Oligonucleotide compositions and methods thereof

Abstract

Among other things, the present disclosure provides oligonucleotide compositions and methods thereof. In some embodiments, the present disclosure provides technologies useful for exon skipping in cell populations, e.g., myocyte stem cells. In some embodiments, the present disclosure provides technologies useful for restoring functioning of myocyte stem cells. In some embodiments, the present disclosure provides technologies useful for regeneration of myocyte stem cells. In some embodiments, the present disclosure provides technologies useful for differentiation of myocyte stem cells. In some embodiments, the present disclosure provides methods for preventing or treating conditions, disorders, or diseases including Duchenne muscular dystrophy.

Description

OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No. 63 / 586,364, filed September 28, 2023, the entirety 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 progressive 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 encompasses the recognition that certain oligonucleotides can be delivered to satellite cells, which may also be referred to as myocyte stem cells. Delivery of oligonucleotides into satellite cells can provide various benefits and advantages, for example, in some embodiments, provided technologies provide DMD exon skipping in satellite cells which have DMD mutations amenable to exon skipping, and can restore or improve satellite cell functions, e.g., self- renewal, differentiation, regeneration (e.g., into myogenic progenitor cells which can generate other muscle cells and myofibers), etc.

[0004] In some embodiments, the present disclosure provides a method for delivering an oligonucleotide into a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide as described herein.

[0005] In some embodiments, the present disclosure provides a method for treating muscular dystrophy, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide, wherein the oligonucleotide is delivered to satellite cells of the subject.

[0006] In some embodiments, the present disclosure provides a method for providing increased level of a muscle function in a subject, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide, wherein the oligonucleotide is delivered to satellite cells of the subject.

[0007] After an oligonucleotide is delivered into satellite cells, it can provide various functions. For example, for satellite cells with a mutation in a DMD gene that is amenable to exon skipping, an oligonucleotide as described herein can provide effective skipping of an exon in a DMD transcript that has a mutation that is amenable to skipping of such an exon, and provide an effective level of functional dystrophin (may be shorter compared to wild type dystrophin) to restore or improve functions of satellite cells including those in development, injury repair, etc. Various DMD mutations amenable to exon skipping are reported. For example, in some embodiments, a DMD mutation is amenable to exon 53 skipping.

[0008] In some embodiments, the present disclosure provides a method for providing DMD exon skipping in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide as described herein.

[0009] In some embodiments, the present disclosure provides a method for restoring DMD RNA reading frame in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide as described herein.

[0010] In some embodiments, the present disclosure provides a method for providing a DMD polypeptide in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide as described herein, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein. In some embodiments, the present disclosure provides a method for providing muscle regeneration in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide wherein the oligonucleotide comprises a PN internucleotidic linkage. As described herein, among other things, provided technologies can deliver oligonucleotide into satellite cells and promote satellite cells self-renewal and differentiation, e.g., through providing exon skipping in DMD transcripts having mutations amenable to such exon skipping. In some embodiments, the present disclosure provides a method for increasing dystrophin level in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide, wherein the DMD polypeptide is truncated compared to wild-type dystrophin. In some embodiments, the present disclosure provides a method for increasing level of a dystrophin polypeptide encoded by an exon-skipped DMD transcript in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide, wherein the DMD polypeptide is truncated compared to wild-type dystrophin.

[0011] In some embodiments, exon skipping (e.g., in cells as described herein) is increased compared to a reference. In some embodiments, production of a dystrophin polypeptide (e.g., in cells described herein), e.g., encoded by an exon skipped DMD transcript, is increased compared to a reference. In some embodiments, level of a dystrophin function (e.g., in cells described herein) is increased compared to a reference, hr some embodiments, differentiation (e.g., satellite cells, myogenic progenitor cells, etc.) isincreased compared to a reference. In some embodiments, myotube formation is increased compared to a reference. In some embodiments, myofiber formation is increased compared to a reference. In some embodiments, muscle regeneration is increased compared to a reference. In some embodiments, a muscle function is increased compared to a reference. In some embodiments, muscle repair is increased compared to a reference.

[0012] In some embodiments, a benefit, an advantage, etc., e.g., an increase, a restoration, an enhancement, etc., is compared to a reference. In some embodiments, a reference is absence of administration or delivery of an oligonucleotide as described herein. In some embodiments, a reference is administration or delivery of a different oligonucleotide (e.g., an approved exon skipping drug (e.g., golodirsen or viltolarsen for exon 53 skipping) by the US FDA for an oligonucleotide for such exon skipping, a reported terminated develop exon skipping candidate oligonucleotide (e.g., suvodirsen for exon 53) for an oligonucleotide for such exon skipping). In some embodiments, a reference is baseline, e.g., prior to administration or delivery of an oligonucleotide as described herein.

[0013] In some embodiments, the present disclosure provides a method for restoring satellite cell function in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide as described herein.

[0014] In some embodiments, the present disclosure provides a method for increasing differentiation of satellite cells in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.

[0015] In some embodiments, the present disclosure provides a method for producing myogenic progenitor cells in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide wherein the oligonucleotide comprises a PN internucleotidic linkage.

[0016] In some embodiments, a provided method comprises formation of formation of activated satellite cells. In some embodiments, upon an event, e.g., injury, quiescent muscle stem cells, e.g., those Pax7+, is activated to form activated satellite cells, e.g., those Pax7+ / Myf5+. In some embodiments, the present disclosure provides DMD exon skipping in these cells to provide dystrophin polypeptide with improved functions. In some embodiments, with such DMD exon skipping satellite cells go through selfrenewal and differentiation, e.g., into myogenic progenitor cells (e.g., those Pax7+ / Myf5+ / MyoDl+).

[0017] In some embodiments, a provided method comprises formation of myogenic progenitor cells (e.g., those Pax7+ / Myf5+ / MyoDl+), e.g., from satellite cells. In some embodiments, a provided method comprises formation of cells, e.g., those MyoDl+ / Myogenin+, from myogenic progenitor cells, e.g., through differentiation. In some embodiments, the present disclosure comprises formation of Myogenin+ / MyHC+cells, e.g., from MyoDlVMyogcnin+cells. In some embodiments, formation of Myogcnin+ / MyHC+cells comprises fusion. In some embodiments, a provided method comprises formation of myotubes. In someembodiments, a provided method comprises formation of myofibers. In some embodiments, a provided method comprises formation of mature myocytes. In some embodiments, a provided method comprises formation of myotubes, myofibers, and mature myocytes. As described herein, formation of various cells or regeneration may comprise division, differentiation, fusion, etc. Certain aspects, processes, cells, functions (e.g., those of various cells including satellite cells), etc. are described in Yucel and Blau, Principles of Regenerative Medicine (3rdedition), Chapter 18 - Skeletal Muscle Stem Cells, pages 273- 293, 2019.

[0018] In some embodiments, a system comprises satellite cells. In some embodiments, oligonucleotides as described herein are delivered into satellite cells. In some embodiments, cells have DMD genes comprising mutations amenable to exon skipping. In some embodiments, satellite cells haveDMD genes comprising mutations amenable to exon skipping. In some embodiments, oligonucleotides as described herein provide DMD exon skipping in various cells. In some embodiments, oligonucleotides as described herein provide DMD exon skipping in satellite cells. In some embodiments, oligonucleotides as described herein provide increased levels of dystrophin polypeptides encoded by exon skipped DMD transcripts which polypeptides can provide high levels of functions compared to those encoded by DMD transcripts without exon skipping. In some embodiments, oligonucleotides as described herein provides increased levels of dystrophin functions. In some embodiments, oligonucleotides as described herein provides sufficiently high levels of exon skipped DMD transcripts, polypeptides encoded thereby, and / or dystrophin functions in satellite cells (and optionally other muscle cells) for satellite cell self-renewal and differentiation for myofiber formation, myofiber repair, muscle regeneration, etc.

[0019] In some embodiments, a system is or comprises a population of cells. In some embodiments, a system is or comprises cell culture. In some embodiments, a system is or comprises a tissue. In some embodiments, a system is or comprises a muscle tissue. In some embodiments, a system comprises skeletal muscle cells. In some embodiments, a system comprises myofiber. In some embodiments, a system is or comprises an organ. In some embodiments, a system is or comprises an animal. In some embodiments, a system is or comprises a subject. In some embodiments, a system is or comprises a sample. In some embodiments, to assess a provided technology, a sample may be taken from a system and such a sample is assessed.

[0020] In some embodiments, a subject has a DMD mutation that is amenable to exon skipping, e.g., exon 53 skipping. In some embodiments, a subject suffers from a muscular dystrophy. In some embodiments, a subject suffers from Duchenne muscular dystrophy.

[0021] In some embodiments, an oligonucleotide is WVE-N531. Among other things, the present disclosure demonstrates that WVE-N531 can be delivered to patient satellite cells.

[0022] In some embodiments, WVE-N531 is administered 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.

[0023] In some embodiments, WVE-N531 is administered in a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutically acceptable earner is or comprises a buffered solution. In some embodiments, it is a phosphate buffered solution. In 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.

[0024] In some embodiments, two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 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, 3 or more doses are administered. In some embodiments, 10 or more doses are administered. In some embodiments, a does is equivalent to about 10 mg / kg WVE-N531 free acid form. In some embodiments, each dose is about the same. In some embodiments, a does is independently equivalent to about 10 mg / kg WVE-N531 free acid form.

[0025] 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, three or more doses arc administered about every two weeks. In some embodiments, doses are first administered with shorter intervals (e.g., about every two weeks) and thenadministered 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, 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.

[0026] 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.

[0027] 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.

[0028] 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, asubject is administered a steroid at least about one month prior to the first dose of WVE-N531 or a composition thereof.

[0029] 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, difenpir amide, 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.

[0030] In some embodiments, provided technologies (oligonucleotides, compositions, methods, doses, dosage regimens, etc.) provide one or more desired biological effects. In some embodiments, provided technologies provides delivery into satellite cells. 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, disease progression is delayed, slowed or 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. Tn 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-mctcr 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 handheldmyometer. 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.

[0031] In some embodiments, provided technologies reduce loss of ambulation relative to baseline.

[0032] In some embodiments, 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. In some embodiments, provided technologies provide increases from baseline in dystrophin (e.g., encoded by exon-skipped DMD transcript) 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 2-60 weeks. In some embodiments, a time period is about 10-60 weeks. In some embodiments, it is about 4 weeks. In some embodiments, it is about 6 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, itis 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, a certain number of doses is about 3 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.

[0033] 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.

[0034] 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).DEFINITIONS

[0035] As used herein, the following definitions shall apply unless otherwise indicated. For purposesof 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.

[0036] 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.

[0037] 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 var ious forms including salt forms, par ticularly 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.

[0038] 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 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%.

[0039] 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 separ ated 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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 lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable bcncfit / risk ratio. Pharmaceutically acceptable salts arc 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).

[0044] 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.

[0045] 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 aits 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.

[0046] 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.

[0047] 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, 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.

[0048] 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.

[0049] 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.

[0050] 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 ai t, 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.

[0051] 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.

[0052] Chirally controlled oligonucleotide composition: The terms “chirally controlled (stereocontrolled or stereodefined) oligonucleotide composition”, “chirally controlled (stereocontrolled or stcrcodcfincd) 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 chirallycontrolled 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.

