein a pharmaceutically acceptable salt form is hexadecasodium salt; (ii) a pharmaceutical composition is a liquid composition comprising dissolved WVE-N531; (iii) a pharmaceutically acceptable carrier is or comprises a phosphate buffered solution; (iv) the components in a pharmaceutical composition are WVE-N531, potassium phosphate monobasic, sodium phosphate dibasic, sodium chloride and water, and hydrochloric acid and / or sodium hydroxide for pH adjustment; (v) a pharmaceutical composition is isotonic; and / or (vi) a pharmaceutical composition has a pH of about 7-8 and / or about 7.3 or 7.

4.

36. The method of any one of the preceding claims, wherein: (i) two or more doses are administered about every four weeks; and / or (ii) two or more doses are administered about monthly.

37. The method of any one of the preceding claims, wherein: (i) two or more consecutive doses are administered about weekly, or about every 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or about every 1, 2, 3, 4, 5, 6, or more months; (ii) 10 or more consecutive doses are administered about weekly, or about every 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or about every 1, 2, 3, 4, 5, 6, or more months; and / or (iii) all doses are administered about weekly, or about every 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or about every 1, 2, 3, 4, 5, 6, or more months.

38. The method of any one of the preceding claims, wherein: (i) two or more consecutive doses are administered about every two weeks; (ii) three consecutive doses are administered about every two weeks; (iii) three or more consecutive doses are administered about every two weeks; (iv) 10 or more consecutive doses are administered about every two weeks; and / or (v) all doses are administered about every two weeks.

39. The method of any one of claims 1-37, wherein: (i) two or more consecutive doses are administered about every four weeks; 161 of 176 13004462v1Attorney Docket No.: 2010581-1542 (ii) 10 or more consecutive doses are administered about every four weeks; (iii) all doses are administered about every four weeks; (iv) two or more consecutive doses are administered about monthly; (v) 10 or more consecutive doses are administered about monthly; and / or (vi) all doses are administered about monthly.

40. The method of any one of claims 37-39, wherein each of the consecutive doses is independently equivalent to about 10 mg / kg WVE-N531 free acid form.

41. The method of any one of claims 1-37, wherein: (i) one or more doses are administered about every two weeks for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks from first dose and then doses are administered about every 3, 4, 5, 6, 7, 8, 9 or 10 weeks; (ii) doses are first administered about every two weeks, and then after about 4 weeks, 8 weeks, 12 weeks, or 16 weeks from first dose, doses are administered about every four weeks; (iii) doses are first administered about every two weeks, and then after about 4 weeks, 8 weeks, 12 weeks, or 16 weeks from first dose, doses are administered about monthly; (iv) two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every two weeks, and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every four weeks; optionally wherein the two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses administered about every two weeks are administered before two or more doses administered about every four weeks; (v) two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every two weeks, and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about monthly; optionally wherein the two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses administered about every two weeks are administered before two or more doses administered about monthly; (vi) 20 or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses are administered about every two weeks, and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every four weeks; optionally wherein the 20 or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses administered about every two weeks are administered before two or more doses administered about every four weeks; and / or (vii) 20 or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses are administered about every two weeks, and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about monthly; optionally wherein 20 or more (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) doses administered about every two weeks are administered before two or more 162 of 176 13004462v1Attorney Docket No.: 2010581-1542 doses administered about monthly.

42. The method of any one of the preceding claims, wherein: (i) the composition has a purity of about 70%-90%, about 70%-85%, or about 70%-80%; and / or the composition has a purity of about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or more; optionally wherein the purity is measured by IP-RP-UPLC using area % at 260 nm, by IP-RP- UPLC using area % at 260 nm and the Set A parameters, and / or by an IP-RP-UPLC method for purity as described herein; (ii) impurities in the composition are no more than about 10%-30%, about 15-30%, or about 20%- 30%; and / or impurities in the composition are no more than about or about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10%; optionally wherein the impurities are measured by IP-RP-UPLC using area % at 260 nm, by IP- RP-UPLC using area % at 260 nm and the Set A parameters, and / or by an IP-RP-UPLC method for purity as described herein; (iii) stereochemical purity of WVE-N531 is about 80% or more, about 85% or more, or about 80%- 90%; optionally wherein stereochemical purity is assessed by dimer modeling; (iv) the amount of WVE-N531 is measured by UV at 260 nm and 27 OD / mg; and / or (v) stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC, by IP-RP-UPLC according to Set B parameters, and / or by an IP-RP-UPLC method for stereochemical identity as described herein.

