Oligonucleotide compositions and methods for exon skipping - Patents.com

Abstract

In particular, the present disclosure provides various techniques involving chiral controlled oligonucleotide compositions and techniques for making and using such oligonucleotide compositions. In some embodiments, the present disclosure provides techniques useful for preventing or treating various conditions, disorders or diseases, such as Duchenne muscular dystrophy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 315,952, filed March 2, 2022, U.S. Provisional Patent Application No. 63 / 397,221, filed August 11, 2022, U.S. Provisional Patent Application No. 63 / 424,418, filed November 10, 2022, and U.S. Provisional Patent Application No. 63 / 433,733, filed December 19, 2022, each of which is incorporated by reference in its entirety herein. [Background technology]

[0002] background Muscular dystrophies (MD) are a group of muscle conditions, diseases or disorders that are reported to cause (increasing) skeletal muscle weakening and breakdown 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 lose the ability to walk. In many cases, muscular dystrophies are fatal. Some forms are also associated with problems in other organs, including the central nervous system. In some embodiments, the muscular dystrophy is Duchenne (Duchenne type) muscular dystrophy (DMD). In some embodiments, the muscular dystrophy is Becker (Becker type) muscular dystrophy (BMD).

[0003] overview The U.S. Food and Drug Administration (FDA) has approved treatments for DMD, including the oligonucleotides golodirsen and viltolarsen, and there remains an unmet need for additional technologies to treat muscular dystrophies, including DMD, particularly as of the time of the present invention, the clinical utility of the approved oligonucleotide drugs has not been established.

[0004] WVE-N531 is an oligonucleotide compound that has demonstrated exon skipping activity and DMD function recovery in vitro and in mouse models. Structurally, WVE-N531 belongs to a different type of oligonucleotide than gologirsen and viltolarsen. For example, while both gologirsen and viltolarsen are phosphorodiamidate morpholino oligomers (PMOs), WVE-N531 is a 2'-F and 2'-OMe modified oligonucleotide, and the internucleotide linkages are, independently, phosphorothioate, n001, and natural phosphate linkages. Furthermore, the chiral bond phosphorus centers are not sterically restricted, and both gologirsen and viltolarsen are provided and administered as stereoirregular compositions in which their chiral bond phosphorus centers randomly exist in two possible configurations (Rp or Sp). The compositions have a molecular weight of 33 million (2 25 ) and 1 million (2 20 ) is a random mixture of diastereomers of more than n001. In contrast, the chiral linkage phosphorus in WVE-N531 is each independently sterically restricted. The linkage phosphorus of each phosphorothioate linkage is Sp, and the linkage phosphorus of each n001 linkage is Rp. As demonstrated herein, the WVE-N531 composition is a chiral controlled composition in which WVE-N531 or a salt thereof is highly enriched over other diastereomers or salts thereof. Furthermore, to the applicant's knowledge prior to this disclosure, oligonucleotides containing phosphorylguanidine linkages such as n001 have not been administered systematically or evaluated in human subjects for therapeutic use. The oligonucleotide, suvodirsen, which is more similar to WVE-N531 than golodirsen and viltolarsen, contains specific chemical moieties, such as 2'-F, 2'-OMe, phosphorothioate linkages, that have also been prepared and administered as a chiral controlled oligonucleotide composition, but it failed in human clinical trials at evaluated doses including about 4.5 mg / kg. Summary of the Invention [Means for solving the problem]

[0005] Among other things, the present disclosure provides techniques for treating muscular dystrophies, such as DMD. In some embodiments, the present disclosure provides doses and dosing regimens that are sufficiently safe for clinical administration to subjects and effective to effect DMD exon 53 skipping, generation of truncated DMD polypeptides that can effect an improvement in the level of one or more functions of the wild-type DMD protein compared to the absence of WVE-N531, restoration or improvement of DMD function, and / or clinical utility. In some embodiments, as demonstrated herein, WVE-N531 has properties and / or activity, e.g., AUC, C, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39 ...0, IL-31, IL-32, IL-34, IL-35, IL-35, IL-36, IL-37, IL-38, IL-39, IL-39, IL-39, max , tissue distribution and / or plasma half-life. The various results presented herein support that WVE-N531 may be safely and effectively utilized to treat conditions, disorders, or diseases associated with DMD mutations amenable to exon 53 skipping, including in human subjects.

[0006] In some embodiments, the disclosure provides a method of treating a muscular dystrophy, e.g., DMD, comprising administering to a subject suffering therefrom WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg (mg per kg of body weight) of WVE-N531 free acid form, wherein the subject has a mutation in the DMD gene amenable to exon 53 skipping. In some embodiments, WVE-N531 is administered as a pharma- ceutically acceptable salt form. In some embodiments, WVE-N531 is administered as a WVE-N531 hexadeca sodium salt form. In some embodiments, WVE-N531 is administered in one or more forms, including one or more pharma- ceutically acceptable salt forms, with the total amount of all WVE-N531 forms being equal 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.

[0007] In some embodiments, the disclosure provides a method of treating a muscular dystrophy, e.g., DMD, comprising administering to a subject suffering therefrom a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form, wherein the subject has a mutation in the DMD gene amenable to exon 53 skipping. In some embodiments, the pharmaceutically acceptable carrier is or comprises a buffer solution. In some embodiments, it is a phosphate buffer solution. In some embodiments, WVE-N531 is dissolved in solution and is present in one or more forms, with the total amount of all WVE-N531 forms being equal 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.

[0008] In some embodiments, the disclosure provides a method of effecting DMD exon 53 skipping in a subject, comprising administering to the subject WVE-N531 or a pharmaceutical composition comprising WVE-N531 and a pharma- ceutical composition comprising WVE-N531 and a pharma- ceutical acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. In some embodiments, the disclosure provides a method of restoring a DMD RNA reading frame in a subject, comprising administering to the subject WVE-N531 or a pharmaceutical composition comprising WVE-N531 and a pharma- ceutical composition comprising WVE-N531 and a pharma- ceutical acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. In some embodiments, the disclosure provides a method of providing a truncated DMD polypeptide in a subject, comprising administering to the subject WVE-N531 or a pharmaceutical composition comprising WVE-N531 and a pharma- ceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. In some embodiments, the truncated (relative to wild-type DMD protein) DMD polypeptide is encoded by a DMD mRNA in which exon 53 has been skipped. In some embodiments, such an mRNA may lack one or more additional exons in addition to exon 53. In some embodiments, such a truncated DMD polypeptide partially or completely provides one or more functions of the wild-type DMD protein.In some embodiments, the disclosure provides a method of producing an increased level of DMD function in a subject, comprising administering to the subject WVE-N531 or a pharmaceutical composition comprising WVE-N531 and a pharma- ceutical carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. In some embodiments, the subject has a mutation in the DMD gene that is susceptible to exon 53 skipping. In some embodiments, the subject is afflicted with or susceptible to muscular dystrophy, e.g., DMD.

[0009] In some embodiments, two or more doses are administered, each independently an amount as described herein, e.g., an amount 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 WVE-N531 free acid form. In some embodiments, about 2-10 doses are administered. In some embodiments, 10 or more doses are administered. In some embodiments, each dose is about the same.