[0053] In some embodiments, the plurality of oligonucleotides in a chirally controlled oligonucleotide composition share the same base sequence, the same, if any, nuclcobasc, sugar, and internucleotidic linkage modifications, and the same stereochemistry (Rp or Sp) independently at linkage phosphorus chir al centersof 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 l%-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 l%-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 l%-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 l%-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 (e.g. , of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share the same constitution, arc 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 chirallycontrolled 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 l%-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 acids 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.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0054] Among other things, the present disclosure provides provided technologies for delivering oligonucleotides and provide exon skipping in satellite cells that have a DMD mutation amenable to suchexon skipping (e.g., exon 53 skipping). In some embodiments, provided technologies are useful for treating muscular dystrophy in a subject having a mutation of the DMD gene that is amenable to exon skipping, e.g., exon 53 skipping. 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.

[0055] In some embodiments, an oligonucleotide is WVE-N531. 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, Δld-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, 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 A48-52. 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 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.

[0056] In some embodiments, the provided technologies provide WVE-N531 in satellite cells. In some embodiments, WVE-N531 in satellite cells is observed in samples from patients after about 3 doses administered about every two weeks, wherein each dose is equivalent to about 10 mg / kg WVE-N531 free acid form.

[0057] In some embodiments, the present disclosure provides methods comprising using WVE-N531 to induce exon 53 skipping in DMD in muscle cells including in satellite cells. In some embodiments, thepresent 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.

[0058] In some embodiments, the present disclosure relates to improved methods for treating muscular dystrophy by inducing exon skipping in muscle cells (e.g., satellite cells, myogenic progenitor cells, etc.) 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.

[0059] In some embodiments, the present disclosure provides a method for increasing satellite cell differentiation 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).

[0060] In some embodiments, the present disclosure provides a method for increasing myogenic progenitor cell formation 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).

[0061] In some embodiments, the present disclosure provides a method for increasing muscle regeneration 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).

[0062] In some embodiments, the present disclosure provides a method for increasing level of exon 53-skipped DMD transcripts in satellite cells 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 satellite cells 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 satellite cells 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 satellite cells 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 exon 53-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 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.

[0063] In some embodiment, about is ±1%. In some embodiment, about is ±2%. In some embodiment, about is ±3%. In some embodiment, about is ±4%. In some embodiment, about is ±5%. In some embodiment, about is ±6%. In some embodiment, about is ±7%. In some embodiment, about is ±8%. In some embodiment, about is ±9%. In some embodiment, about is ±10%.

[0064] 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 someembodiments, 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+.

[0065] In some embodiments, a dose of WVE-N531 is about 1-20 mg / kg. In some embodiments, it is about 1-5 mg / kg. In some embodiments, it is about 5-20 mg / kg. In some embodiments, it is about 5-10 mg / kg. In some embodiments, it is about 10-20 mg / kg. In some embodiments, it is about 10-15 mg / kg. In some embodiments, it is about 15-20 mg / kg. In some embodiments, it is about 1 mg / kg. In some embodiments, it is about 1 mg / kg. In some embodiments, it is about 2 mg / kg. In some embodiments, it is about 3 mg / kg. In some embodiments, it is about 4 mg / kg. In some embodiments, it is about 5 mg / kg. In some embodiments, it is about 6 mg / kg. In some embodiments, it is about 7 mg / kg. In some embodiments, it is about 8 mg / kg. In some embodiments, it is about 9 mg / kg. In some embodiments, it is about 10 mg / kg. In some embodiments, it is about 11 mg / kg. In some embodiments, it is about 12 mg / kg. In some embodiments, it is about 13 mg / kg. In some embodiments, it is about 14 mg / kg. In some embodiments, it is about 15 mg / kg. In some embodiments, it is about 16 mg / kg. In some embodiments, it is about 17 mg / kg. In some embodiments, it is about 18 mg / kg. In some embodiments, it is about 19 mg / kg. In some embodiments, it is about 20 mg / kg.

[0066] 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.

[0067] 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, 2930, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 or more) doses of WVE-N531 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 arc administered. In some embodiments, about 70 or more doses arc administered. In some embodiments, about 80 or more doses are administered. In some embodiments, about 90 or moredoses 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 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.

[0068] In some embodiments, WVE-N531 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.

[0069] In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 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. In some embodiments, three or more consecutive doses are administered about every two weeks.

[0070] 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%.

[0071] Unless specified otherwise, amounts, concentrations, doses, etc., of WVE-N531 are of the free acid form.

[0072] Amounts, concentrations, doses, etc. arc typically purity corrected. In some embodiments, purity is measured by IP-RP-UPLC as described herein based on % area.

[0073] Among other things, provided technologies provide various advantages compared to golodirsen and / or viltolarsen, e.g., effective delivery to satellite cells, 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

[0074] 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: Pl 1532; 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.

[0075] 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-gly coprotein 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.

[0076] 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.

[0077] 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.

[0078] 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 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.

[0079] In some embodiments, a dystrophin mRNA, gene or protein is a revertant version.

[0080] 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.

[0081] Satellite cells or myocyte stem cells are reported to play an important role in regeneration as source of myogenic progenitor cells that differentiate into myoblasts, myotubes, and eventually mature myofibers. In addition to myofibers, dystrophin has been reported to be highly expressed in satellite cells where it, among other things, regulates satellite cell fate and myogenesis. According to some reports, dystrophin binds to the cell polarity effector Mark2 in activated satellite cells which contributes to apicobasal orientation of the mitotic spindle and asymmetric cell division that enables satellite cells to maintain their reserve pool and contribute to myogenic progenitors (see, e.g., Kodippili K, Rudnicki MA. Satellite cell contribution to disease pathology in Duchenne muscular dystrophy. Front Physiol. 2023;14: 1180980). It has been reported that, for DMD patients, in the absence of dystrophin in satellite cells, cellular polarity is disrupted resulting in abnormal cell division, reduction in myogenic progenitors and reduced muscle regeneration (see, e.g., Dumont NA, Wang YX, von Maltzahn J, Pasut A, Bentzinger CF, Brun CE, Rudnicki MA. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division. Nat Med. 2015 Dec;21(12):1455-63. doi: 10.1038 / nm.3990). Among other things, the present disclosure, provides technologies to deliver oligonucleotides to satellite cells to increase or restore satellite cell functions. In some embodiments, dystrophin restoration in these cells serve to replenish damaged myofibers. Various reported technologies are reported to target differentiated myofibers and are notreported to address dystrophin deficient satellite cells. In some embodiments, provided technologies increase levels of dystrophin polypeptides and / or functions in satellite cells.Muscular Dystrophy

[0082] 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).

[0083] 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 aims, neck, and other areas. Calves are often enlarged. Symptoms usually appear before age six and may appear in early 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 hyper lordosis, 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.

[0084] 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 ct al. 1990 Nature, 343, 180-182.

[0085] 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.

[0086] 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

[0087] Among other things, the present disclosure provides technologies for exon skipping including in satellite cells that have a DMD mutation amenable to exon skipping. In some embodiments, provided technologies provide exon skipping in satellite cells that can increase or restore levels of satellite cell functions. In some embodiments, provided technologies provide exon skipping in satellite cells that can increase or restore satellite cell self-renewal and differentiation.

[0088] 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.

[0089] 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 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.

[0090] 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 c.g., produced from a dystrophin transcript with an out-of-framc deletion.

[0091] In some embodiments, an internally truncated DMD protein produced from a dystrophintranscript 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.

[0092] 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 provided improved functions compared to without exon 53 skipping.

[0093] 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.Oligonucleotides

[0094] In some embodiments, oligonucleotides are delivered into satellite cells having a DMD mutation amenable to exon skipping. In some embodiments, as demonstrated herein, when an oligonucleotide is administered or delivered to a subject having a DMD mutation amenable to exon skipping, such an oligonucleotide can enter satellite cells. Various oligonucleotides can be assessed for entry into satellite cells in accordance with the present disclosure.

[0095] In some embodiments, structural elements, e.g., sugar modifications, nucleobase modifications, internucleotidic linkage modifications including internucleotidic linkage types, linkage phosphorus stereochemistry, etc., can impact oligonucleotide delivery or distribution into satellite cells. In some embodiments, incorporation of PN linkages facilitate oligonucleotide delivery or distribution into satellite cells.

[0096] Various internucleotidic linkages may be utilized in oligonucleotides in accordance with the present disclosure. In some embodiments, an oligonucleotide comprises one or more types of internucleotidic linkage. In some embodiments, an oligonucleotide comprises two or more types of internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least three types of internucleotidic linkages. In some embodiments, a linkage contains a linkage phosphorus atom bonded toan oxygen atom which oxygen atom is not bonded to or is not part of a backbone sugar (“a PO linkage”, e.g., a natural phosphate linkage). In some embodiments, a linkage contains a linkage phosphorus atom bonded to a sulfur atom which sulfur atom is not bonded to or is not part of a backbone sugar (“a PS linkage”, e.g., a phosphorothioate internucleotidic linkage). In some embodiments, a linkage contains a linkage phosphorus atom bonded to a nitrogen atom which nitrogen atom is not bonded to or is not part of a backbone sugar (“a PN linkage”, e.g., nOOl). In some embodiments, an oligonucleotide comprises one or more PS linkages. In some embodiments, an oligonucleotide comprises one or more PO linkages. In some embodiments, an oligonucleotide comprises one or more PN linkages. In some embodiments, an oligonucleotide comprises one or more PS and one or more PO linkages. In some embodiments, an oligonucleotide comprises one or more PS and one or more PN linkages. In some embodiments, an oligonucleotide comprises one or more PS, one or more PN and one or more PO linkages. In some embodiments, a PS linkage is a phosphorothioate linkage. In some embodiments, each PS linkage is independently a phosphorothioate linkage. In some embodiments, a PO linkage is a natural phosphate linkage. In some embodiments, each PO linkage is independently a natural phosphate linkage. In some embodiments, a PN linkage is a phosphoryl guanidine linkage. In some embodiments, each PN linkage is independently a phosphoryl guanidine linkage. In some embodiments, each PN linkage is independently a nOOl internucleotidic linkage.

[0097] In some embodiments, oligonucleotides of the present disclosure can be stereodefined. In some embodiments, an oligonucleotide comprises a PN linkage. In some embodiments, an oligonucleotide comprises a Rp PN linkage. In some embodiments, an oligonucleotide comprises a Sp PN linkage. In some embodiments, an oligonucleotide comprises a phosphoryl guanidine internucleotidic linkage. In some embodiments, an oligonucleotide comprises a Rp phosphoryl guanidine internucleotidic linkage. In some embodiments, an oligonucleotide comprises a Sp phosphoryl guanidine internucleotidic linkage. In some embodiments, an oligonucleotide comprises a n001 internucleotidic linkage. In some embodiments, an oligonucleotide comprises a Rp n001 internucleotidic linkage. In some embodiments, an oligonucleotide comprises a Sp n001 internucleotidic linkage. In some embodiments, an oligonucleotide comprises a PS internucleotidic linkage. In some embodiments, an oligonucleotide comprises a Rp PS internucleotidic linkage. In some embodiments, an oligonucleotide comprises a Sp PS internucleotidic linkage. In some embodiments, an oligonucleotide comprises a phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide comprises a Rp phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide comprises a Sp phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide comprises a PO internucleotidic linkage. In some embodiments, an oligonucleotide comprises a natural phosphate linkage.