43. The method of any one of the preceding claims, wherein: (i) a WVE-N531 drug substance is manufactured by a process described herein or characterized, released, or stored by one or more method described herein; optionally wherein the WVE-N531 drug substance is hexadecasodium salt; (ii) a WVE-N531 drug product is manufactured by a process described herein or characterized, released, or stored by one or more method described herein; and / or (iii) a pharmaceutical composition is manufactured by a process described herein or characterized, released, or stored by one or more method described herein.

44. The method of any one of the preceding claims, wherein WVE-N531 is administered intravenously.

45. The method of any one of the preceding claims, wherein a DMD mutation is Δ3-52, Δ4-52, Δ5-52, Δ6-52, Δ9-52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17-52, Δ19-52, Δ21-52, Δ23-52, Δ24- 52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32-52, Δ33-52, Δ34-52, Δ35-52, Δ36- 52, Δ37-52, Δ38-52, Δ39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ45-52, Δ47-52, Δ48-52, Δ49-52, Δ50- 52, Δ51-52, Δ52-52, Δ52, Δ54-58, Δ54-61, Δ54-63, Δ54-64, Δ54-66, Δ54-76, or Δ54-77, optionally 163 of 176 13004462v1Attorney Docket No.: 2010581-1542 wherein: (i) the DMD mutation is Δ3-52, Δ4-52, Δ5-52, Δ6-52, Δ9-52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17-52, Δ19-52, Δ21-52, Δ23-52, Δ24-52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32-52, Δ33-52, Δ34-52, Δ35-52, Δ36-52, Δ37-52, Δ38-52, Δ39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ45-52, Δ47-52, Δ48-52, Δ49-52, Δ50-52, Δ51-52, Δ52-52, or Δ52; or (ii) the DMD mutation is Δ45-52, Δ48-52, Δ49-52, Δ50-52, or Δ52-52.

46. The method of any one of the preceding claims, wherein the mutation comprises or is Δ52, optionally wherein the mutation is Δ45-52, Δ47-52, Δ48-52, Δ49-52, Δ50-52, or Δ52-52.

47. The method of any one of the preceding claims, wherein the muscular dystrophin is DMD.

48. The method of any one of the preceding claims, wherein: (i) exon 53 is skipped in DMD mRNA; (ii) level of exon 53-skipped DMD mRNA is increased; and / or (iii) about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more of DMD mRNA is exon 53-skipped DMD mRNA after administration for a certain time period or after a certain number of doses.

49. The method of any one of the preceding claims, wherein: (i) a truncated DMD polypeptide is produced compared to a wild-type DMD protein; and / or (ii) level of a truncated DMD polypeptide is increased; optionally wherein the truncated DMD polypeptide performs one or more functions of a wild-type DMD protein.

50. The method of any one of the preceding claims, wherein the method provide increases from baseline in dystrophin levels of about 1% or more of normal levels after administration for a certain time period or after a certain number of doses, optionally wherein: (i) the increases from baseline in dystrophin levels is of about 2%, 5%, 5.3%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or more of normal levels; (ii) the increase is measured after administration for about 12, 13, 14, 24, 25, 26, 36, 37, 38, 48, 49, 50, 72, 73, 74, 96, 97, or 98 weeks from first dose; and / or (iii) the increase is measured after 6, 7, 8, 12, 13, 14, 24, 25, 30, 35, 40, 45, or 48 doses.

51. The method of any one of the preceding claims, wherein a DMD function is increased or restored.

52. The method of any one of the preceding claims, wherein level of muscle inflammation in the subject is decreased, optionally wherein: (i) level of muscle inflammation is assessed using a biopsy sample from the subject; (ii) level of muscle inflammation is assessed as described in Figure 7; (iii) level of muscle inflammation is assessed after administration of 24 doses of WVE-N531 and 164 of 176 13004462v1Attorney Docket No.: 2010581-1542 the level of muscle inflammation is decreased as compared to after administration of 12 doses of WVE- N531; and / or (iv) level of muscle inflammation is assessed after administration for about 48 weeks from first dose and the level of muscle inflammation is decreased as compared to after administration for about 24 weeks from first dose.