[0010] In some embodiments, two or more doses (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more doses) are administered about every week. In some embodiments, two or more doses (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 doses (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, a dose is administered about once a week. In some embodiments, a dose is administered about every two weeks. In some embodiments, a dose is administered about every three weeks. In some embodiments, a dose is administered about every four weeks. In some embodiments, a dose is administered about once a month. In some embodiments, a dose is administered about every 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, three or more doses are administered about every two weeks. In some embodiments, doses are initially administered at short intervals (e.g., about every 2 weeks), followed by 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, or 10 doses, are administered about every 2 weeks, after which one or more doses are each independently administered about 3, 4, 5, 6, 7, 8, 9, or 10 weeks from the previous dose. In some embodiments, about 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses are administered about every 2 weeks, followed by one or more doses about every 3, 4, 5, 6, 7, 8, 9, or 10 weeks or about every month. 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 weeks or more from the first dose, followed by doses 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 weeks or more from the first dose, followed by doses administered about every four weeks. In some embodiments, doses are administered every two weeks for about 4, 8, 12, or 16 weeks or more from the first dose, followed by doses administered about every month.In some embodiments, doses are initially administered about every 2 weeks, followed by about 4, 8, 12, or 16 weeks after the first dose, whereupon doses are administered about every 4 weeks or about every month, in some embodiments, each dose is independently about the same, e.g., about 10 mg / kg.

[0011] In some embodiments, WVE-N531 or compositions thereof are administered intravenously. In some embodiments, WVE-N531 or compositions thereof are administered intramuscularly. In some embodiments, the dose is administered as an intravenous infusion of about 20 to 60, e.g., about 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the infusion is administered at 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.

[0012] In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is a male subject. In some embodiments, the subject is 5 years of age or older. In some embodiments, the subject is 18 years of age or younger. In some embodiments, the subject meets one or more or all of the inclusion criteria described in the Examples. In some embodiments, the subject is selected based on one or more or all of the inclusion and exclusion criteria described in the Examples.

[0013] In some embodiments, WVE-N531 is administered at a dose that can achieve a clinically relevant maximum plasma concentration.

[0014] In some embodiments, the subject is administered a steroid at least about 6 months (or 24 weeks) prior to the first dose of WVE-N531 or a composition thereof. In some embodiments, the subject is administered a corticosteroid at least about 6 months (or 24 weeks) prior to the first dose of WVE-N531 or a composition thereof. In some embodiments, the subject is on a stable corticosteroid therapy regimen. In some embodiments, the corticosteroid is deflazacort. In some embodiments, the subject is administered a steroid at least about 1 month prior to the first dose of WVE-N531 or a composition thereof.

[0015] In some embodiments, the provided techniques (oligonucleotides, compositions, methods, doses, administration regimens, etc.) provide one or more desired biological effects. In some embodiments, the provided techniques provide exon 53 skipping. In some embodiments, the provided techniques provide exon 53 skipping at a clinically meaningful level. In some embodiments, the provided techniques increase the level of an internally truncated but functional DMD polypeptide, for example, encoded by an exon 53-skipped DMD mRNA. In some embodiments, the provided techniques provide one or more restored DMD functions, for example, via a truncated but functional DMD polypeptide. In some embodiments, the provided techniques provide a clinical benefit. In some embodiments, disease progression is delayed, slowed, or prevented. In some embodiments, disease progression is delayed, slowed, or prevented as assessed by the 10-meter walk test. In some embodiments, the provided techniques reduce the decline in lung function compared to baseline. In some embodiments, muscle weakness in a subject is delayed, slowed, or prevented. In some embodiments, muscle mass loss in the subject is delayed, slowed or prevented. In some embodiments, the subject improves in muscle dystrophin assessment. In some embodiments, the subject improves in DMD assessment. In some embodiments, the improvement is achieved in one or more functional assessments including North Star Ambulatory Assessment (NSAA) 2.0, Performance of Upper Limb (PUL) 2.0, 4-stair climbing, handheld muscle strength measurement and pulmonary function test. In some embodiments, such assessments are performed in such order. In some embodiments, the assessment is or includes lower limb motor function by timed tests (including 10 meter walk / run time, 4-stair climbing time and floor rise time). In some embodiments, the assessment is or includes upper limb proximal muscle strength assessed by handheld muscle strength measurement. In some embodiments, the assessment is or includes pulmonary function tests (peak flow rate [PFR], peak expiratory flow rate [CPF] and FVC).In some embodiments, improvement is achieved independently in one or more or all of these assessments. In some embodiments, the assessments are those disclosed in the Examples. In some embodiments, the improvement is compared to baseline. In some embodiments, the improvement is compared to prior to administration of WVE-N531. In some embodiments, the improvement is compared to predicted levels, severity and / or function with WVE-N531 administration. In some embodiments, the improvement is compared to the absence of WVE-N531 administration. In some embodiments, the improvement is compared to administration of a reference composition. In some embodiments, the reference composition is equivalent to the administered WVE-N531 composition, but contains no or a reduced amount of WVE-N531.

[0016] In some embodiments, the techniques provided reduce loss of ambulation compared to baseline.

[0017] In some embodiments, the provided technology results in an increase in dystrophin levels from baseline of about 1% or more of normal levels after administration over a certain period of time 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, the period is about 10-60 weeks. In some embodiments, it is about 4 weeks. In some embodiments, it is about 8 weeks. In some embodiments, it is about 10 weeks. In some embodiments, it is about 12 weeks. In some embodiments, it is about 13 weeks. In some embodiments, it is about 14 weeks. In some embodiments, it is about 15 weeks. In some embodiments, it is about 16 weeks. In some embodiments, it is about 20 weeks. In some embodiments, it is about 24 weeks. In some embodiments, it is about 25 weeks. In some embodiments, it is about 28 weeks. In some embodiments, it is about 30 weeks. In some embodiments, it is about 32 weeks. In some embodiments, it is about 35 weeks.In some embodiments, it is about 36 weeks. In some embodiments, it is about 37 weeks. In some embodiments, it is about 38 weeks. In some embodiments, it is about 40 weeks. In some embodiments, it is about 45 weeks. In some embodiments, it is about 48 weeks. In some embodiments, it is about 49 weeks. In some embodiments, it is about 50 weeks. In some embodiments, it is about 72 weeks. In some embodiments, it is about 73 weeks. In some embodiments, it is about 74 weeks. In some embodiments, it is about 96 weeks. In some embodiments, it is about 97 weeks. In some embodiments, it is about 98 weeks or more. In some embodiments, it is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 months. In some embodiments, the specific number of doses is about 5 doses. In some embodiments, it is about 6 doses. In some embodiments, it is about 7 doses. In some embodiments, it is about 8 doses. In some embodiments, it is about 9 doses. In some embodiments, it is about 10 doses. In some embodiments, it is about 11 doses. In some embodiments, it is about 12 doses. In some embodiments, it is about 13 doses. In some embodiments, it is about 14 doses. In some embodiments, it is about 15 doses. In some embodiments, it is about 16 doses. In some embodiments, it is about 17 doses. In some embodiments, it is about 18 doses. In some embodiments, it is about 19 doses. In some embodiments, it is about 20 doses. In some embodiments, it is about 21 doses. In some embodiments, it is about 22 doses. In some embodiments, it is about 23 doses. In some embodiments, it is about 24 doses. In some embodiments, it is about 25 doses. In some embodiments, it is about 30 doses. In some embodiments, it is about 35 doses.In some embodiments, it is about 40 doses. In some embodiments, it is about 45 doses. In some embodiments, it is about 48 doses. In some embodiments, it is about 49 doses. In some embodiments, it is about 50 doses or more. In some embodiments, WVE-N531 is administered every two weeks.