[0098] In some embodiments, an oligonucleotide comprises a modified sugar. In some embodiments,each sugar in an oligonucleotide is modified. In some embodiments, an oligonucleotide comprises a 2’-F modified sugar. In some embodiments, an oligonucleotide comprises a 2’ -OR modified sugar, wherein R is optionally substituted Ci-Ce aliphatic. In some embodiments, an oligonucleotide comprises a 2’-0Me modified sugar. In some embodiments, an oligonucleotide comprises a 2’ -MOE modified sugar-.

[0099] Among other things, the present disclosure provides oligonucleotides with high purity. In some embodiments, diastereomeric purity of each chiral linkage phosphorus is independently about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, it is independently at least about 95%. In some embodiments, it is independently at least about 97%. In some embodiments, diastereomeric purity of an oligonucleotide is about or at least about least (DS)nc, wherein DS is about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more, and nc is the number of chirally controlled internucleotidic linkages in the oligonucleotide (e.g., 1-50, 1- 40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 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 or more).

[0100] In some embodiments, DS is about or at least about 95%. In some embodiments, DS is about or at least about 97%. In some embodiments, DS is about or at least about 98%. In some embodiments, DS is about or at least about 99%. In some embodiments, nc is about 5-20. In some embodiments, nc is about or at least about 10. In some embodiments, nc is about 10-20. In some embodiments, nc is about or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0101] In some embodiments, the present disclosure provides chirally controlled oligonucleotide composition. In some embodiments, an oligonucleotide is provided in a chirally controlled oligonucleotide composition. In some embodiments, an oligonucleotide is administered in a chirally controlled oligonucleotide composition. In some embodiments, an oligonucleotide is administered in a pharmaceutical composition which is a chirally controlled oligonucleotide composition.

[0102] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising an oligonucleotide, wherein the composition is enriched, relative to a substantially racemic preparation of the oligonucleotide, for the oligonucleotide.

[0103] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein oligonucleotides of the plurality share the same constitution, and about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of all oligonucleotides in the composition that share the same constitution are oligonucleotides of the plurality. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in the composition that share the same base sequence arc oligonucleotides of the plurality. In some embodiments, a percentage is about or at least about (DS)nc, wherein DS is about or at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, and nc is the number of chirally controlled internucleotidic linkages an oligonucleotide of the plurality (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 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 or more).

[0104] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising an oligonucleotide, wherein at least about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%. 65%, 70%, 75%, 80%. 85%, 90%, or 95%, of all oligonucleotides in the composition that share the same constitution are the oligonucleotide. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%. 70%. 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in the composition that share the same base sequence as the oligonucleotide are the oligonucleotide. In some embodiments, a percentage is about or at least about (DS)nc, wherein DS is about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, and nc is the number of chirally controlled internucleotidic linkages an oligonucleotide of the plurality (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 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 or more).

[0105] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides, wherein oligonucleotides of the plurality share:1 ) a common base sequence; and2) the same linkage phosphorus stereochemistry independently at 1, 2, 3, 4. 5, 6, 7. 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chiral internucleotidic linkages; wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that share the common base sequence are oligonucleotides of the plurality.

[0106] In some embodiments, oligonucleotides of plurality share the same linkage phosphorus stereochemistry independently at each chiral internucleotidic linkage. In some embodiments, a level is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. 75%. 80%. 85%. 90%, 95% or more of all oligonucleotides that share the same base sequence as an oligonucleotide having the structure or a pharmaceutically acceptable salt thereof. In some embodiments, a level is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of all oligonucleotides that share the same constitution as an oligonucleotide having the structure or a pharmaceutically acceptable salt thereof.

[0107] In some embodiments, a level, purity, percentage, etc. is based on UV peak area at 260 nm.

[0108] In some embodiments, a pharmaceutical composition is a chirally controlled oligonucleotide composition.Pharmaceutical Compositions

[0109] In some embodiments, the present disclosure provides pharmaceutical compositions comprising an oligonucleotide and a pharmaceutical earner. In some embodiments, for example, for therapeutic and clinical purposes, oligonucleotides of the present disclosure are provided as pharmaceutical compositions. In some embodiments, a provided oligonucleotide is administered as a pharmaceutical composition.

[0110] As appreciated by those skilled in the art, oligonucleotides can be provided in various forms. In some embodiments, oligonucleotides can be in acid forms, e.g., for natural phosphate linkages, in the form of -OP(O)(OH)O-; for phosphorothioate intemucleotidic linkages, in the form of -OP(O)(SH)O-; etc. In some embodiments, oligonucleotides can be in salt forms, e.g., for natural phosphate linkages, in the form of -OP(O)(ONa)O- in sodium salts; for phosphorothioate intemucleotidic linkages, in the form of -OP(O)(SNa)O- in sodium salts; etc. Unless otherwise noted, oligonucleotides of the present disclosure can exist in acid, base and / or salt forms. In some embodiments, a composition comprises one or more forms of an oligonucleotide. In some embodiments, a composition comprises one or more salt forms of an oligonucleotide. In some embodiments, a composition comprises one or more pharmaceutically acceptable salt forms of an oligonucleotide. In some embodiments, oligonucleotides in compositions, e.g., pharmaceutical compositions, exists in pharmaceutically acceptable salt forms. In some embodiments, a salt of a provided oligonucleotide comprises two or more cations; for example, in some embodiments, up to the number of negatively charged acidic groups (e.g., phosphate, phosphorothioate, etc.) in an oligonucleotide. As appreciated by those skilled in the art, oligonucleotides described herein may be provided and / or utilized in a salt form, particularly a pharmaceutically acceptable salt form.

[0111] In some embodiments, a pharmaceutical composition of an oligonucleotide is chirally controlled.WVE-N53I

[0112] In some embodiments, an oligonucleotide that is delivered into satellite cells is WVE-N531.

[0113] WVE-N531 may be described as (as those skilled in the art appreciate, 5’ to 3’ unless specified otherwise):m: 2’-OMe modified nucleoside; f: 2’-F modified nucleoside;AS': Sp phosphorothioate linkage; and n001 R : Rp A-( 1 ,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage.

[0114] WVE-N531 may be named by accepted oligonucleotide nomenclature:

[0115] 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.

[0116] Molecular formulae and molecular weights of WVE-N531 and its hexadecasodium salt is described below:Molecular Formula (free acid form): C206H250F17N78O109P19S14Molecular Weight (free acid form): 6923.02 g / molMolecular Formula (hexadecasodium salt form): C206H234F17N7sO109P19S14Na16Molecular Weight (hexadecasodium salt form): g / mol

[0117] WVE-N531 has 17 stereodefined internucleotide linkages, 14 of which are identified as Sp phosphorothioate linkages, and three as / ?p 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 A-(l,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage, and phosphate linkages, respectively. A A-(1,3-dimethylimidazolidin-2- ylidenyl ) phosphoramidate linkage may be referred to as a nOOl linkage.

[0118] The structure of WVE-N531 may be depicted as Formula I:Formula I

[0119] WVE-N531 hexadecasodium salt may be described as Formula I-a:

[0120] In Formulae I and I-a, due to the sizes of the structures, the structures are presented as 4 rows, in 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 beindependently replaced with cations, e.g., in the hexadecasodium salt, each replaced with sodium.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] In some embodiments, WVE-N531 is provided and administered as chirally controlled composition in which WVE-N531 is enriched over other diastereomers.

[0125] 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.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.

[0126] 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 some embodiments, 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.

[0127] 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.

[0128] 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.

[0129] Among other things, the present disclosure demonstrates that WVE-N531 can be delivered into satellite cells. In some embodiments, the present disclosure provides a method for treating Duchenne muscular dystrophy, comprising administering to a subject suffering therefrom three or more doses of WVE-N531, wherein each dose is independently equivalent to about 10 mg / kg WVE-N531 free acid form, and WVE-N531 is delivered into one or more satellite cells of the subject after one or more (e.g., about or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) doses of WVE-N531each independently equivalent to about 10 mg / kg WVE-N531 free acid form. As those skilled in the art appreciate, delivery into satellite cells may be assessed by assessing a sample from a subject and / or results from properly designed clinical trials.Manufacturing

[0130] Various methods can be utilized for production of oligonucleotides and compositions and can be utilized in accordance with the present disclosure. For example, traditional phosphoramidite chemistry (e.g., phosphoramidites comprising — CH2CH2CN and -N(i-Pr)2) can be utilized to prepare stereorandom oligonucleotides and compositions, and certain reagents and chirally controlled technologies can be utilized to prepare chirally controlled oligonucleotide compositions, e.g., as described in US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 223056, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612, WO 2020 / 191252, WO 2021 / 071858, and / or WO 2022 / 099159.

[0131] Certain technologies utilized for preparing WVE-N531 are described herein as examples.

[0132] 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 or a pharmaceutically acceptable salt form thereof and a pharmaceutically acceptable earner. In some embodiments, the present disclosure provides WVE-N531 drug products.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] A useful manufacturing process for WVE-N531 hcxadccasodium salt as a drug substance and certain results are described below as an example.A Useful Manufacturing Process

[0137] 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 nOOl), 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 NEtrHF (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, e.g., WVE-N531 hexadecasodium salt, in some embodiments, as drug substance.A useful cycle for phosphoro thioate assemblyA useful cycle for n001 assemblyA useful cycle for phosphate assembly

[0138] The steps to manufacture WVE-N531 drug substance are described in further detail below.Stage 1: Synthesis

[0139] Synthesis of the oligonucleotide is carried out on Controlled Pore Glass (CPG) solid support functionalized with 5’-ODMTr-2’-F-dC(W4-Ac), on an automated oligonucleotide synthesizer. All reactions take place on the solid support packed in a column.

[0140] 1. Detrity lation

[0141] To initiate the synthesis, the CPG-5’-ODMTr-2’-F-dC(A4-Ac) solid support 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.

[0142] 2. Coupling

[0143] Elongation of the growing oligonucleotide chain is achieved by reaction of 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)-lH-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 nOOl 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.

[0144] 3. Capping- 1'Capping-1'R = Ph2MeSi orphSO2Cap B: Acetic anhydride / 2 ,6-lutidine / Acetonitrile|00145| Capping 1 can protect a secondary amine which results from a oxazaphospholidine ring 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 12,6- lutidine I acetonitrile (20:30:50, v:v:v) through the synthesis column. Excess reagents are flushed from the synthesis column with acetonitrile.

[0146] 4. Oxidation, Thiolation or Imidation

[0147] 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. Thiolation by treatment with xanthane hydride (XH) in pyridine : acetonitrile (l:l,v:v) resulting in the generation of a stereodefined P(V) phosphorothioate triester linkage.3. Imidation by treatment with 2-azido-l,3-dimethylimidazolinium hexafluorophosphate (ADIH) in acetonitrile resulting in the generation of a stereodefined P(V) phosphorimidate triester linkage.