53. The method of any one of the preceding claims, wherein level of muscle necrosis in the subject is decreased, optionally wherein: (i) level of muscle necrosis is assessed using a biopsy sample from the subject; (ii) level of muscle necrosis is assessed as described in Figure 7; (iii) level of muscle necrosis is assessed after administration of 24 doses of WVE-N531 and the level of muscle necrosis is decreased as compared to after administration of 12 doses of WVE-N531; and / or (iv) level of muscle necrosis is assessed after administration for about 48 weeks from first dose and the level of muscle necrosis is decreased as compared to after administration for about 24 weeks from first dose.

54. The method of any one of the preceding claims, wherein level of muscle fibrosis in the subject is decreased, optionally wherein: (i) level of muscle fibrosis is assessed using a biopsy sample from the subject; (ii) level of muscle fibrosis is assessed using trichrome staining of a biopsy sample from the subject; (iii) level of muscle fibrosis is assessed after administration of 24 doses of WVE-N531 and the level of muscle fibrosis is decreased as compared to after administration of 12 doses of WVE-N531; and / or (iv) level of muscle fibrosis is assessed after administration for about 48 weeks from first dose and the level of muscle fibrosis is decreased as compared to after administration for about 24 weeks from first dose.

55. The method of any one of the preceding claims, wherein muscle fibrosis in the subject is reversed.

56. The method of any one of the preceding claims, wherein level of one or more inflammatory biomarkers in the subject is decreased, optionally wherein: (i) one or more inflammatory biomarkers comprise one or more inflammatory cytokines; (ii) one or more inflammatory biomarkers comprise monocyte chemoattractant protein-1 (MCP-1) and / or interleukin-6 (IL-6); (iii) one or more inflammatory biomarkers comprise one or more circulating inflammatory biomarkers; (iv) level of one or more inflammatory biomarkers is assessed using a serum sample from the subject; (v) level of one or more inflammatory biomarkers is assessed after administration of 12 or 24 doses 165 of 176 13004462v1Attorney Docket No.: 2010581-1542 of WVE-N531 and the level of one or more inflammatory biomarkers is decreased as compared to baseline, or wherein level of one or more inflammatory biomarkers is assessed after administration of 24 doses of WVE-N531 and the level of one or more inflammatory biomarkers is decreased as compared to after administration of 12 doses of WVE-N531; and / or (vi) level of one or more inflammatory biomarkers is assessed after administration for about 24 or 48 weeks from first dose and the level of one or more inflammatory biomarkers is decreased as compared to baseline, or wherein level of one or more inflammatory biomarkers is assessed after administration for about 48 weeks from first dose and the level of one or more inflammatory biomarkers is decreased as compared to after administration for about 24 weeks from first dose.

57. The method of any one of the preceding claims, wherein: (i) loss of ambulation in the subject is reduced; (ii) disease progression in the subject is delayed or slowed; (iii) muscle health in the subject is improved; (iv) transition from regenerative muscle to mature muscle in the subject is increased; (v) muscle weakness in the subject is delayed or slowed; (vi) loss of muscle mass in the subject is delayed or slowed; and / or (vii) loss of pulmonary function in the subject is delayed or slowed.

58. The method of any one of the preceding claims, wherein the subject improves in a muscular dystrophy assessment, one or more functional assessments, a 10 meter walk test, North Star Ambulatory Assessment (NSAA) 2.0, Performance of the Upper Limb (PUL) 2.0, one or more lower limb motor function by timed function tests, four-stair climb, time to rise from the floor, upper limb proximal strength, handheld myometry, grip strength, grip strength in both hands, one or more pulmonary function tests, and / or one or more of peak flow rate [PFR], cough peak flow [CPF], and FVC; optionally wherein: (i) the improvement in grip strength is from baseline; (ii) grip strength is assessed using handheld myometry; and / or (iii) grip strength is assessed after administration of 24 doses of WVE-N531 and the improvement in grip strength is as compared to after administration of 12 doses of WVE-N531.

59. The method of any one of the preceding claims, wherein the increase or improvement is assessed in a population of subjects.

60. The method of any one of the preceding claims, comprising administering WVE-N531 to a subject according to a regimen, wherein the regimen comprises 12 or more doses of WVE-N531 administered about every two weeks, wherein each dose is independently equivalent to about 10 mg / kg WVE-N531 free acid form; 166 of 176 13004462v1Attorney Docket No.: 2010581-1542 optionally wherein the first dose of the 12 or more doses is the first dose of the regimen.