[0018] In some embodiments, the provided techniques cause no or low levels of adverse events, or no or low levels of severe and / or serious adverse events, hi some embodiments, adverse events associated with the provided techniques are tolerable or manageable.

[0019] In some embodiments, the disclosure provides techniques for producing WVE-N531. In some embodiments, the disclosure provides techniques for producing WVE-N531 drug substance. In some embodiments, the disclosure provides techniques for producing WVE-N531 formulations. In some embodiments, the disclosure provides techniques for characterizing WVE-N531 drug substance and / or formulations. In some embodiments, the disclosure provides shipping specifications for characterizing WVE-N531 drug substance and / or formulations. In some embodiments, the disclosure provides WVE-N531, compositions thereof, drug substances thereof and / or formulations thereof produced by the processes provided. [Brief description of the drawings]

[0020] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] 1 is an exemplary flow diagram for a WVE-N531 formulation manufacturing process. [Figure 2A] WVE-N531 provides robust exon 53 skipping in non-human primates. Example regimen. Monkey animals received 6x weekly IV infusions of PBS or 3, 7 or 25 mg / kg WVE-N531 (n=2 per dose). W: week. Necropsy was performed on day 38. [Figure 2B]WVE-N531 provides robust exon 53 skipping in non-human primates. Exon 53 skipping assessed by RT-PCR. Healthy animals have normal levels of dystrophin, but exon skipping can be assessed by detection of skipped transcripts. [Diagram 3] Schematic diagram of an example study design. Abbreviations: DSMB = Data Safety Monitoring Board, N = number of patients. [Figure 4] WVE-N531 demonstrates elevated plasma concentrations in human subjects compared to suvodirsen. Human subjects received an IV infusion of 1, 3 or 6 mg / kg WVE-N531 or 5 mg / kg suvodirsen. After infusion, plasma concentrations (μg / ml) of WVE-N531 or suvodirsen were measured over time for at least 42 days. [Diagram 5] WVE-N531 demonstrates elevated plasma concentrations in human subjects. Human subjects received IV infusions of 1, 3, 6, or 10 mg / kg WVE-N531. After infusion, plasma concentrations (μg / ml) of WVE-N531 were measured over time for at least 105 days. [Figure 6] WVE-N531 demonstrates elevated plasma concentrations in human subjects compared to suvodirsen. Human subjects received an IV infusion of 1, 3, 6, or 10 mg / kg WVE-N531 or 5 mg / kg suvodirsen. After infusion, plasma concentrations (μg / ml) of WVE-N531 or suvodirsen were measured over time for at least 21 days. [Figure 7] Schematic diagram of an example study design. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] definition As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS system, Handbook of Chemistry and Physics, 75th Ed. Furthermore, 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, MB and March, J., John Wiley & Sons, New York: 2001.

[0022] As used herein in this disclosure, unless otherwise clear from the 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," "including," "including" (whether or not used in conjunction with "without limitation") and "including" (whether or not used in conjunction with "without limitation") may be understood to encompass the itemized components or steps, whether presented by themselves or 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) when ranges are given, the endpoints are included.

[0023] Unless otherwise specified, the description of oligonucleotides and their elements (e.g., base sequence, sugar modification, internucleotide bond, stereochemistry of the linking phosphorus, etc.) is in the order of 5' to 3'. Unless otherwise specified, the oligonucleotides described herein may be provided and / or utilized in various forms, including salt forms, particularly pharma-ceutically acceptable salt forms. As one skilled in the art will understand after reading this disclosure, in some embodiments, the oligonucleotides may be provided as salts, such as sodium or potassium salts. As one skilled in the art will understand, in some embodiments, individual oligonucleotides within a composition may be considered to be of the same composition and / or structure even within such a composition (e.g., liquid composition), and certain such oligonucleotides may be in different forms (e.g., dissolved and oligonucleotide chains may exist as anionic forms, for example, when in a liquid composition), including salt forms at a particular moment. For example, one of skill in the art will understand that at a given pH, individual internucleotide bonds along an oligonucleotide chain may be in the acid (H) form or one of several possible salt forms (e.g., sodium salts or salts of different cations depending on which ions may be present in the preparation or composition), and will understand that so long as their acid forms (e.g., replacing all cations, if present, with H) are of the same composition and / or structure, such individual oligonucleotides may be considered to be of the same composition and / or structure, as appropriate.

[0024] Approximately: As used herein, the terms "about" and "approximately" can be understood to allow for standard variations as understood by those of ordinary skill in the art. In some embodiments, the term "approximately" or "about" in reference to a number is generally interpreted to include numbers that fall within a range of 5%, 10%, 15%, 20%, 25%, or 30% in either direction of that number (greater or less than that number) unless otherwise specified or otherwise evident from the context. In some embodiments, the range is ±5%. In some embodiments, the range is ±10%.

[0025] Dosing regimen: As used herein, "dosing regimen" or "treatment regimen" refers to a set of unit doses (typically two or more doses) administered to a subject individually, typically spaced apart in time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each of which is separated from the other by the same length of time, and in some embodiments, a dosing regimen includes multiple doses and at least two different time periods separating the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dose amount. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first dose at a first dose value, followed by one or more additional doses at a second dose value that is different from the first dose value. In some embodiments, a dosing regimen includes a first dose at a first dose value, followed by one or more additional doses at a second dose value that is the same as the first dose value.

[0026] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a controlled therapeutic effect when administered to a relevant population.

[0027] Pharmaceutically acceptable: As used herein, the phrase "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that 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.

[0028] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutical acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent that encapsulates a material, which is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject.

[0029] Pharmaceutically acceptable salts: The term "pharmaceutical acceptable salts" as used herein refers to salts of such compounds that are suitable for use in connection with medicines, i.e., salts that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. provide a detailed description of pharmaceutical acceptable salts in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, provided compounds contain two or more acidic groups, e.g., provided oligonucleotides may contain two or more acidic groups (e.g., at natural phosphate linkages and / or modified internucleotide linkages). In some embodiments, pharmaceutical acceptable salts of such compounds, or salts in general, contain two or more cations that may be the same or different. In some embodiments, in a pharma- ceutically acceptable salt (or salt in general), each acidic group having sufficient acidity is present independently in its salt form (e.g., in an oligonucleotide comprising a natural phosphate linkage and a phosphorothioate internucleotide linkage, each of the natural phosphate linkage and the phosphorothioate internucleotide linkage is present independently in its salt form). In some embodiments, a pharma- ceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide. In some embodiments, a pharma- ceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide, in which each acidic linkage, e.g., each natural phosphate linkage and phosphorothioate internucleotide linkage, is present in sodium salt form (all sodium salts).

[0030] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a provided compound or composition is administered, e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes, in accordance with the present disclosure. In some embodiments, the subject may be suffering from and / or susceptible to a disease, disorder and / or condition. In some embodiments, the subject is a human subject.

[0031] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the entire or nearly entire extent or degree of a property or characteristic of interest. Those skilled in the art of biology will understand that biological and chemical events rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, as used herein, the term "substantially" is used to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0032] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0033] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition is an individual who is at a higher risk of developing the disease, disorder, and / or condition than members of the general population. 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.

[0034] Systemic: The phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally," as used herein, have their art-recognized meanings referring to the administration of a compound or composition such that it enters the recipient's system.