[0148] In each case, after the step, excess reagents are flushed from the synthesis column with acetonitrile.

[0149] 5. Capping-2

[0150] 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]: A-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.Cap B: Acetic anhydride 12,6-1 utidine I AcetonitrileCap A: / V-Methylimidazole / Acetonitrile

[0151] Oligonucleotide Assembly and Final Step

[0152] 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 dctritylation step.Stage 2: Cleavage and Deprotection

[0153] 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 process.

[0154] Step 1: (L)-PSM Chiral Auxiliary and Cyanoethyl Removal with Diethylamine Treatment

[0155] The crude oligonucleotide on the solid support is treated with a solution of diethylamine in ACN which can simultaneously remove cyanoethyl protecting groups from the phosphate triester linkages, and (L)-PSM chiral auxiliary from the phosphorimidate triester to generate phosphodiester and phosphoramidate diester linkages, respectively.

[0156] Step 2: (L)-DPSE Chiral Auxiliary Removal with Fluoride Treatment

[0157] The crude oligonucleotide on the solid support is treated with a temperature-controlled solution of NEt3-3HF in a mixture of dimethyl sulfoxide (DMSO), NEtj and H2O to effect removal of the chiral auxiliary. This process converts a stereodefined phosphorothioate triester to a stereodefined phosphorothioate diester as shown above.

[0158] Step 3: Cleavage and Global Nucleobase Deprotection

[0159] The crude oligonucleotide is then cleaved from the solid support by treatment with ammonium hydroxide 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”).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.

[0160] 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

[0161] 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)|00162| 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

[0163] 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

[0164] Certain useful starting materials are listed below:Abbreviations: CoA = Certificate of Analysis; NF = National Formulary; CIP = clean in place; USP = United States Pharmacopoeia; ACS = American Chemical Society

[0165] The starting materials include 9 phosphoramidites, a Controlled Pore Glass (CPG) solid support and 2-azido-1,3-dimcthylimidazolinium hcxafluorophosphatc. The reactive cxocyclic 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 A 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 in2 / g in surface area Hg intrusion (mercury intrusion), and 70 - 80 pmol / g assessed by DMT ligand assay (spectrophotometric at 498 nm). 2-Azido-l,3-dimethylimidazolinium hexafluorophosphate has a purity of about 98.0% or more (HPLC) and nitrogen content of about 23.70 - 24.80 % (elemental analysis).

[0166] Stereochemistry can be established through control of the starling 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 by3IP 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.

[0167] 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. Duringpurification, 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 ding 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

[0168] Among other things, the present disclosure provides technologies for assessing WVE-N531 or compositions thereof.

[0169] 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 (ESLMS) 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 ESLMS / 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 ESLMS / MS analysis, then the identities and locations of all 20 nucleobases have been demonstrated and the sequence was confirmed.

[0170] 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 A-(l,3- dimethylimidazolidin-2-ylidenyl) phosphoramidate, and phosphate linkages, respectively. WVE-N531 or a salt thereof also comprises 2’-fluoro (F) and 2’- O-Mcthyl (OMc) modifications.

[0171] Establishment of stereochemical identity comprises control of stalling materials for synthesis.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.

[0172] 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 (*H,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.19F NMR data for a preparation of WVE-N531 drug substance (hexadecasodium salt). Acquired and recorded at 348K.

[0173] 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.

[0174] FTIR spectrum of solid WVE-N531 hexadecasodium salt as a drag substance was recorded. The major absorbance bands at 1643 cm1and 1682 cm1are consistent with amide C=O, C=N and C=C stretch vibrations. Broad peaks in the 3200 -3300 cm1region are also consistent with oligonucleotide functional groups.

[0175] 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 drag substance lot, in agreement with the theoretical sodium content value of 5.1% (w / w) (hexadecasodium salt).

[0176] 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.

[0177] The molar extinction coefficient of WVE-N531 drug substance was experimentally determined in water to be 186486 M 'em1using 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.

[0178] 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.

[0179] 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.

[0180] Various preparations of WVE-N531 drug substance have met specified limits for residual solvents, including acetonitrile, toluene and pyridine (1CH Class 2), and elemental impurities, including various elements in ICH Class 1, 2A, 3, etc.

[0181] The fidelity of transfer of chiral information (diastereoselectivity) from each stereodefined phosphoramidite to its stereodefined Sp phosphorothioate or Rp 7V-(l,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.

[0182] 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.

[0183] The model studies demonstrate excellent stereochemical fidelity at each location, 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).

[0184] 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 stcrcopurity is about 81% or more. In some embodiments, a stcrcopurity 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 stcrcopurity is about 80%-90%. In some embodiments, a stcrcopurity is about 83%-87%.

[0185] 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),bioburden (total microbial aerobic count, total yeast and bolds count, etc. by USP <61) and / or Ph. Eur 2.6.12), etc.

[0186] Useful parameters for identity (molecular weight) by LC MS are described below:In 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.

[0187] 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] inwater) and mobile phase B (acetonitrile). A summary of method parameters is provided below as an example.

[0188] 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% acetonitile 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 diastereomerseach 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.).

[0189] 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.

[0190] 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 using a Waters BEH Cl 8 Column. A useful procedure is described below as an example (Protocol B). The separ ation is accomplished using a gr adient 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.).

[0191] 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, 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 DS 17, 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 DSl 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 rs free of two of the rest of WVE-N531 and DS1 to DS17. In some embodiments, a composition rs 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 someembodiments, a composition is free of five of the rest of WVE-N531 and DS1 to DS 17. In some embodiments, a composition is free of six of the rest of WVE-N531 and DS1 to DS 17. 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 DS 12. 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 comprises DS 16. In some embodiments, a composition comprises DS 17. In some embodiments, at leastWVE-N531 is absent from a composition. In some embodiments, at least DS 1 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 DS 10 is absent from a composition. In some embodiments, at least DS 11 is absent from a composition. In some embodiments, at least DS 12 is absent from a composition. In some embodiments, at least DS 13 is absent from a composition. In some embodiments, at least DS 14 is absent from a composition. In some embodiments, at least DS 15 is absent from a composition. In some embodiments, at least DS 16 is absent from a composition. In someembodiments, at least DS 17 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 DS 17 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.

[0192] 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 DS 17. In some embodiments, a composition is free of four of DS1 to DS 17. 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 DS 17. In some embodiments, a composition is free of seven of DS1 to DS 17. 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 DS 17. In some embodiments, a composition is free of 10 of DS1 to DS 17. 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 DS 17. In some embodiments, a composition is free of 16 of DS1 to DS 17. In some embodiments, a composition is free of 17 of DS1 to DS17. 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 DS 10 is absent from a composition. In some embodiments, at least DS 11 is absent from a composition. In some embodiments, at least DS 12 is absent from a composition. In some embodiments, at least DS 13 is absent from a composition. In some embodiments, at least DS 14 is absent from a composition. In some embodiments, at least DS 15 is absent from a composition. In some embodiments, at least DS 16 is absent from a composition. In some embodiments, at least DS 17 is absent from a composition.

[0193] Similar to WVE-N531, each of DS1 to DS17 may independently be in various forms. In someembodiments, 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 DI to DS 17 are dissolved and may dissociate to yield cations and anions.

[0194] 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 hairier with high levels of abrasion and puncture resistance.

[0195] 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 -20°C ± 5°C and after 14 days of storage at an accelerated condition of 5°C ± 3°C.Drug Product|00196| 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 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.

[0197] 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 drag product is or comprises aWVE- N531 drag 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.

[0198] Certain WVE-N531 drag products and useful manufacturing technologies are described below as examples.

[0199] In some embodiments, a WVE-N531 pharmaceutical composition, e.g., a drug product ispackaged 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.

[0200] An example composition of WVE-N531 drug product is presented below:Abbreviations: USP = United States Pharmacopeia, NF = National Formulary, Ph. Eur. = European Pharmacopoeia, JP = Japanese Pharmacopoeia, BP = British Pharmacopoeia; qs = quantity sufficient, N / A = Not Applicable

[0201] 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.

[0202] 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.

[0203] 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 made by pooling multiple drug substance lots.

[0204] 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.

[0205] A flow diagram for a WVE-N531 drug product manufacturing process is provided in Figure 1 as an example.

[0206] 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 lx and 2x strengths are prepared where the lx 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.

[0207] If a single lot of drug substance is used for formulation, the release purity result is used as the purity collection factor. If multiple lots of drug substance are to be pooled, then a purity collection factor that is based on the quantity of active ingredient from each drug substance lot is determined.1002081 In some embodiments, water for injection (WEI) 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.

[0209] Based on the results of WVE-N531 drug substance concentration and solution mass, the required volume of lx PBS to accomplish WVE-N531 drug substance concentration of 6.0 mg / mL (WVE- N531 free acid form) is calculated. The required amount of 1 x PBS is then added directly to the formulation vessel, and the contents of the vessel are mixed to ensure solution homogeneity.

[0210] 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.

[0211] In some embodiments, an in-process pre-fil trati on bioburden sample is collected prior to sterile filtration. In some embodiments, the final formulation is sterile filtered through two 0.2 pm filters in series prior to filling. In some embodiments, sterile filters arc checked for filter integrity by bubble point method prior to and post filtration.

[0212] 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 vials are visually inspected prior to release testing, bulk packaging, and / or stability studies.

[0213] 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.

[0214] 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 lx PBS to be added for the final concentration which is measured prior to sterile filtration and filling.

[0215] 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 / orPh. 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.

[0216] A WVE-N531 drug product may comprise similar impurities as a WVE-N531 drug substance at levels as described herein.

[0217] In some embodiments, a container closure system for WVE-N531 drug product comprises orconsists 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.

[0218] In some embodiments, a storage condition for WVE-N531 drug product is -20 ± 5°C. In some embodiments, a condition of 5°C ± 3°C, e.g., for relatively short term storage.