61. The method of any one of the preceding claims, comprising administering WVE-N531 to a subject according to a regimen, wherein the regimen comprises 24 or more doses of WVE-N531 administered about every two weeks, wherein each dose is independently equivalent to about 10 mg / kg WVE-N531 free acid form, optionally wherein the first dose of the 24 or more doses is the first dose of the regimen.

62. The method of any one of the preceding claims, wherein no serious adverse effects are observed and / or no discontinuation due to adverse effects are observed.

63. The method of any one of the preceding claims, comprising: (i) assessing serum creatine kinase level in a subject after one or more doses of WVE-N531 are administered or delivered, optionally wherein serum creatine kinase level in a subject after one or more doses of WVE-N531 is reduced, and administration or delivery of WVE-N531 is continued; (ii) assessing serum AST level in a subject after one or more doses of WVE-N531 are administered or delivered, optionally wherein serum AST level in a subject after one or more doses of WVE-N531 is reduced, and administration or delivery of WVE-N531 is continued; (iii) assessing level of muscle inflammation in a subject after one or more doses of WVE-N531 are administered or delivered; (iv) assessing level of muscle necrosis in a subject after one or more doses of WVE-N531 are administered or delivered; (v) assessing level of muscle fibrosis in a subject after one or more doses of WVE-N531 are administered or delivered; (vi) assessing TTR of a subject after one or more doses of WVE-N531 are administered or delivered; (vii) assessing NSAA total score of a subject after one or more doses of WVE-N531 are administered or delivered; (viii) assessing grip strength of a subject after one or more doses of WVE-N531 are administered or delivered; and / or (ix) assessing level of one or more inflammatory biomarkers in a subject after one or more doses of WVE-N531 are administered or delivered, optionally wherein: (a) one or more inflammatory biomarkers comprise one or more inflammatory cytokines; (b) one or more inflammatory biomarkers comprise monocyte chemoattractant protein-1 (MCP-1) and / or interleukin-6 (IL-6); and / or (c) one or more inflammatory biomarkers comprise one or more circulating inflammatory biomarkers. 167 of 176 13004462v1Attorney Docket No.: 2010581-1542 64. The method of any one of claims 59-63, wherein each subject in the population has a mutation in the DMD gene that is amenable to exon 53 skipping; or wherein each subject in the population has a mutation in the DMD gene that is amenable to exon 53 skipping, is suffering from DMD, and is ambulatory.

65. The method of claim 64, wherein the DMD mutation is: (i) Δ3-52, Δ4-52, Δ5-52, Δ6-52, Δ9-52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17- 52, Δ19-52, Δ21-52, Δ23-52, Δ24-52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32- 52, Δ33-52, Δ34-52, Δ35-52, Δ36-52, Δ37-52, Δ38-52, Δ39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ45- 52, Δ47-52, Δ48-52, Δ49-52, Δ50-52, Δ51-52, Δ52-52, Δ52, Δ54-58, Δ54-61, Δ54-63, Δ54-64, Δ54-66, Δ54-76, or Δ54-77; (ii) Δ45-52, Δ4-52, Δ5-52, Δ6-52, Δ9-52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17- 52, Δ19-52, Δ21-52, Δ23-52, Δ24-52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32- 52, Δ33-52, Δ34-52, Δ35-52, Δ36-52, Δ37-52, Δ38-52, Δ39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ45- 52, Δ47-52, Δ48-52, Δ49-52, Δ50-52, Δ51-52, Δ52-52, or Δ52; or (iii) Δ45-52, Δ48-52, Δ49-52, Δ50-52, or Δ52-52.

66. The method of any one of claims 59-65, wherein: (i) one or more subjects in the population independently have a mutation in the DMD gene that is amenable to exon 53 skipping, wherein the mutation is Δ52 mutation; (ii) one or more subjects in the population independently have a mutation in the DMD gene that is amenable to exon 53 skipping, wherein the mutation is Δ45-52; (iii) one or more subjects in the population independently have a mutation in the DMD gene that is amenable to exon 53 skipping, wherein the mutation is Δ47-52; (iv) one or more subjects in the population independently have a mutation in the DMD gene that is amenable to exon 53 skipping, wherein the mutation is Δ48-52; (v) one or more subjects in the population independently have a mutation in the DMD gene that is amenable to exon 53 skipping, wherein the mutation is Δ49-52; (vi) one or more subjects in the population independently have a mutation in the DMD gene that is amenable to exon 53 skipping, wherein the mutation is Δ50-52; (vii) one or more subjects in the population independently have a mutation in the DMD gene that is amenable to exon 53 skipping, wherein the mutation is Δ52-52; (viii) one or more subjects in the population receive no doses of WVE-N531 before the regimen is started; and / or (ix) one or more subjects in the population have independently received one or more doses of WVE- N531 before the regimen is started. 168 of 176 13004462v1Attorney Docket No.: 2010581-1542 67. The method of any one of claims 59-66, wherein: (i) none in the population have received a dose of WVE-N531 in the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months prior to the start of the regimen; (ii) none in the population have received a dose of WVE-N531 in the 18 weeks prior to the start of the regimen; and / or (iii) none in the population have received a dose of WVE-N531 in the 12 months prior to the start of the regimen.