[0035] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect upon administration to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0036] Therapeutically effective amount: In some embodiments, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that induces a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be appreciated by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that relieves, ameliorate, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose, and in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. In some embodiments, the single dose is an infusion, which may take up to an hour or more.

[0037] Treat: As used herein, the terms "treat", "treatment" or "treating" refer to any method used to partially or completely alleviate, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who show only early signs of the disease, disorder, and / or condition, e.g., to reduce the risk of developing pathologies associated with the disease, disorder, and / or condition.

[0038] Chiral controlled oligonucleotide composition: The terms "chiral controlled (stereocontrolled or stereoregulated) oligonucleotide composition", "chiral controlled (stereoregulated or stereoregulated) nucleic acid composition" and the like, as used herein, refer to a composition comprising a plurality of oligonucleotides (or nucleic acids, chiral controlled oligonucleotides or chiral controlled nucleic acids) that 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 (a specific type of oligonucleotide), where the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotide linkages (chiral controlled internucleotide linkages, whose chiral linkage phosphorus is Rp or Sp, not a random Rp and Sp mixture as in non-chiral controlled internucleotide linkages). The level of the plurality of oligonucleotides (or nucleic acids) in the chiral controlled oligonucleotide composition is not random (predefined, controlled). Chiral controlled oligonucleotide compositions are typically prepared through the stereoselective formation of one or more chiral internucleotide linkages by chiral controlled oligonucleotide preparations (e.g., using asymmetric auxiliaries as exemplified in the present disclosure, as compared to non-chiral controlled (sterically irregular, non-stereoselective, racemic) oligonucleotide syntheses, such as conventional phosphoramidite-based oligonucleotide syntheses that do not attempt to purposefully control stereoselectivity using asymmetric auxiliaries or chiral catalysts). Chiral controlled oligonucleotide compositions are enriched for a plurality of oligonucleotides, as compared to substantially racemic preparations of oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications.In some embodiments, the chiral 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, which are enriched for oligonucleotides of a particular oligonucleotide type, as compared to a substantially racemic preparation of oligonucleotides having the same base sequence, pattern of backbone linkages, and pattern of backbone phosphorus modifications. As one skilled in the art will readily appreciate, such enrichment can be characterized in that a higher level of the bound phosphorus at each chiral controlled internucleotide linkage has the desired configuration, as compared to a substantially racemic preparation. In some embodiments, each chiral controlled internucleotide linkage independently has a diastereomeric purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for the chiral bound phosphorus. In some embodiments, each independently has a diastereomeric purity of at least 90%. In some embodiments, each independently has a diastereomeric purity of at least 95%. In some embodiments, each independently has a diastereomeric purity of at least 97%. In some embodiments, each independently has a diastereomeric purity of at least 98%. In some embodiments, the oligonucleotides have the same structure. In some embodiments, the oligonucleotides have the same structure and stereochemistry and are structurally identical.

[0039] In some embodiments, multiple oligonucleotides in a chiral controlled oligonucleotide composition independently share the same base sequence, the same nucleobase, sugar and internucleotide linkage modifications, if present, and the same stereochemistry (Rp or Sp) at the linking phosphorus chiral center of one or more chiral controlled internucleotide linkages, although the stereochemistry of a particular linking phosphorus chiral center may differ. In some embodiments, about 0.1% to 100%, (e.g., about 1% to 100%, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 50%, 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 50%, 60%, 70%, 80%, 90%, 95%, 50 ... %, 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%) are a plurality of oligonucleotides. In some embodiments, about 0.1% to 100%, (e.g., about 1% to 100%, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or About or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% are a plurality of oligonucleotides.In some embodiments, about 0.1% to 100%, (e.g., about 1% to 100%, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 10 ... 00%, 50% to 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%) is a plurality of oligonucleotides.In some embodiments, all of the oligonucleotides in a chiral controlled oligonucleotide composition, or all of the oligonucleotides in a composition (e.g., of multiple oligonucleotide types or of one oligonucleotide type) that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications, or all of the oligonucleotides in a composition (e.g., of multiple oligonucleotide types or of one oligonucleotide type) that share a common base sequence, a common pattern of base modifications, a common pattern of sugar modifications, a common pattern of internucleotide linkage types, and / or a common pattern of internucleotide linkage ... the same About 0.1% to 100%, (e.g., about 1% to 100%, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 50%, 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 50%, 50%, 60%, 70%, 80%, 90%, 95%, 50%, 50%, 60%, 70%, 80%, 90%, 95%, 50%, 50%, 60%, 70%, 80%, 90%, 95%, 50%, 50%, 60%, 70%, 80%, 90%, 95%, 50%, 50%, 50%, 60%, 70%, 80%, 90%, 95 ... 0%, 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% are multiple oligonucleotides. In some embodiments, the percentage is at least (DP). NCIand DP is a percentage selected from 85% to 100%, and NCI is the number of chiral controlled internucleotide linkages. 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 the diastereomeric purity of the chiral controlled internucleotide linkages at the chiral center of the bound phosphorus. In some embodiments, the diastereopurity of the chiral center at the linking phosphorus of the internucleotide linkage can be assessed using an appropriate dimer that typically includes such an internucleotide linkage and two nucleoside units linked by the internucleotide 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 internucleotide linkages.In some embodiments, the plurality of oligonucleotides is about 0.1% to 100% (e.g., about 1% to 100%, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 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 the chiral internucleotide linkages share the same stereochemistry. In some embodiments, each chiral internucleotide linkage is a chiral controlled internucleotide linkage and the composition is a fully chiral controlled oligonucleotide composition. In some embodiments, not all chiral internucleotide linkages are chiral controlled internucleotide linkages and the composition is a partially chiral controlled oligonucleotide composition. In some embodiments, the chiral controlled oligonucleotide composition comprises a predetermined level of individual oligonucleotide or nucleic acid types. For example, in some embodiments, a chiral controlled oligonucleotide composition comprises one oligonucleotide type at a predetermined level (e.g., as described above). In some embodiments, a chiral controlled oligonucleotide composition comprises two or more oligonucleotide types, each independently at a predetermined level. In some embodiments, a chiral controlled oligonucleotide composition comprises a plurality of oligonucleotide types, each independently at a predetermined level. In some embodiments, a chiral controlled oligonucleotide composition is a composition of oligonucleotides of an oligonucleotide type, the composition comprising a plurality of oligonucleotides of that oligonucleotide type at a predetermined level.

[0040] Detailed Description of Specific Embodiments In particular, the present disclosure provides a method of treatment for treating muscular dystrophy, for example by administering to a subject an amount of WVE-N531 as described herein, the subject having a mutation in the DMD gene that is susceptible to exon 53 skipping. Dystrophin plays an important role in muscle function and various muscle-related diseases are characterized by mutant forms of this gene. In certain embodiments, the methods described herein can be used to induce exon skipping in transcripts, for example, mRNA of mutant forms of the human dystrophin gene, such as muscular dystrophin, for example, the mutated dystrophin gene forms found in DMD and BMD. In some embodiments, the provided technology provides a DMD mRNA in which exon 53 has been skipped. In some embodiments, the present disclosure provides a DMD polypeptide encoded by such a DMD mRNA. In some embodiments, the generation of such a DMD polypeptide restores, in some embodiments, partially, and in some embodiments, completely, one or more DMD functions.