[0219] Among other things, the present disclosure provides the following Example Embodiments:1. A method for delivering an oligonucleotide into a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.2. A method for providing DMD exon skipping in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.3. A method for restoring DMD RNA reading frame in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.4. A method for providing a DMD polypeptide in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide, wherein the DMD polypeptide is truncated compared to wild-type dystrophin.5. A method for increasing dystrophin level in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide, wherein the DMD polypeptide is truncated compared to wild-type dystrophin.6. A method for increasing level of a dystrophin polypeptide encoded by an exon-skipped DMD transcript in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide, wherein the DMD polypeptide is truncated compared to wild-type dystrophin.7. A method for restoring satellite cell function in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.8. A method for producing myogenic progenitor cells in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.9. A method for providing muscle regeneration in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.10. A method for providing myofiber repair in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.11. The method of any one of Embodiments 8-10, wherein the system comprises satellite cells.12. The method of Embodiment 11, wherein the oligonucleotide is delivered into satellite cells.13. A method for increasing differentiation of satellite cells in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.14. The method of any one of the preceding Embodiments, comprising formation of myoblasts.15. The method of any one of the preceding Embodiments, comprising formation of myogenic progenitor cells.16. The method of any one of the preceding Embodiments, comprising formation of myogenic progenitor cells from satellite cells.17. The method of any one of the preceding Embodiments, comprising formation of myotubes.18. The method of any one of the preceding Embodiments, comprising formation of myotubes from myogenic progenitor cells.19. The method of any one of the preceding Embodiments, comprising formation of mature myofibers.20. The method of any one of the preceding Embodiments, wherein the satellite cells are Pax7+.21. The method of any one of the preceding Embodiments, comprising formation of activated satellite cells.22. The method of any one of the preceding Embodiments, comprising satellite cell self-renewal.23. The method of any one of the preceding Embodiments, comprising formation of myogenic progenitor cell from satellite cell.24. The method of any one of the preceding Embodiments, comprising myogenic progenitor cell differentiation.25. The method of any one of the preceding Embodiments, comprising cell fusion.26. The method of any one of the preceding Embodiments, comprising myotube formation.27. The method of any one of the preceding Embodiments, comprising myofiber formation.28. The method of any one of the preceding Embodiments, comprising myofiber repair.29. The method of any one of the preceding Embodiments, comprising formation ofPax7+ / Myf5+ / MyoDl+cells.30. The method of any one of the preceding Embodiments, comprising formation ofPax7+ / Myf5+ / MyoDl+cells from activated satellite cells.31. The method of any one of the preceding Embodiments, comprising formation ofMyoDl+ / Myogenin+cells.32. The method of any one of the preceding Embodiments, comprising formation ofMyoDl+ / Myogenin+cells from Pax7+ / Myf57MyoDl+cells.33. The method of any one of the preceding Embodiments, comprising formation ofMyogenin+ / MyHC+cells.34. The method of any one of the preceding Embodiments, comprising formation ofMyogenin+ / MyHC+cells from MyoDl+ / Myogenin+cells.35. The method of any one of Embodiments 14-34, wherein the formation comprises cell division.36. The method of any one of Embodiments 14-35, wherein the formation comprises differentiation.37. The method of any one of Embodiments 14-36, wherein the formation comprises fusion.38. The method of any one of the preceding Embodiments, wherein the cells have a mutation of the DMD gene that is amenable to exon skipping.39. The method of any one of the preceding Embodiments, wherein the cells have a mutation of the DMD gene that is amenable to exon 53 skipping.40. The method of any one of the preceding Embodiments, wherein the system is or comprises a population of cells (e.g., cell culture).41. The method of any one of the preceding Embodiments, wherein the system is or comprises a tissue.42. The method of any one of the preceding Embodiments, wherein the system is or comprises a muscle tissue.43. The method of any one of the preceding Embodiments, wherein the system comprises skeletal muscle cells.44. The method of any one of the preceding Embodiments, wherein the system comprises myofiber.45. The method of any one of the preceding Embodiments, wherein the system is or comprises an organ.46. The method of any one of the preceding Embodiments, wherein the system is or comprises an animal.47. The method of any one of the preceding Embodiments, wherein the system is or comprises a subject.48. The method of any one of the preceding Embodiments, wherein the system is a patient susceptible to or suffering from DMD.49. A method for treating muscular dystrophy, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide, wherein the oligonucleotide is delivered to satellite cells of the subject.50. A method for providing increased level of a muscle function in a subject, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide, wherein the oligonucleotide is delivered to satellite cells of the subject.51. A method for providing increased level of myofiber repair in a subject, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide, wherein the oligonucleotide is delivered to satellite cells of the subject.52. A method for providing increased level of a DMD function in a subject, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide, wherein the oligonucleotide is delivered to satellite cells of the subject.53. The method of any one of Embodiments 48-52, wherein the subject suffers from a musculardystrophy.54. The method of any one of Embodiments 48-52, wherein the subject suffers from Duchenne muscular dystrophy.55. The method of any one of the preceding Embodiments, wherein the subject has a mutation of the DMD gene that is amenable to exon skipping.56. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide is delivered to a satellite cell.57. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a PN linkage.58. The oligonucleotide of any one of the preceding Embodiments, wherein a PN linkage is Rp.59. The oligonucleotide of any one of the preceding Embodiments, wherein a PN linkage is Sp.60. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a phosphoryl guanidine internucleotidic linkage.61. The oligonucleotide of any one of the preceding Embodiments, wherein a phosphoryl guanidine internucleotidic linkage is Rp .62. The oligonucleotide of any one of the preceding Embodiments, wherein a phosphoryl guanidine internucleotidic linkage is Sp.63. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a nOOl internucleotidic linkage.64. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a Rp nOOl internucleotidic linkage.65. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a Sp nOOl internucleotidic linkage.66. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a PS linkage.67. The oligonucleotide of any one of the preceding Embodiments, wherein a PS linkage is Rp .68. The oligonucleotide of any one of the preceding Embodiments, wherein a PS linkage is Sp.69. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a phosphorothioate internucleotidic linkage.70. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a Rp phosphorothioate internucleotidic linkage.71. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a Sp phosphorothioate internucleotidic linkage.72. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotidecomprises a PO linkage.73. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a natural phosphate linkage.74. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a 2'-F modified sugar.75. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a block of two or more consecutive 2’-F modified sugars.76. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a 2’-OR modified sugar-, wherein R is optionally substituted Ci-Ce aliphatic.77. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a 2’-0Me modified sugar.78. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide is administered or delivered in a chirally controlled oligonucleotide composition.79. The method of any one of the preceding Embodiments, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping.80. The method of any one of the preceding Embodiments, wherein the oligonucleotide is WVE-N531.81. The method of Embodiment 80, wherein WVE-N531 is administered or delivered 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.82. The method of any one of Embodiments 80-81, wherein WVE-N531 is administered or delivered in one or more forms.83. The method of any one of the preceding Embodiments, wherein WVE-N531 is administered or delivered in one or more pharmaceutically acceptable salt forms.84. The method of any one of Embodiments 82-83, wherein one form is WVE-N531 hexadecasodium salt.85. The method of any one of Embodiments 82-84, wherein each dose of WVE-N531 is independently administered in a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable earner.86. The method of any one of Embodiments 80-85, wherein a dose of WVE-N531 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.87. The method of any one of Embodiments 80-86, wherein a dose of WVE-N531 is equivalent to about 10 mg / kg WVE-N531 free acid form.88. The method of any one of Embodiments 80-87, 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.89. The method of any one of the preceding Embodiments, wherein about 3 or more doses are administered.90. The method of any one of the preceding Embodiments, wherein about 10 or more doses are administered.91. The method of any one of Embodiments 88-90, wherein each dose is independently administered in a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier.92. The method of any one of Embodiments 88-91, wherein each dose has about the same amount of WVE-N531.93. The method of any one of Embodiments 88-92, 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.94. The method of any one of Embodiments 88-92, wherein WVE-N531 in each dose is equivalent to about 10 mg / kg WVE-N531 free acid form.95. The method of any one of Embodiments 88-92, wherein WVE-N531 in each dose is equivalent to about 20 mg / kg WVE-N531 free acid form.96. The method of any one of Embodiments 92-95, wherein about is ±1%.97. The method of any one of Embodiments 92-95, wherein about is ±2%.98. The method of any one of Embodiments 92-95, wherein about is ±3%.99. The method of any one of Embodiments 92-95, wherein about is ±4%.100. The method of any one of Embodiments 92-95, wherein about is ±5%.101. The method of any one of Embodiments 92-95, wherein about is ±6%.102. The method of any one of Embodiments 92-95, wherein about is ±7%.103. The method of any one of Embodiments 92-95, wherein about is ±8%.104. The method of any one of Embodiments 92-95, wherein about is ±9%.105. The method of any one of Embodiments 92-95, wherein about is ±10%.106. The method of any one of Embodiments 80-105, wherein WVE-N531 is administered or delivered in a pharmaceutical composition which is a liquid composition comprising dissolved WVE-N531.107. The method of any one of Embodiments 80-106, wherein a pharmaceutically acceptable carrier in the pharmaceutical composition is or comprises a phosphate buffered solution.108. The method of any one of Embodiments 80-107, 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.109. The method of any one of Embodiments 80-108, wherein a pharmaceutical composition is isotonic.110. The method of any one of Embodiments 80-109, wherein a pharmaceutical composition has a pHof about 7-8.111. The method of any one of Embodiments 80-110, wherein two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 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.112. The method of any one of Embodiments 80-111, wherein three or more consecutive doses are administered about every two weeks.113. The method of any one of Embodiments 80-112, wherein WVE-N531 has a purity of about 70%- 90%.114. The method of Embodiment 113, wherein the purity is measured by IP-RP-UPLC using area % at 260 nm.115. The method of Embodiment 113, wherein the purity is measured by IP-RP-UPLC using area % at 260 nm and the Set A parameters.116. The method of Embodiment 113, wherein the purity is measured by an IP-RP-UPLC method for purity as described herein.117. The method of any one of Embodiments 80- 116, wherein impurities in the composition are no more than about 10%-30%.118. The method of Embodiment 117, wherein the impurities are measured by IP-RP-UPLC using area % at 260 nm.119. The method of Embodiment 117, wherein the impurities are measured by IP-RP-UPLC using area % at 260 nm and the Set A par ameters.120. The method of Embodiment 117, wherein the impurities are measured by an IP-RP-UPLC method for purity as described herein.121. The method of any one of Embodiments 80-120, wherein stereochemical purity of the oligonucleotide is about 80% or more.122. The method of any one of Embodiments 80-121 , wherein the amount of WVE-N531 is measured by UV at 260 nm and 27 OD / mg.123. The method of any one of Embodiments 80-122, wherein a WVE-N531 drug substance is manufactured by a process described herein.124. The method of any one of Embodiments 80-123, wherein a WVE-N531 drug substance is characterized by one or more method described herein.125. The method of any one of Embodiments 80-124, wherein stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC.126. The method of any one of Embodiments 80-125, wherein stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC according to Set B parameters.127. The method of any one of Embodiments 80-126, wherein stereochemical identity of WVE-N531 is confirmed by an IP-RP-UPLC method for stereochemical identity as described herein.128. The method of any one of Embodiments 80-127, wherein a WVE-N531 drug substance is released by one or more method described herein.129. The method of any one of Embodiments 80-128, wherein a WVE-N531 drug substance is stored by one or more method described herein.130. The method of any one of Embodiments 123-129, wherein the WVE-N531 drug substance is hexadecasodium salt.131. The method of any one of Embodiments 80-130, wherein a WVE-N531 drug product is manufactured by a process described herein.132. The method of any one of Embodiments 80-131, wherein a WVE-N531 drug product is characterized by one or more method described herein.133. The method of any one of Embodiments 80-132, wherein a WVE-N531 drug product is released by one or more method described herein.134. The method of any one of Embodiments 80-133, wherein WVE-N531 is administered intravenously.135. The method of any one of the preceding Embodiments, wherein a DMD mutation is A3-52, A4-52, A5-52, Δ6-52, Δ9-52, Δ10-52, Δll-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17-52, Δ19-52, Δ21-52, A23- 52, Δ24-52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32-52, Δ33-52, A34-52, A35- 52, Δ36-52, Δ37-52, Δ38-52, Δ39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ45-52, Δ47-52, A48-52, Δ49- 52, Δ50-52, Δ51-52, Δ52, Δ54-58, Δ54-61, Δ54-63, Δ54-64, Δ54-66, A54-76, or A54-77.136. The method of any one of the preceding Embodiments, wherein the mutation comprises A52.137. The method of any one of the preceding Embodiments, wherein the mutation is A52.138. The method of Embodiment 136, wherein the mutation is A45-52.139. The method of Embodiment 136, wherein the mutation is A47-52.140. The method of Embodiment 136, wherein the mutation is A48-52.141. The method of Embodiment 136, wherein the mutation is A49-52.142. The method of Embodiment 136, wherein the mutation is A50-52.143. The method of Embodiment 136, wherein the mutation is A51-52.144. The method of any one of the preceding Embodiments, wherein exon 53 is skipped in DMD mRNA.145. The method of any one of the preceding Embodiments, wherein level of exon 53-skipped DMD mRNA is increased.146. The method of any one of the preceding Embodiments, wherein level of exon 53-skippcd DMD mRNA in satellite cells is increased.147. The method of any one of the preceding Embodiments, wherein level of exon 53-skipped DMD mRNA in myogenic progenitor cells is increased.148. 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.149. The method of any one of the preceding Embodiments, wherein a truncated DMD polypeptide is produced compared to wild-type dystrophin.150. The method of any one of the preceding Embodiments, wherein a truncated DMD polypeptide is produced in satellite cells compared to wild-type dystrophin.151. The method of any one of the preceding Embodiments, wherein a truncated DMD polypeptide is produced in myogenic progenitor cells compared to wild-type dystrophin.152. The method of any one of the preceding Embodiments, wherein level of a truncated DMD polypeptide in is increased.153. The method of any one of the preceding Embodiments, wherein level of a truncated DMD polypeptide in satellite cells is increased.154. The method of any one of the preceding Embodiments, wherein level of a truncated DMD polypeptide in myogenic progenitor cells is increased.155. The method of any one of Embodiments 149-154, wherein the truncated DMD polypeptide performs one or more functions of wild-type dystrophin.156. The method of any one of the preceding Embodiments, wherein the method provide increases from baseline in dystrophin levels of about 1 %-20% or more of normal levels after administration for a certain time period or after a certain number of doses.157. The method of any one of the preceding Embodiments, wherein the method provide increases from baseline in dystrophin levels of about 1 %-20% or more of normal levels in satellite cells after administration for a certain time period or after a certain number of doses.158. The method of any one of Embodiments 156-157, wherein the increase is measured after administration for about 3-98 weeks from first dose.159. The method of any one of Embodiments 156-157, wherein the increase is measured after administration for about 4 weeks from first dose.160. The method of any one of Embodiments 156-157, wherein the increase is measured after administration for about 6 weeks from first dose.161. The method of any one of Embodiments 156-160, wherein the increase is measured after about 1- 48 doses.162. The method of any one of Embodiments 156-160, wherein the increase is measured after 3 doses.163. The method of any one of the preceding Embodiments, wherein an assessment is performed after about 12 doses.164. The method of any one of the preceding Embodiments, wherein an assessment is performed after about 24 doses.165. The method of any one of Embodiments 162-164, wherein the doses are administered or delivered about every two weeks.166. The method of any one of Embodiments 162-165, wherein each of the doses is equivalent to about 10 mg / kg WVE-N531 free acid form.167. The method of any one of the preceding Embodiments, wherein a muscle function is increased.168. The method of any one of the preceding Embodiments, wherein a muscle function is restored.169. The method of any one of the preceding Embodiments, wherein a DMD function is increased.170. The method of any one of the preceding Embodiments, wherein a DMD function is restored.171. The method of any one of the preceding Embodiments, wherein loss of ambulation in a subject is reduced.172. The method of any one of the preceding Embodiments, wherein disease progression in the subject is delayed or slowed.173. The method of any one of the preceding Embodiments, wherein muscle weakness in the subject is delayed or slowed.174. The method of any one of the preceding Embodiments, wherein loss of muscle mass in the subject is delayed or slowed.175. The method of any one of the preceding Embodiments, wherein loss of pulmonary function in the subject is delayed or slowed.176. The method of any one of the preceding Embodiments, wherein the subject improves in a muscular dystrophy assessment.177. The method of any one of the preceding Embodiments, wherein the subject improves in a 10 meter walk test.178. The method of any one of the preceding Embodiments, wherein the subject improves in one or more functional assessments.179. The method of any one of the preceding Embodiments, wherein the subject improves in North Star Ambulatory Assessment (NSAA) 2.0.180. The method of any one of the preceding Embodiments, wherein the subject improves in Performance of the Upper Limb (PUL) 2.0.181. 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.182. The method of any one of the preceding Embodiments, wherein the subject improves in four-stair climb.183. The method of any one of the preceding Embodiments, wherein the subject improves in time to rise from the floor.184. The method of any one of the preceding Embodiments, wherein the subject improves in upper limb proximal strength.185. The method of any one of the preceding Embodiments, wherein the subject improves in handheld myometry.186. The method of any one of the preceding Embodiments, wherein the subject improves in one or more pulmonary function tests.187. The method of any one of the preceding Embodiments, wherein the subject improves in one or more of peak flow rate [PER], cough peak flow [CPE], and FVC.188. The method of any one of the preceding Embodiments, wherein an improvement is compared to baseline.189. The method of any one of the preceding Embodiments, wherein an improvement is compared to absence of WVE-N531 administration.190. The method of any one of the preceding Embodiments, wherein an improvement is compared to administration of a reference composition.191. The method of any of the previous Embodiments, wherein the subject is a pediatric subject.192. The method of any one of the preceding Embodiments, wherein the subject is administered a steroid at least about one month prior to the first dose of WVE-N531.193. The method of any one of the preceding Embodiments, wherein the subject is administered a steroid at least about six months prior to the first dose of WVE-N531.194. The method of any one of the preceding Embodiments, wherein the subject is administered a corticosteroid at least about one month prior to the first dose of WVE-N531.195. The method of any one of the preceding Embodiments, wherein the subject is administered a corticosteroid at least about six months prior to the first dose of WVE-N531.196. The method of any one of Embodiments 194-195, wherein the corticosteroid is deflazacort.197. The method of any one of the preceding Embodiments, wherein the subject is administered a glucocorticoid at least about one month prior to the first dose of WVE-N531.198. The method of any one of the preceding Embodiments, wherein the subject is administered a glucocorticoid at least about six months prior to the first dose of WVE-N531.199. The method of any one of Embodiments 197-198, wherein the corticosteroid is prednisone.200. The method of any one of the preceding Embodiments, comprising administering or delivering oneor more other therapeutic agents.201. The method of any one of the preceding Embodiments, comprising administering or delivering an agent selected from veliflapon, difenpiramide, amopyroquine, cloricromen, alpelisib, benorilate, xipamide, fabomotizole and altizide, or a pharmaceutically acceptable salt thereof.202. The method of any one of Embodiments 200-201, wherein the oligonucleotide and an agent is administered or delivered concurrently.203. The method of any one of Embodiments 200-201, wherein the oligonucleotide and an agent is administered or delivered in the same composition.204. The method of any one of Embodiments 200-201 , wherein the oligonucleotide is administered prior to an agent.205. The method of Embodiment 204, wherein the system or subject is under the effect of the oligonucleotide when the agent is administered or delivered.206. The method of any one of Embodiments 200-201, wherein the oligonucleotide is administered subsequently to an agent.207. The method of Embodiment 206, wherein the system or subject is under the effect of the agent when the oligonucleotide is administered or delivered.208. An oligonucleotide or a pharmaceutical composition thereof, for use in a method of any one of the preceding Embodiments.209. An oligonucleotide or a pharmaceutical composition thereof, 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.210. The oligonucleotide or a phar maceutical composition thereof of any one of Embodiments 208-209, wherein the oligonucleotide is WVE-N531.211. An oligonucleotide, for manufacturing of a medicament for use in a method of any one of the preceding Embodiments.212. An oligonucleotide, for manufacturing of a medicament 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.213. The oligonucleotide of any one of Embodiments 211-212, wherein the oligonucleotide is WVE- N531.214. Use of an oligonucleotide or a pharmaceutical composition, for a method of any one of the preceding Embodiments.215. Use of an oligonucleotide or a pharmaceutical composition, 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.216. Use of an oligonucleotide, for manufacturing a medicament for use in a method of any one of thepreceding Embodiments.217. Use of an oligonucleotide, for manufacturing a medicament 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.218. The use of any one of Embodiments 214-217, wherein the oligonucleotide is WVE-N531.EXEMPLIFICATION