68. The method of any one of the preceding claims, wherein each dose of WVE-N531 is independently administered as a pharmaceutical composition comprising WVE-N531 dissolved in a pharmaceutically acceptable carrier.

69. The method of claim 68, wherein: (i) WVE-N531 exists in the pharmaceutical composition as one or more pharmaceutically acceptable salt forms, optionally wherein a pharmaceutically acceptable salt form is hexadecasodium salt; (ii) a pharmaceutically acceptable carrier is or comprises a phosphate buffered solution; (iii) the components in the pharmaceutical composition are WVE-N531, potassium phosphate monobasic, sodium phosphate dibasic, sodium chloride and water, and hydrochloric acid and / or sodium hydroxide for pH adjustment; (iv) the pharmaceutical composition is isotonic; and / or (v) pH of the pharmaceutical composition is about 7-8 or about 7.3 or about 7.

4.

70. The method of any one of the preceding claims, wherein about is ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7 %, ±8%, ±9%, or ±10%.

71. The method of any one of the preceding claims, wherein an improvement is compared to baseline, absence of WVE-N531 administration, administration of a reference composition, and / or a natural history cohort.

72. The method of any one of the preceding claims, wherein a reference composition is comparable to an administered WVE-N531 composition but does not contain WVE-N531.

73. The method of any of the previous claims, wherein: (i) the subject is administered a steroid, optionally wherein the steroid is a glucocorticoid, a corticosteroid, prednisone, or deflazacort; (ii) the subject is administered a steroid at least about one month prior to the first dose of WVE- N531, optionally wherein the steroid is a glucocorticoid, a corticosteroid, prednisone, or deflazacort; (iii) the subject is administered a steroid at least about six months prior to the first dose of WVE- N531, optionally wherein the steroid is a glucocorticoid, a corticosteroid, prednisone, or deflazacort; (iv) the subject is administered a corticosteroid at least about one month prior to the first dose of 169 of 176 13004462v1Attorney Docket No.: 2010581-1542 WVE-N531, optionally wherein the corticosteroid is deflazacort; and / or (v) the subject is administered a corticosteroid at least about six months prior to the first dose of WVE-N531, optionally wherein the corticosteroid is deflazacort.

74. The method of any one of the preceding claims, comprising administering or delivering one or more other therapeutic agents and / or administering or delivering an agent selected from veliflapon, difenpiramide, amopyroquine, cloricromen, alpelisib, benorilate, xipamide, fabomotizole and altizide, or a pharmaceutically acceptable salt thereof.

75. The method of claim 74, wherein: (i) the oligonucleotide and an agent is administered or delivered concurrently and / or the oligonucleotide and an agent is administered or delivered in the same composition; (ii) the oligonucleotide is administered prior to an agent, optionally wherein the system or subject is under the effect of the oligonucleotide when the agent is administered or delivered; or (iii) the oligonucleotide is administered subsequently to an agent, optionally wherein the system or subject is under the effect of the agent when the oligonucleotide is administered or delivered.