[0041] WVE-N531 can induce exon 53 skipping and restore dystrophin protein.Mutations in DMD gene that are susceptible to exon 53 skipping have been reported to occur in approximately 8% of subjects with DMD.As those skilled in the art will understand, various DMD mutations that are susceptible to exon 53 skipping have been reported, and various techniques are available to determine whether a mutation is susceptible to exon 53 skipping. In some embodiments, Δ3 to 52, Δ4 to 52, Δ5 to 52, Δ6 to 52, Δ9 to 52, Δ10 to 52, Δ11 to 52, Δ13 to 52, Δ14 to 52, Δ15 to 52, Δ16 to 52, Δ17 to 52, Δ19 to 52, Δ21 to 52, Δ23 to 52, Δ24 to 52, Δ25 to 52, Δ26 to 52, Δ27 to 52, Δ28 to 52, Δ29 to 52, Δ30 to 52, Δ31 to 52, Δ32 to 52, Δ33 to 52, Δ34 to 52, DMD mutations such as 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 are susceptible to exon 53 skipping. In some embodiments, the mutation is Δ45-52. In some embodiments, the mutation is Δ47-52. In some embodiments, the mutation is Δ48-52. In some embodiments, the mutation is Δ49-52. In some embodiments, the mutation is Δ50-52. In some embodiments, the mutation is Δ51 to 52. In some embodiments, the mutation comprises Δ52. In some embodiments, the mutation is Δ52.In some embodiments, WVE-N531 mediates effective exon 53 skipping in DMD to restore the reading frame, such that a short but at least partially functional dystrophin can be generated (the resulting dystrophin protein is not necessarily a "wild-type" form of dystrophin, but is a functional form of dystrophin, albeit truncated, resulting in increased levels of one or more dystrophin functions compared to the absence of WVE-N531 / exon 53 skipping).

[0042] In some embodiments, WVE-N531 provides high muscle concentrations, e.g., in various muscles, for example, about or at least about 5 μg / g, 8 μg / g, 10 μg / g, 15 μg / g, 20 μg / g, 25 μg / g, 30 μg / g, 33 μg / g, 35 μg / g, 40 μg / g, 45 μg / g, 50 μg / g, 55 μg / g, 60 μg / g, 65 μg / g, 70 μg / g, 75 μg / g, 80 μg / g, 85 μg / g, 90 μg / g, etc. In some embodiments, such muscle concentrations are provided at a time point about 6 weeks after the first dose and / or after about 3 doses, each dose being about 10 mg / kg of WVE-N531. In some embodiments, WVE-N531 effects exon 53 skipping at a level of, for example, about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, etc. In some embodiments, exon skipping is effected at a time point about 6 weeks after the first dose and / or after about 3 doses, each dose being about 10 mg / kg of WVE-N531 (a dose significantly less than golodirsen and / or viltolarsen).

[0043] In some embodiments, the present disclosure provides a method comprising using WVE-N531 to induce exon 53 skipping in DMD in muscle cells. In some embodiments, the present disclosure provides a method comprising using WVE-N531 to increase the level of functional dystrophin protein in muscle cells, which is useful for the prevention and treatment of muscular dystrophies, particularly those forms of muscular dystrophies that are susceptible to exon 53 skipping. In particular, the methods described herein provide improved treatment options for subjects with muscular dystrophies, offering significant and practical advantages over other methods, such as those using other oligonucleotide therapeutics.

[0044] In some embodiments, the present disclosure relates to an improved method for treating muscular dystrophy by inducing exon skipping in a subject. In some embodiments, exon 53 skipping in DMD in a subject is induced by administering an effective amount of WVE-N531 as described herein. In some embodiments, the present disclosure relates to an improved method for treating muscular dystrophy by restoring / increasing the level of dystrophin polypeptide and / or its activity in a subject (e.g., by restoring / increasing the level of functional dystrophin protein).

[0045] In some embodiments, the disclosure provides a method of treating DMD in a subject having a mutation in the DMD gene that is susceptible to exon 53 skipping, comprising administering WVE-N531 (optionally as part of a composition, e.g., a pharmaceutical formulation or dosage form) at an effective dose equivalent to, e.g., 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 WVE-N531 free acid form at a suitable frequency (e.g., every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks).

[0046] In some embodiments, the disclosure provides a method of treating DMD in an affected subject, wherein the subject has a mutation in the DMD gene that is susceptible to exon 53 skipping, comprising administering to the subject one or more doses of WVE-N531, each dose 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 of WVE-N531 free acid form.

[0047] In some embodiments, the disclosure provides a method of effecting DMD exon skipping in a subject, comprising administering to the subject one or more doses of WVE-N531, each dose 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 of WVE-N531 free acid form. In some embodiments, the disclosure provides a method of increasing levels of exon 53-skipped DMD transcripts in a subject, comprising administering to the subject one or more doses of WVE-N531, each dose 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 of WVE-N531 free acid form. In some embodiments, the disclosure provides a method of restoring a DMD RNA reading frame in a subject, the method comprising administering to the subject one or more doses of WVE-N531, each dose 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 of WVE-N531 free acid form. In some embodiments, the disclosure provides a method of increasing levels of a truncated DMD polypeptide in a subject, comprising administering to the subject one or more doses of WVE-N531, each dose 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 of WVE-N531 free acid form.In some embodiments, the disclosure provides a method of producing an increased level of DMD function in a subject, comprising administering to the subject one or more doses of WVE-N531, each dose being 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 of WVE-N531 free acid form. In some embodiments, the truncated DMD polypeptide is encoded by a DMD mRNA in which exon 53 has been skipped. In some embodiments, such an mRNA may lack one or more additional exons in addition to exon 53. In some embodiments, the subject has a mutation in the DMD gene that is susceptible to exon 53 skipping. In some embodiments, the subject is afflicted with or susceptible to muscular dystrophy, e.g., DMD. In some embodiments, each dose of WVE-N531 is administered in a pharmaceutical composition independently comprising WVE-N531 and a pharma- ceutically acceptable carrier, and the 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 of WVE-N531 free acid form.

[0048] WVE-N531 may be administered in a variety of forms. For example, in many embodiments, WVE-N531 is administered in a salt form. In some embodiments, a single form is present in the composition. In some embodiments, a single form is present in a dose. In some embodiments, the form is a pharma- ceutically acceptable salt form. In some embodiments, the form is WVE-N531 hexadeca sodium salt. In some embodiments, two or more forms are present in the composition. In some embodiments, two or more forms are present in a dose. In some embodiments, each of the two or more forms is, independently, a pharma- ceutically acceptable salt form. In some embodiments, one of them is WVE-N531 hexadeca sodium salt. In some embodiments, the composition is a liquid composition comprising WVE-N531 dissolved, for example, in phosphate buffered saline. In some embodiments, there is one or more types of cations in the solution. In some embodiments, each is, independently, H + or a metal cation. In some embodiments, the metal cation is Na + In some embodiments, the metal cation is K + In some embodiments, each metal cation is independently Na + Or K + It is.