[0220] The foregoing has been a description of certain non-limiting embodiments of the disclosure. Accordingly, it is to be understood that embodiments of the disclosure herein described are merely illustrative of applications of principles of the disclosure. Reference herein to details of illustrated embodiments is not intended to limit the scope of any claims.

[0221] Functions and advantage of certain embodiments of the present disclosure may be more fully understood from the examples described below. The following example(s) are intended to illustrate certain benefits of such embodiments.EXAMPLE 1. WVE-N531 can be safely delivered into satellite cells.

[0222] WVE-N531 uptake by skeletal muscle stem cells was assessed using muscle specimens obtained from DMD patients enrolled in clinical trial WVE-N531-001 phase IB (also identified by ClinicalTrials.gov ID NCT04906460; patients received single escalating doses of 1, 3, 6 and 10 mg / kg (administered >4 weeks apart), and following a period of ~1-2 months, three doses of WVE-N531 at 10 mg / kg every other week, followed by muscle biopsy 2 weeks after the last dose). Among other things, three assays were utilized including PAX7 (stem cell marker) immunohistochemistry (singleplex), in-situ hybridization (RNAScope, ACDBio) for WVE-N531 (singleplex), and dual staining using RNAScope and immunohistochemistry.

[0223] PAX7 Immunohistochemistry Singleplex.

[0224] Frozen sections were kept at -80 °C freezer until removal for IHC. Upon freezer removal, slides were immediately placed in 4 °C 10% NBF for 1 hour in 4 °C refrigerator. After fixation, slides were run through PBS (2 min) and a series of 50% ethanol, 70% ethanol, 100% ethanol (2 changes) for 5 minutes each solution. Slides were then dried for 5 minutes before loading into a Ventana Ultra (automated immunostainer). Pax7 (Novus BP2-32894, Lot 5051-1P230) was run on the Ventana Ultra at 5 ug / ml concentration. IgG Ms (Vector 1-2000) was run with same conditions as Pax7 for IgG Ms Control. Pax7 IHC slides (along with IgG Ms controls) were run with Roche ChromoMap and Mouse OmniMap Kit (760- 150). Antigen Retrieval consisted of CC1 (Cell Conditioning 1) application for 64 minutes, after which antibody was applied for 1 hr with no heat and Ms-HRP for 16 minutes. Hematoxylin (760-2021) for 8 minutes and Bluing Reagent (760-2037) for 4 minutes finished up the assay. Slides were then washed, dried in a 60 °C oven for 30 minutes and coverslipped with ClearMount (Statlab MMC0126EA) beforereview.

[0225] RNAScope Singleplex.