76. A method of treating muscular dystrophy in a human subject in need thereof, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping, the method comprising administering to the subject about 10 mg / kg of WVE-N531 about every two weeks (Q2W), wherein WVE-N531 is fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU* SfC, wherein: m represents 2’-OMe modified nucleoside; f represents 2’-F modified nucleoside; *S represents Sp phosphorothioate linkage; and n001R represents Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage; or a method of treating muscular dystrophy in a human subject in need thereof, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping, the method comprising administering to the subject about 10 mg / kg of WVE-N531 about every four weeks (Q4W), wherein WVE-N531 is fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU* SfC, wherein: m represents 2’-OMe modified nucleoside; f represents 2’-F modified nucleoside; *S represents Sp phosphorothioate linkage; and n001R represents Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage; or a method of treating muscular dystrophy in a human subject in need thereof, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping, the method comprising administering 170 of 176 13004462v1Attorney Docket No.: 2010581-1542 to the subject about 10 mg / kg of WVE-N531 about monthly, wherein WVE-N531 is fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU* SfC, wherein: m represents 2’-OMe modified nucleoside; f represents 2’-F modified nucleoside; *S represents Sp phosphorothioate linkage; and n001R represents Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage; or a method of slowing progression of muscular dystrophy in a human subject suffering therefrom, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping, the method comprising administering to the subject about 10 mg / kg of WVE-N531 about every two weeks (Q2W), wherein WVE-N531 is fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU* SfC, wherein: m represents 2’-OMe modified nucleoside; f represents 2’-F modified nucleoside; *S represents Sp phosphorothioate linkage; and n001R represents Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage; or a method of slowing progression of muscular dystrophy in a human subject suffering therefrom, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping, the method comprising administering to the subject about 10 mg / kg of WVE-N531 about every four weeks (Q4W), wherein WVE-N531 is fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU* SfC, wherein: m represents 2’-OMe modified nucleoside; f represents 2’-F modified nucleoside; *S represents Sp phosphorothioate linkage; and n001R represents Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage; or a method of slowing progression of muscular dystrophy in a human subject suffering therefrom, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping, the method comprising administering to the subject about 10 mg / kg of WVE-N531 about monthly, wherein WVE- N531 is fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU* SfC, wherein: m represents 2’-OMe modified nucleoside; 171 of 176 13004462v1Attorney Docket No.: 2010581-1542 f represents 2’-F modified nucleoside; *S represents Sp phosphorothioate linkage; and n001R represents Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage.

77. The method of any one of the preceding claims, wherein the subject is a male.

78. The method of any one of the preceding claims, wherein the subject is ambulatory.

79. The method of any one of claims 1-77, wherein the subject is non-ambulatory.

80. The method of any one of the preceding claims, wherein: (i) the subject is a pediatric subject; (ii) the subject is a child; (iii) the subject is less than about 18 years of age; (iv) the subject is less than about 12 years of age; and / or (v) the subject is about 5-11 years of age.

81. The method of any one of claims 1-79, wherein the subject is an adult.

82. The method of any one of the preceding claims, wherein the subject exhibits one or more symptoms of muscular dystrophy and / or the subject exhibits one or more symptoms of DMD.

83. The method of any one of the claims 1-81, wherein the subject does not exhibit symptoms of muscular dystrophy and / or the subject does not exhibit symptoms of DMD.

84. A composition comprising WVE-N531.

85. The composition of claim 84, wherein: (i) a form of WVE-N531 in the composition is a pharmaceutically acceptable salt form; (ii) each form of WVE-N531 in the composition is independently a salt form; (iii) each form of WVE-N531 in the composition is independently a pharmaceutically acceptable salt form; (iv) a form of WVE-N531 in the composition is WVE-N531 hexadecasodium salt; (v) the composition is a drug substance or wherein the composition is a drug product; (vi) the composition is a liquid composition wherein WVE-N531 is dissolved; (vii) the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, optionally wherein the pharmaceutically acceptable carrier is or comprises a phosphate buffered solution; (viii) the components in the composition are WVE-N531, potassium phosphate monobasic, sodium phosphate dibasic, sodium chloride and water, and hydrochloric acid and / or sodium hydroxide for pH adjustment; (ix) the composition is isotonic; (x) the composition has a pH of about 7-8, about 7.3, or about 7.4; 172 of 176 13004462v1Attorney Docket No.: 2010581-1542 (xi) the concentration of WVE-N531 is equivalent to about 5-45 mg / mL, about 25-40 mg / mL, about 5-7 mg / mL, or about 6 mg / mL WVE-N531 free acid form, optionally wherein the composition is packaged into a vial, wherein the volume of the composition in the vial is at least 6 mL, at least 6-7 mL, and / or at least 6-6.5 mL; (xii) the composition has a purity of about 70%-90%, about 70-85%, or about 70%-80%; and / or wherein the composition has a purity of about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or more; optionally wherein the purity is measured by IP-RP-UPLC using area % at 260 nm, by IP-RP-UPLC using area % at 260 nm and the Set A parameters, and / or by an IP-RP-UPLC method for purity as described herein; (xiii) impurities in the composition are no more than about 10%-30%, about 15%-30%, or about 20%-30%; and / or wherein impurities in the composition are no more than about or about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10%; optionally wherein the impurities are measured by IP-RP-UPLC using area % at 260 nm, by IP-RP-UPLC using area % at 260 nm and the Set A parameters, and / or by an IP-RP-UPLC method for purity as described herein; (xiv) stereochemical purity of WVE-N531 is about 80% or more, about 85% or more, or about 80%-90%; and / or wherein stereochemical purity is assessed by dimer modeling; (xv) amount of WVE-N531 is measured by UV at 260 nm and / or measured by UV at 260 nm and 27 OD / mg; (xvi) the WVE-N531 drug substance in the composition is manufactured by a process described herein or characterized, released, or stored by one or more method described herein; optionally wherein the WVE-N531 drug substance is hexadecasodium salt; (xvii) the composition is a WVE-N531 drug product; (xviii) WVE-N531 drug product is manufactured by a process described herein or characterized, released, or stored by one or more method described herein; (xix) a pharmaceutical composition is manufactured by a process described herein or characterized, released, and / or stored by one or more method described herein; and / or (xx) the composition does not contain DS1, DS2, DS3, DS4, DS5, DS6, DS7, DS8, DS9, DS10, DS11, DS12, DS13, DS14, DS15, DS16, and / or DS17.