[0049] In some embodiments, the dose is about 1-20 mg / kg. In some embodiments, the dose is about 1-5 mg / kg. In some embodiments, the dose is about 5-20 mg / kg. In some embodiments, the dose is about 5-10 mg / kg. In some embodiments, the dose is about 10-20 mg / kg. In some embodiments, the dose is about 10-15 mg / kg. In some embodiments, the dose is about 15-20 mg / kg. In some embodiments, the dose is about 1 mg / kg. In some embodiments, the dose is about 1 mg / kg. In some embodiments, the dose is about 2 mg / kg. In some embodiments, the dose is about 3 mg / kg. In some embodiments, the dose is about 4 mg / kg. In some embodiments, the dose is about 5 mg / kg. In some embodiments, the dose is about 6 mg / kg. In some embodiments, the dose is about 7 mg / kg. In some embodiments, the dose is about 8 mg / kg. In some embodiments, the dose is about 9 mg / kg. In some embodiments, the dose is about 10 mg / kg. In some embodiments, the dose is about 11 mg / kg. In some embodiments, the dose is about 12 mg / kg. In some embodiments, the dose is about 13 mg / kg. In some embodiments, the dose is about 14 mg / kg. In some embodiments, the dose is about 15 mg / kg. In some embodiments, the dose is about 16 mg / kg. In some embodiments, the dose is about 17 mg / kg. In some embodiments, the dose is about 18 mg / kg. In some embodiments, the dose is about 19 mg / kg. In some embodiments, the dose is about 20 mg / kg.

[0050] In some embodiments, the dose is administered as a pharmaceutical composition comprising WVE-N531 and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition is a solution of WVE-N531, e.g., as described herein, in a buffer. In some embodiments, the pharma- ceutically acceptable carrier is isotonic. In some embodiments, it is a solution of WVE-N531 in phosphate buffered saline, as described herein.

[0051] In some embodiments, two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more) doses are administered. In some embodiments, about 10 or more doses are administered. In some embodiments, about 20 or more doses are administered. In some embodiments, about 30 or more doses are administered. In some embodiments, about 40 or more doses are administered. In some embodiments, about 50 or more doses are administered. In some embodiments, about 60 or more doses are administered. In some embodiments, about 70 or more doses are administered. In some embodiments, about 80 or more doses are administered. In some embodiments, about 90 or more doses are administered. In some embodiments, about 100 or more doses are administered. In some embodiments, about 200 or more doses are administered. In some embodiments, about 500 or more doses are administered. In some embodiments, each dose 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 as described herein and a pharma- ceutically acceptable carrier. 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, the pharmaceutical compositions administered for two or more doses are about the same with respect to components and their relative amounts. In some embodiments, the pharmaceutical compositions administered for all doses are about the same with respect to components and their relative amounts. In some embodiments, one or more earlier doses are independently less than one or more later doses. In some embodiments, one or more earlier doses are independently more than one or more later doses.

[0052] In some embodiments, doses are administered about once a week, 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 is administered about every 2 weeks, and each dose is independently as described herein. In some embodiments, WVE-N531 is administered about every 4 weeks, and each dose is independently as described herein. In some embodiments, WVE-N531 is administered about every 4 weeks or more, and each dose is independently as described herein. In some embodiments, the interval between each dose (the time between a dose and the dose before or after it) is about the same. In some embodiments, the interval between one or more earlier doses is independently longer than one or more later ones. In some embodiments, the interval between one or more earlier doses is independently shorter than one or more later ones.

[0053] In some embodiments, "about" is + / - 1% (99% to 101% of the 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%.

[0054] Unless otherwise specified, amounts, concentrations, dosages, etc. of WVE-N531 are of the free acid form.

[0055] Amounts, concentrations, doses, etc. are typically corrected for purity. In some embodiments, purity is measured by IP-RP-UPLC as described herein based on % area.

[0056] Vyondys 53™ (gologirsen) is a phosphorodiamidate morpholino oligomer (PMO) subclass oligonucleotide intended to increase dystrophin production via exon skipping. Gologirsen was approved in the United States in 2019 for the treatment of patients with confirmed mutations in the DMD gene that are amenable to exon 53 skipping. Approval was based on a mean change from baseline in dystrophin levels of 0.92% (standard deviation [SD] 1.01) of normal levels after 48 weeks of treatment at 30 mg / kg / week. Viltepso™ (viltolarsen), another PMO subclass exon skipping ASO, was approved in the United States in 2020 for the treatment of DMD patients amenable to exon 53 skipping. Approval was also based on an observed increase in dystrophin production with a mean increase of 5.3% (SD 4.5) of normal levels measured after 25 weeks of treatment at 80 mg / kg / week. Both golodirsen and viltolarsen were approved through the accelerated approval mechanism in the United States based on changes in surrogate endpoints of dystrophin production, and clinical benefit of either drug has not yet been demonstrated in patients with DMD at the time of approval. Continued approval in the United States is contingent on clinical benefit upon confirmation in a confirmatory trial. At the time of this invention, neither drug has been approved in the United Kingdom or the European Union (EU).

[0057] In particular, the provided technology offers various advantages compared to golodirsen and / or viltolarsen, such as, for example, a smaller single dose, a smaller total dose, less frequent dosing, improved exon 53 skipping in DMD, higher levels of functional DMD polypeptide, and improved clinical outcomes.

[0058] Dystrophin In some embodiments, the dystrophin (DMD) gene or product thereof or a variant or portion thereof is DMD, BMD, CMD3B, DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270, DXS272, MRX85 or dystrophin; external ID: OMIM:300377 MGI:94909;HomoloGene:20856;GeneCards:DMD;In Human:Entrez:1756;Ensembl:ENSG00000198947;UniProt:P11532;RefSeq(mRNA):NM_000109;NM_004006;NM_004007;NM_004009;NM_004010;RefSeq(protein):NP_000100;NP_003997;NP_004000;NP_004001;NP_004002;Location(UCSC):Chr X:31.1-33.34Mb; 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:82.95-85.21Mb.

[0059] Dystrophin is reported to be found in muscle cells and is very important for strengthening and protecting muscle fibers.Normal dystrophin protein is reported to be part of a protein complex called dystrophin-glycoprotein complex, which provides structural stability to skeletal muscle and protects muscle from damage during contraction and relaxation, according to various reports.In addition, dystrophin is reported to be essential for cell survival through transmembrane signaling function and regulation of vasomotor response to physical activity.

[0060] DMD gene reportedly contains 79 exons distributed over 2.3 million bp of genetic space on X chromosome, but only about 14,000 bp (<1%) is reported to be used for translation into protein (coding sequence). Intronic sequences, which are about 99.5% of gene sequence, are reported to be spliced ​​from the initial heteronuclear RNA transcript of 2.3 million bp to produce a mature 14,000 bp mRNA that contains all the important information for dystrophin protein production. In some embodiments, subjects with DMD have a mutation in the DMD gene that prevents proper assembly of wild-type DMD mRNA and / or production of wild-type dystrophin protein, and subjects with DMD often show significant dystrophin deficiency in their muscles.

[0061] In some embodiments, dystrophin transcripts, such as mRNAs or proteins, include those associated with or generated from alternative splicing. For example, at least 16 alternative transcripts of the dystrophin gene have been reported following analysis of the splicing patterns of the DMD gene in skeletal muscle, brain and heart tissue.

[0062] Dystrophin has been reported to have several isoforms. In some embodiments, dystrophin refers to a particular isoform. At least three full-length dystrophin isoforms have been reported, each controlled by a tissue-specific promoter. The muscle isoform is reported to be expressed primarily in skeletal muscle, but also in smooth and cardiac muscles; brain dystrophin is reported to be specific to cortical neurons, but can also be found in cardiac and cerebellar neurons; and Purkinje cell types are reported to account for nearly all of the 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.

[0063] In some embodiments, the dystrophin mRNA, gene or protein is a revertant version.