[0226] Customized probe to detect WVE-N531 (single Z probe, ACDBio) and positive control probes to detect human UBC (ubiquitin C, ACDBio catalogue numbers 312029 (human)) were used to confirm preservation of sample mRNA quality. Negative control probe to detect bacterial DapB (dihydrodipicolinate reductase, ACDBio) was used to rule out non-specific binding. Muscle sample obtained from WVE-N531 treated and non-treated (control) monkeys were used for protocol development and were then ran along with patient samples as positive control tissues to confirm that assay was reproducible. Unstained slides with frozen muscle sections were stored in slide boxes in zip-lock bags until use at -80 °C. On the day of experiment, slides were removed from -80 °C storage and placed immediately into cold (4 °C) 10% NBF for 1 hour in 4 °C refrigerator. Slides were then dehydrated in graded ethanols (50%, 70%, 100% (x2), 5 minute each at room temperature and then run on the Ventana Ultra (see next section) at probe (WVE-N531) dilution of 1:10, 1:50, and 1:100.

[0227] Ventana Ultra mRNA Universal Protocol.

[0228] The program mRNAscope Universal (v5.00.0000) was used to run VS RNAscope. Protease was run for 16 minutes at 37 °C and RNAscope probes were hybridized at 43 °C for 2 hrs. The program has 7 AMP steps (TSA Amplification), 3 AMPs are modifiable; Amp 1 and Amp2 were run at 39 °C and Amp3 was run for 24 minutes. After amplification steps and Past Red steps for colorization were completed. Hematoxylin (760-2021 ) was administered for 12 minutes and Bluing Reagent (760-2037) for 4 minutes to finish up the assay. After slides were washed in running water and Dawn soap, they were dried in a 60 °C oven for 30 minutes and coverslipped with Ecomount Mounting Media (Biocare EM897L) for review.

[0229] With all dilutions strong signal was detected in samples obtained from patients treated with WVE-N531 using WVE-N531 and UBC probes, whereas no signal was detected in muscle samples stained with DapB probes.

[0230] Dual WVE-N531 RNAScope and PAX7 immunohistochemistry.

[0231] Frozen sections were kept in -80 °C freezer until removal for IHC. Upon freezer removal, slides were immediately placed in 4 °C 10% NBF for 1 hour in 4 °C refrigerator. After fixation, slides were run through PBS (2 min) and series of 50% ethanol, 70%- ethanol, 100$% ethanol (2 changes) for 5 minutes each solution. Slides were then dried for 5 minutes before loading into a Ventana Ultra. ISH was performed with Advanced Cell Diagnostics (ACD) RNAscope VS Universal AP Reagent Kit (ACD 323250) using Ventana mRNA Universal Procedure v5. RNAscope Probe hybridization was for 2 hours at 43 °C.

[0232] As those skilled in the art appreciate, in some instances, a protocol may be modified. In someembodiments, protocol modifications included elimination of Cell Conditioning and Protease steps. Conditions for RNAscope steps that could be modified also included running Amp 1 and Amp 2 at 39 °C and setting time for Amp 5 for 24 minutes. As RNAscope ISH and antibody 1HC were included in a single program, IHC was sequentially run after RNAscope was completed.

[0233] PAX7 (Novus BP2-32894, Lot 5051-1P230) was run on the Ultra at 5 ug / ml concentration. IgG Ms (Vector 1-2000) was run with same conditions as Pax7 for IgG Ms Control. Pax? IHC slides (along with IgG Ms controls) were run with Roche ChromoMap and Mouse OmniMap Kit (760-150). PAX7 antibody was applied for 1 hr with no heat and Ms-HRP for 16 mins. Hematoxylin (760-2021) for 8 minutes and Bluing Reagent (760-2037) for 4 minutes finished up the assay. Slides were then washed, dried in a 60 C oven for 30 minutes and coverslipped with Ecomount Mounting Media (Biocare EM897L) for review.

[0234] All 3 patient samples contained variable numbers of PAX7 positive cells. Specific signal was detected in nucleus of cells that were located on the periphery of myocytes or surrounding necrotic myofibers (stem cells). Nonspecific staining, unrelated to PAX7 antibody, was observed in cytoplasm of some endothelial and smooth muscle cells. All 3 patient samples were stained positive for WVE-N531 which was observed in myocytes (sarcoplasm and nucleus), interstitium, and macrophages.

[0235] Among other things, the present disclosure demonstrated that WVE-N531 was uptaken by satellite cells. Various numbers and percentages of PAX7 positive myocyte stem cells (nuclear signal) contained WVE-N531 in nucleus and cytoplasm in samples from three patients who received three doses of WVE-N531 administered intravenously every two weeks and each independently equivalent to about 10 mg / kg WVE-N531 free acid form. Patients can receive more doses of WVE-N531, e.g., each independently equivalent to about 10 mg / kg WVE-N531 free acid form about every two weeks.

[0236] Stem cell distribution data from Part B of clinical trial NCT04906460 (certain information of which is described at www.clinicaltrials.gov) in a population of patients receiving 10 mg / kg (unless noted otherwise, equivalent to free acid form) WVE-N531 about every two weeks for 24 weeks confirmed that WVE-N531 was delivered into myocyte nuclei in all participants and into myogenic stem cells in the majority of participants. Further, multiple indicators of improvement in muscle health were observed, including an increase in the percentage of myocytes with internalized nuclei and an improvement in myofiber size and diameter.

[0237] In some embodiments, WVE-N531 may be administered less frequently than about every two weeks (e.g., about monthly). In some embodiments, one or more doses of WVE-N531 are administered about every 4 weeks. In some embodiments, one or more doses of WVE-N531 are administered about monthly. In some embodiments, each dose of WVE-N531 is independently equivalent to about 10 mg / kg WVE-N531 free acid form.

[0238] Certain information of the Part B clinical trial is presented below.

[0239] Certain participation criteria are described below.

[0240] Ages eligible for study: 5 years to 18 years (child, adult).

[0241] Sexes Eligible for Study: Male.

[0242] Accepts Healthy Volunteers: No.

[0243] Inclusion Criteria:1. Part A patients may be screened for Part B upon completion of a washout period of >18 weeks from last dose in Part A. New patients may also be screened for Part B2. Diagnosis of DMD based on clinical phenotype .3. Documented mutation in the DMD gene associated with DMD that is amenable to exon 53 intervention4. Score of >1 on item 1 or 2 of the shoulder component of the Performance of the Upper Limb (PUL).5. Ambulatory or non-ambulatory male6. Stable pulmonary and cardiac function, as measured by the following:1. Reproducible percent predicted forced vital capacity (FVC) >50%;2. Left ventricular ejection fraction (LVEF) >55% in patients <10 years of age and >45% in patients >10 years of age, as measured (and documented) by echoc ardiogr am (ECHO) or cardiac magnetic resonance imaging (MRI), within 6 months prior to enrollment into the study.7. Adequate muscle at Screening to perform open muscle biopsies.8. Currently on a stable corticosteroid therapy regimen, defined as initiation of systemic corticosteroid therapy that occurred >6 months prior to Screening and no changes in dose <3 months prior to Screening visit.

[0244] Exclusion Criteria:1. Clinically significant medical finding on the physical examination other than DMD that, in the judgment of the Investigator, will make the patient unsuitable for participation in, and / or completion of the study procedures.2. Major surgery within 3 months prior to Day 1 or planned major surgery for any time during the study.3. Diagnosis of active alcohol, cannabinoid, or other substance use disorder (except nicotine) within 6 months prior to the Screening visit.

[0245] WVE-N531 were administered intravenously 10 mg / kg Q2W (every 2 weeks). Key assessments include:Safety and tolerabilityMuscle biopsies after 24 weeks of treatment- PK: Drug tissue concentrations- PD: Exon-skipping, Dystrophin level (% of normal) as assessed by Western BlotFunctional outcome measures11 participants enrolled, including two from prior Part A clinical trial- Pre-specified analyses in ambulatory patients

[0246] Certain results from an assessment after 24-week administration are presented below.

[0247] Baseline patient characteristics: Eleven boys amenable to exon 53 skipping (age 5-11; 10 ambulatory and 1 non-ambulatory) were enrolled in the ongoing, open-label FORWARD-53 trial.

[0248] Dystrophin was observed to be localized to sarcolemma membrane (immunohistochemistry staining of anti-dystrophin antibody of a representative patient). WVE-N531 was localized in myofiber nuclei and myogenic stem cells. WVE-N531 was detected in myocyte nuclei in all participants and in myogenic stem cells in the majority of participants. Myogenic stem cells are the progenitor cells for new myoblasts, which give rise to new myocytes and ultimately aid in skeletal muscle regeneration. WVE- N531 uptake in myofiber nuclei was observed (In-situ hybridization for WVE-N531). WVE-N531 uptakein myogenic stem cells was observed (Dual staining utilizing in-situ hybridization for WVE-N531 and PAX7 immunohistochemistry for stem cells). Among other things, restoring dystrophin in muscle stem cells can enable repair of muscle fibers. Without the intention to be limited by any particular theory, Applicant notes that 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, and restoring dystrophin in muscle stem cells can enable them to function properly, allowing quiescent cells to “wake up,” differentiate, and initiate repair of muscle fibers. In some embodiments, provided technologies provide, promote and / or enhance muscle stem cell activation, renewal and / or differentiation, and / or muscle repair. Dual staining utilizing in-situ hybridization for WVE-N531 and PAX7 immunohistochemistry for stem cells was utilized.

Claims

CLAIMS1. A method for treating Duchenne muscular dystrophy, comprising administering to a subject suffering therefrom three or more doses of WVE-N531, wherein: each dose is independently equivalent to about 10 mg / kg WVE-N531 free acid form;WVE-N531 is delivered into one or more satellite cells of the subject after no more than three doses of WVE-N531 each independently equivalent to about 10 mg / kg WVE-N531 free acid form, ; andWVE-N531 may be described as, wherein m represents a 2’-0Me modified nucleoside; f represents a 2’-F modified nucleoside;*5 represents a ftp phosphorothioate linkage; and n001 ft represents a ftp A-( 1 ,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage.

2. A method for delivering WVE-N531 into a satellite cell (myocyte stem cell) in a subject, comprising administering to the subject three or more doses of WVE-N531, wherein: each dose is independently equivalent to about 10 mg / kg WVE-N531 free acid form;WVE-N531 may be described aswherein m represents a 2’-0Me modified nucleoside; f represents a 2’-F modified nucleoside;*5 represents a -S’p phosphorothioate linkage; and n001 ft represents a ftp A-( 1 ,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage.

3. A method for delivering an oligonucleotide into a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide; or a method for providing DMD exon skipping in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide; or a method for restoring DMD RNA reading frame in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide; or a method for providing a DMD polypeptide in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide, wherein the DMD polypeptide is truncated compared to wild-type dystrophin. a method for increasing dystrophin level in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide, wherein the DMD polypeptide istruncated compared to wild-type dystrophin; or a method for increasing level of a dystrophin polypeptide encoded by an exon-skipped DMD transcript in a satellite cell in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide, wherein the DMD polypeptide is truncated compared to wild-type dystrophin; or a method for restoring satellite cell function in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide; or a method for producing myogenic progenitor cells in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide; or a method for providing muscle regeneration in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide; or a method for providing myofiber repair in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.