86. A composition comprising one of WVE-N531 and DS1 to DS17, wherein the composition is free of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) of the rest of WVE-N531 and DS1 to DS17.

87. The composition of claim 86, wherein the composition comprises WVE-N531, DS1, DS2, DS3, DS4, DS5, DS6, DS7, DS8, DS9, DS10, DS11, DS12, DS13, DS14, DS15, DS16, or DS17. 173 of 176 13004462v1Attorney Docket No.: 2010581-1542 88. The composition of any one of any one of claims 84-87, wherein about is ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7 %, ±8%, ±9%, or ±10%.

89. The composition of any one of any one of claims 84-88, for use in a method of any one of claims 1-83.

90. WVE-N531, for use in a method of any one of claims 1-83; or WVE-N531, for manufacturing a medicament for a method of any one of claims 1-83; or use of WVE-N531 for a method of any one of claims 1-83; or use of WVE-N531 for manufacturing a medicament for a method of any one of claims 1-83.

91. A pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, for use in a method of any one of claims 1-83; or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, for manufacturing a medicament for a method of any one of claims 1-83; or a composition of any one of claims 84-88, for use in a method of any one of claims 1-83; or a composition of any one of claims 84-88, for manufacturing a medicament for a method of any one of claims 1-83; or use of a composition of any one of claims 84-88 for a method of any one of claims 1-83; or use of a composition of any one of claims 84-88 for manufacturing a medicament for a method of any one of claims 1-83.

92. A method for manufacturing a WVE-N531 composition according to a method described in the specification.

93. The method of claim 92, comprising utilizing IP-RP-UPLC to assess purity and / or impurities in the manufactured WVE-N531 composition and release the preparation if the purity and / or impurities meet certain criteria; and / or wherein the composition is a drug substance or drug product.

94. A method for releasing a WVE-N531 preparation, comprising utilizing IP-RP-UPLC to assess purity and / or impurities in the WVE-N531 preparation and release the preparation if the purity and / or impurities meet certain criteria.

95. A method for assessing purity of WVE-N531 utilizing IP-RP-UPLC.

96. The method of any one of claim 93-95, wherein: (i) the IP-RP-UPLC utilized one or more parameters described in the specification; (ii) the IP-RP-UPLC utilized one or more parameters of Set A; or (iii) the IP-RP-UPLC utilized the Set A parameters.

97. The method of any one of claims 92-96, wherein stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC. 174 of 176 13004462v1Attorney Docket No.: 2010581-1542 98. A method for confirming stereochemical identity of WVE-N531 utilizing IP-RP-UPLC.

99. The method of any one of claims 92-98, wherein stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC according to Set B parameters or wherein stereochemical identity of WVE- N531 is confirmed by an IP-RP-UPLC method for stereochemical identity as described herein; and / or wherein the composition is any one of claims 84-88.

100. A compound, oligonucleotide, composition, method, process, use, dose or dosage regimen of any one of Embodiments 1-723. 175 of 176 13004462v1

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