[0064] Various mutations in the DMD gene have been reported to be capable of and / or to cause muscular dystrophy. Mutations in the form of large deletions (one or more exons) have been reported to account for approximately two-thirds of all DMD gene mutations, and the remaining mutations have been reported to result from duplications and small deletions, insertions, point mutations, or splicing mutations. In certain reports, the deletions are usually clustered in the hotspot region between exons 45 and 55, preventing the translation of dystrophin. The absence or lack of dystrophin protein resulting from DMD gene mutations has been reported to disrupt the dystrophin-glycoprotein complex, leading to increased muscle membrane fragility, chronic muscle damage, inflammation, replacement of muscle fibers with fat and fibrous tissue, and subsequent loss of muscle function.

[0065] Muscular dystrophies Muscular dystrophy (MD) is any of a group of muscle conditions, diseases, or disorders that cause (increasing) skeletal muscle weakening and breakdown 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 lose the ability to walk. In many cases, muscular dystrophy is fatal. Some forms are also associated with problems in other organs, including the central nervous system. In some embodiments, the muscular dystrophy is Duchenne (Duchenne type) muscular dystrophy (DMD). In some embodiments, the muscular dystrophy is Becker (Becker type) muscular dystrophy (BMD).

[0066] In some embodiments, the symptoms of Duchenne muscular dystrophy are muscle wasting and associated weakness, affecting voluntary muscles first, especially those in the hips, pelvic region, thighs, shoulders, and calves. Weakness may occur later in the arms, neck, and other areas. The calves are often enlarged. Symptoms usually appear before age 6 and may appear in early infancy. Other physical symptoms include awkward walking, stepping, or running (in some cases, subjects tend to walk on their front feet due to increased calf muscle tone), frequent falls, fatigue, motor skill difficulties (e.g., running, hopping, jumping), lumbar hyperlordosis (possibly leading to shortening of the hip flexors), overall posture and / or walking, stepping, or running abnormalities, Achilles tendon muscle contracture and hamstring dysfunction, progressive difficulty walking, and deformed muscle fibers. , pseudohypertrophy (hypertrophy) of the tongue and calf muscles, higher risk of neurobehavioral disorders (e.g., ADHD), learning disabilities (e.g., dyslexia) and non-progressive deficits in certain cognitive abilities (e.g., short-term verbal memory) that are believed to be the result of a lack or dysfunction of dystrophin in the brain, eventual loss of the ability to walk (usually by age 12), skeletal deformities (including scoliosis in some cases) and difficulty rising from a lying or sitting position. In some embodiments, the provided technology delays the onset of, slows the progression of, reduces the severity of, and / or prevents one or more symptoms.

[0067] In some embodiments, Becker muscular dystrophy (BMD) is caused by mutations that result in shortened but in-frame transcripts, resulting in the production of truncated but partially functional proteins. Such partially functional proteins have been reported to retain important amino-terminal, cysteine-rich and C-terminal domains, but generally lack elements of the central rod domain that have been reported to be of little functional significance. England et al. 1990 Nature, 343, 180-182.

[0068] In some embodiments, the BMD phenotype ranges from mild DMD to essentially asymptomatic, depending on the exact mutation and the level of dystrophin produced. Yin et al. 2008 Hum. Mol. Genet. 17: 3909-3918.

[0069] In some embodiments, dystrophic subjects with out-of-frame mutations are generally diagnosed with the more severe form of Duchenne muscular dystrophy, and dystrophic subjects with in-frame mutations are generally diagnosed with the less severe form of Becker muscular dystrophy.

[0070] Exon skipping as a treatment for muscular dystrophies Exon skipping can induce cellular machinery to skip one or more targeted exons and restore the reading frame, resulting in the production of an internally truncated but functional dystrophin polypeptide. In various exon skipping approaches, oligonucleotides can bind to target complementary sequences in messenger ribonucleic acid (mRNA) and engage the splicing machinery to exclude the exon from the final transcript. In subjects with DMD, this approach may slow disease progression by converting severe DMD symptoms to milder symptoms, such as those seen in patients with Becker muscular dystrophy (BMD), and patients can walk longer and have a longer life expectancy.

[0071] Various DMD genotypes are susceptible to exon skipping, including exon 53 skipping. Exon skipping can restore the DMD mRNA reading frame, resulting in the production of a shorter but functional dystrophin protein. Exon 53 skipping is applicable to patients with deletions in the DMD gene, including, but not limited to, 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%-10% of patients with DMD can be treated by exon 53 skipping. Long-term 36-month natural history data in a cohort of patients with deletions susceptible to exon 44, 45, 51, or 53 skipping showed that patients susceptible to exon 53 skipping in particular had earlier onset of decline and a higher risk of dramatic functional decline (e.g., loss of ambulation) compared to other subgroups.

[0072] In some embodiments, exon skipping restores or maintains the proper reading frame and / or creates an artificial internally truncated DMD protein that results in at least partially improved or fully restored biological activity. In some embodiments, an internally truncated DMD protein generated from a dystrophin transcript having a skipped exon is more functional than, for example, a terminally truncated DMD protein generated from a dystrophin transcript having an out-of-frame deletion.

[0073] In some embodiments, internally truncated DMD proteins generated from dystrophin transcripts with skipped exons are more resistant to nonsense-mediated decay that can degrade terminally truncated DMD proteins generated from dystrophin transcripts with, for example, out-of-frame deletions.

[0074] In some embodiments, a DMD subject is analyzed for DMD genotype prior to administration of WVE-N531 and identified as having a mutation in the DMD gene that is amenable to exon 53 skipping. In some embodiments, the mutation in the DMD gene that is amenable to exon 53 skipping is or includes one or more nucleobase changes (e.g., replaced by a different nucleobase), insertions and / or deletions, etc., and exon 53 skipping can generate a DMD polypeptide that can provide improved function compared to one without exon 53 skipping.

[0075] Prior to this disclosure, Emflaza® (deflazacort) is a corticosteroid approved in the United States for the treatment of DMD. Emflaza has been shown to improve muscle strength after a 12-week treatment period at a dose of 0.9 mg / kg / day (mean change of 0.15 points as measured by the Medical Research Council sale). A long-term placebo-controlled study in children with DMD aged 6-12 years showed no significant effect of deflazacort on mean muscle strength scores at year 2. Emflaza is not approved for the treatment of DMD in the European Union (EU), while corticosteroids such as deflazacort are commonly used off-label in patients with DMD in the EU to slow the decline in muscle strength and prolong gait and respiratory function. However, long-term use of corticosteroids has been associated with severe side effects such as immunosuppression and increased risk of infection, altered endocrine function, hypertension, cataracts, bone demineralization, gastrointestinal perforation, and developmental delay in children. Furthermore, corticosteroids do not correct the underlying genetic defect in DMD.

[0076] Vyondys 53™ (gologirsen) is a phosphorodiamidate morpholino oligomer (PMO) subclass oligonucleotide intended to increase dystrophin production via exon 53 skipping. Gologirsen was approved in the United States in 2019 for the treatment of patients with confirmed mutations in the DMD gene that are amenable to exon 53 skipping. Approval was based on a mean change from baseline in dystrophin levels of 0.92% (standard deviation [SD] 1.01) of normal levels after 48 weeks of treatment at 30 mg / kg / week. Viltepso™ (viltolarsen), another PMO subclass exon skipping oligonucleotide, was approved in the United States and Japan in 2020 for the treatment of DMD patients with mutations amenable to exon 53 skipping. Approval was also based on an observed increase in dystrophin production with a mean increase of 5.3% (SD 4.5) of normal levels measured after 25 weeks of treatment at 80 mg / kg / week. Both golodirsen and viltolarsen were approved through the accelerated approval mechanism in the United States based on an increase in a surrogate endpoint of dystrophin production, and neither drug has been shown to provide clinical benefit in patients with DMD. Continued approval in the United States is contingent on clinical benefit upon verification in a confirmatory trial. Neither drug has been approved in the United Kingdom (UK) or EU prior to this disclosure.