4. The method of claim 3, wherein the system comprises satellite cells.

5. The method of claim 4, wherein the oligonucleotide is delivered into satellite cells.

6. A method for increasing differentiation of satellite cells in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide.

7. The method of any one of the preceding claims, comprising formation of myoblasts, myogenic progenitor cells, myotubes, and / or mature myofibers.

8. The method of any one of the preceding claims, wherein the satellite cells are Pax7+.

9. The method of any one of the preceding claims, comprising formation of activated satellite cells, satellite cell self-renewal, formation of myogenic progenitor cell from satellite cell, myogenic progenitor cell differentiation, cell fusion, myotube formation, myofiber formation, myofiber repair, formation of Pax7+ / Myf5+ / MyoDl+cells, formation of Pax7+ / Myf5+ / MyoDl+cells from activated satellite cells, formation of MyoDl+ / Myogenin+cells, formation of MyoDl+ / Myogenin+cells from Pax7+ / Myf5+ / MyoD1+cells, formation of Myogenin+ / MyHC+cells, and / or formation of Myogenin+ / MyHC+cells from MyoDl+ / Myogenin+cells.

10. The method of any one of claims [0016] -9, wherein the formation comprises cell division, differentiation, and / or fusion.

11. The method of any one of the preceding claims, wherein the cells have a mutation of the DMD gene that is amenable to exon skipping.

12. The method of any one of the preceding claims, wherein the cells have a mutation of the DMD gene that is amenable to exon 53 skipping.

13. The method of any one of the preceding claims, wherein the system is or comprises a populationof cells (e.g., cell culture), a muscle tissue, skeletal muscle cells, myofiber, an organ, an animal or a subject.

14. The method of any one of the preceding claims, wherein the system is a patient susceptible to or suffering from DMD.

15. A method for treating muscular dystrophy, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide, wherein the oligonucleotide is delivered to satellite cells of the subject.

16. A method for providing increased level of a muscle function in a subject, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide, wherein the oligonucleotide is delivered to satellite cells of the subject; or a method for providing increased level of myofiber repair in a subject, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide, wherein the oligonucleotide is delivered to satellite cells of the subject; or a method for providing increased level of a DMD function in a subject, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide, wherein the oligonucleotide is delivered to satellite cells of the subject.

17. The method of any one of claims 14-16, wherein the subject suffers from a muscular dystrophy.

18. The method of any one of claims 14-16, wherein the subject suffers from Duchenne muscular dystrophy.

19. The method of any one of the preceding claims, wherein the subject has a mutation of the DMD gene that is amenable to exon skipping.

20. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide is delivered to a satellite cell.

21. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a PN linkage.

22. The oligonucleotide of any one of the preceding claims, wherein a PN linkage is Rp.

23. The oligonucleotide of any one of the preceding claims, wherein a PN linkage is Sp.

24. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a phosphoryl guanidine internucleotidic linkage.

25. The oligonucleotide of any one of the preceding claims, wherein a phosphoryl guanidine internucleotidic linkage is Rp .

26. The oligonucleotide of any one of the preceding claims, wherein a phosphoryl guanidine internucleotidic linkage is Sp.

27. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises anOOl internucleotidic linkage.

28. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a Rp n001 internucleotidic linkage.

29. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a Sp n001 internucleotidic linkage.

30. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a phosphorothioate internucleotidic linkage.

31. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a Rp phosphorothioate internucleotidic linkage.

32. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a .S'p phosphorothioate internucleotidic linkage.

33. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a natural phosphate linkage.

34. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a 2’-F modified sugar.

35. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a block of two or more consecutive 2’-F modified sugars.

36. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a 2’-OR modified sugar, wherein R is optionally substituted C1-C6 aliphatic.

37. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises a 2’-0Me modified sugar.

38. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide is administered or delivered in a chirally controlled oligonucleotide composition.

39. The method of any one of the preceding claims, wherein the subject has a mutation of the DMD gene that is amenable to exon 53 skipping.

40. The method of any one of the preceding claims, wherein the oligonucleotide is WVE-N531.

41. The method of claim 40, wherein WVE-N531 is administered or delivered 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.

42. The method of any one of claims 40-41, wherein WVE-N531 is administered or delivered in one or more forms.

43. The method of any one of the preceding claims, wherein WVE-N531 is administered or delivered in one or more pharmaceutically acceptable salt forms.

44. The method of any one of claims 42-43, wherein one form is WVE-N531 hexadecasodium salt.

45. The method of any one of claims 42-44, wherein each dose of WVE-N531 is independently administered in a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier.

46. The method of any one of claims 40-45, wherein a dose of WVE-N531 is equivalent to about 10 mg / kg WVE-N531 free acid form.

47. The method of any one of claims 40-46, 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.

48. The method claim 47, wherein each dose is independently administered in a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier.

49. The method of any one of claims 47-48, 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.

50. The method of any one of claims 47-49, wherein WVE-N531 in each dose is equivalent to about 10 mg / kg WVE-N531 free acid form.

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

52. The method of any one of claims 40-[0063], wherein WVE-N531 is administered or delivered in a pharmaceutical composition which is a liquid composition comprising dissolved WVE-N531.

53. The method of any one of claims 40-52, wherein a pharmaceutically acceptable carrier in the pharmaceutical composition is or comprises a phosphate buffered solution.

54. The method of any one of claims 40-53, wherein two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 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.

55. The method of any one of claims 40-54, wherein three or more consecutive doses are administered about every two weeks.

56. The method of any one of claims 40-55, wherein WVE-N531 has a purity of about 70%-90%.

57. The method of claim 56, wherein the purity is measured by IP-RP-UPLC using area % at 260 nm and the Set A parameters.

58. The method of claim 56, wherein the purity is measured by an IP-RP-UPLC method for purity as described herein.

59. The method of any one of claims 40-58, wherein stereochemical purity of the oligonucleotide is about 80% or more.

60. The method of any one of claims 40-59, wherein the amount of WVE-N531 is measured by UV at 260 nm and 27 OD / mg.

61. The method of any one of claims 40-60, wherein a WVE-N531 drug substance is manufactured by a process described herein.

62. The method of any one of claims 40-61, wherein WVE-N531 is administered intravenously.

63. The method of any one of the preceding claims, wherein a DMD mutation is A3-52, A4-52, A5-52, A6-52, Δ9-52, Δ10-52, Δll-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17-52, Δ19-52, Δ21-52, A23-52, A24- 52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32-52, Δ33-52, Δ34-52, A35-52, Δ36- 52, Δ37-52, Δ38-52. A39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ45-52, Δ47-52, Δ48-52, A49-52, Δ50- 52, Δ51-52, Δ52, Δ54-58, Δ54-61, Δ54-63, Δ54-64, Δ54-66, Δ54-76, or A54-77.

64. The method of any one of the preceding claims, wherein the DMD mutation is Δ52, Δ45-52, Δ 47- 52, Δ 48-52, Δ 49-52, Δ50-52, or A51-52.

65. The method of any one of the preceding claims, wherein exon 53 is skipped in DMD mRNA.

66. The method of any one of the preceding claims, wherein level of exon 53-skipped DMD mRNA is increased, and / or wherein level of exon 53-skipped DMD mRNA in satellite cells is increased, and / or wherein level of exon 53-skipped DMD mRNA in myogenic progenitor cells is increased.

67. The method of any one of the preceding claims, 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.

68. The method of any one of the preceding claims, wherein a truncated DMD polypeptide is produced compared to wild-type dystrophin, and / or a truncated DMD polypeptide is produced in satellite cells compared to wild-type dystrophin, and / or wherein a truncated DMD polypeptide is produced in myogenic progenitor cells compared to wild-type dystrophin.

69. The method of any one of the preceding claims, wherein level of a truncated DMD polypeptide in is increased, and / or wherein level of a truncated DMD polypeptide in satellite cells is increased, and / or wherein level of a truncated DMD polypeptide in myogenic progenitor cells is increased.

70. The method of any one of claims 68-69, wherein the truncated DMD polypeptide performs one or more functions of wild-type dystrophin.

71. The method of any one of the preceding claims, wherein the method provide increases from baseline in dystrophin levels of about 1 %-20% or more of normal levels after administration for a certain time period or after a certain number of doses, and / or wherein the method provide increases from baseline in dystrophin levels of about 1 %-20% or more of normal levels in satellite cells after administration for a certain time period or after a certain number of doses.

72. The method of claim 71 , wherein the increase is measured after administration for about 3-98 weeks from first dose.

73. The method of any one of claims 71-72, wherein the increase is measured after about 1-48 doses.

74. The method of claim 73, wherein the doses are administered or delivered about every two weeks.

75. The method of any one of claims 73-74, wherein each of the doses is equivalent to about 10 mg / kg WVE-N531 free acid form.

76. The method of any one of the preceding claims, wherein a muscle function is increased or restored, and / or wherein a DMD function is increased or restored.

77. The method of any one of the preceding claims, wherein loss of ambulation in a subject is reduced; and / or wherein disease progression in the subject is delayed or slowed; and / or wherein muscle weakness in the subject is delayed or slowed; and / or wherein loss of muscle mass in the subject is delayed or slowed; and / or wherein loss of pulmonary function in the subject is delayed or slowed; and / or wherein the subject improves in a muscular dystrophy assessment; and / or wherein the subject improves in a 10 meter walk test; and / or wherein the subject improves in one or more functional assessments; and / or wherein the subject improves in North Star Ambulatory Assessment (NSAA) 2.0; and / or wherein the subject improves in Performance of the Upper Limb (PUL) 2.0; and / or wherein the subject improves in one or more lower limb motor function by timed function tests; and / or wherein the subject improves in four-stair climb; and / or wherein the subject improves in time to rise from the floor; and / or wherein the subject improves in upper limb proximal strength; and / or wherein the subject improves in handheld myometry; and / or wherein the subject improves in one or more pulmonary function tests; and / or wherein the subject improves in one or more of peak flow rate [PFR], cough peak flow [CPF], and FVC.

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

79. The method of any of the previous claims, wherein the subject is a pediatric subject.

80. The method of any one of claims 40-79, wherein the subject is administered a steroid at least about six months prior to the first dose of WVE-N531.

81. The method of any one of the preceding claims, comprising administering or delivering one or more other therapeutic agents.

82. An oligonucleotide or a pharmaceutical composition thereof, for use in a method of any one of the preceding claims, or an oligonucleotide or a pharmaceutical composition thereof, for treating DMD in asubject suffering therefrom, wherein the subject has a mutation in the DMD gene that is amenable to exon 53 skipping, optionally wherein the oligonucleotide is WVE-N531.

83. An oligonucleotide, for manufacturing of a medicament for use in a method of any one of the preceding claims, or for manufacturing of a medicament 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, optionally wherein the oligonucleotide is WVE-N531.

84. Use of an oligonucleotide or a pharmaceutical composition, for a method of any one of the preceding claims, or use of an oligonucleotide or a pharmaceutical composition, 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, or use of an oligonucleotide, for manufacturing a medicament for use in a method of any one of the preceding claims, or use of an oligonucleotide, for manufacturing a medicament 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, optionally wherein the oligonucleotide is WVE-N531.

85. A method, composition, or use of any one of Embodiments 1-218.

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