[0077] Similarly, some patients may have mutations that make them eligible for treatment with both exon 53 skipping oligonucleotides and Translarna™ (ataluren) or Exondys 51® (eteplirsen). Ataluren is a small molecule intended to enhance ribosomal readthrough of nonsense mutations that are not approved for use in the United States (US). It has received conditional marketing authorization in the EU for the treatment of DMD resulting from nonsense mutations in the dystrophin gene in ambulatory patients aged 5 years and older. Clinical efficacy of ataluren has not been established in nonambulatory patients and additional evidence is required to support its continued approval in the EU. It is expected that the use of ataluren will be limited to a small percentage of the DMD population, given the low prevalence of nonsense mutations and its limited efficacy in ambulatory patients. Eteplirsen is a PMO subclass oligonucleotide intended to increase dystrophin production via exon skipping. Eteplirsen was approved in the US for the treatment of patients with confirmed mutations in the DMD gene that are amenable to exon 51 skipping. Approval was based on detection of a mean increase in dystrophin protein levels of 0.93% of normal muscle dystrophin levels and a median increase in truncated dystrophin expression of 0.1% in the only study in which dystrophin expression in response to eteplirsen treatment could be estimated. The drug had not been approved in the EU prior to this disclosure.

[0078] Plasma C of golodirsen max and the area under the plasma concentration-time curve (AUC) increased proportionally with dose, the half-life was approximately 3 hours, and no plasma accumulation was observed. Based on Phase 1 studies of viltolarsen, the urinary excretion rate showed elimination of 70%-80% of the total drug administered within 24 hours, indicating a short half-life (<24 hours). Renal function in all patients was within the normal range. Eteplirsen is rapidly eliminated from plasma, with the majority of drug elimination occurring within 24 hours. Twenty-four hours after the end of the infusion, the mean concentration of eteplirsen was 1.2 times the maximum (peak) concentration of the drug in plasma (C maxRenal clearance of eteplirsen accounts for more than 60% of the administered dose, with a terminal half-life (t 1 / 2 No accumulation of eteplirsen has been observed during once-weekly dosing.

[0079] In particular, the present disclosure provides techniques for treating DMD, inducing DMD exon 53 skipping, increasing levels of exon 53-skipped DMD transcripts, generating or increasing levels of truncated DMD polypeptides from exon 53 skipping, increasing levels of one or more DMD functions, etc., and / or restoring one or more DMD functions, etc., comprising administering to a subject WVE-N531 or a composition thereof according to the present disclosure, wherein the subject has a mutated DMD susceptible to exon 53 skipping. In some embodiments, the provided techniques provide various advantages over one or more existing approved drugs, such as improved safety, tolerability, clearance, accumulation, exon skipping, clinical efficacy, and / or one or more other pharmacokinetic or pharmacodynamic aspects.

[0080] WVE-N531 WVE-N531 may be described herein (5' to 3' unless otherwise specified, as one of skill in the art would understand): fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC m: 2'-OMe modified nucleoside, f: 2'-F modified nucleoside, *S: Sp phosphorothioate bond, and n001R: Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate bond.

[0081] WVE-N531 may be named according to accepted oligonucleotide nomenclature: (Sp)-2'-fluoro-2'-deoxy-P-thio-cytidylyl-(O 3’→O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-uridylyl-(O 3’ →O 5’ )-(Rp)-2'-Fluoro-2'-deoxy-P-((1,3-dimethylimidazolidin-2-ylidenyl)amino)-cytidylyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-cytidyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-guanylyl-(O 3’ →O 5’ )-(Rp)-2'-Fluoro-2'-deoxy-P-((1,3-dimethylimidazolidin-2-ylidenyl)amino-guanylyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-uridylyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-uridylyl-(O 3’ →O 5’ )-2'-O-Methylcytidyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-uridylyl-(O 3’ →O 5’ )-(Sp)-2'-O-methyl-P-thio-guanylyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-adenylyl-(O 3’ →O 5’ )-2'-O-Methyladenylyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-guanylyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-guanylyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-uridylyl-(O 3’ →O 5’)-(Rp)-2'-Fluoro-2'-deoxy-P-((1,3-dimethylimidazolidin-2-ylidenyl)amino)-guanylyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-uridylyl-(O 3’ →O 5’ )-(Sp)-2'-Fluoro-2'-deoxy-P-thio-uridylyl-(O 3’ →O 5’ )-2'-fluoro-2'-deoxycytidine.

[0082] In some embodiments, WVE-N531 is provided in a composition, e.g., a pharmaceutical composition, and / or is administered as one or more pharma- ceutically acceptable salts. In some embodiments, the disclosure provides a pharma- ceutically acceptable salt of WVE-N531. In some embodiments, the pharma- ceutically acceptable salt is a sodium salt. In some embodiments, the disclosure provides a WVE-N531 hexadeca sodium salt. In some embodiments, the WVE-N531 drug substance is a WVE-N531 hexadeca sodium salt.

[0083] The molecular formula and molecular weight of WVE-N531 and its hexadeca sodium salt are set forth below: Molecular formula (free acid form): C 206 H 250 F 17 N 78 O 109 P 19 S 14 Molecular weight (free acid form): 6923.02 g / mol Molecular formula (hexadeca sodium salt form): C 206 H 234 F 17 N 78 O 109 P 19 S 14 Na 16 Molecular weight (hexadeca sodium salt form): 7274.73 g / mol.

[0084] WVE-N531 has 17 sterically restricted internucleotide linkages, 14 of which are identified as Sp phosphorothioate linkages and 3 as Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkages. The internucleotide linkages can be shown as 5'-SSRSSRSSOSSSOSSSRSS-3', where "S", "R" and "O" represent the Sp phosphorothioate linkage, the Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage and the phosphate linkage, respectively. The N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage can be referred to as the n001 linkage.

[0085] The structure of WVE-N531 is represented by Formula I: [ka] It can be shown as:

[0086] WVE-N531 hexadeca sodium salt has the formula Ia: [ka] It can be written as:

[0087] In formulas I and Ia, due to the size of the structure, the structure is shown in four lines, each of which has five nucleotide units. As shown in these formulas, each bond in WVE-N531 is independently an Sp phosphorothioate bond, an Rp n001 bond, or a natural phosphate bond, and in WVE-N531 salts, the acidic hydrogen atoms of the phosphorothioate bond and the phosphate bond can be independently replaced with a cation, e.g., in the hexadeca sodium salt, each is replaced with sodium.

[0088] Aqueous solutions of WVE-N531 hexadeca sodium salt are typically clear to pale yellow solutions and may have a concentration of about 150 mg / mL or greater as determined by UV. In some embodiments, the pH of WVE-N531 hexadeca sodium salt in purified water ranges from 6.0 to 8.0.

[0089] The extinction coefficient of WVE-N531 is 186,486 M at 260 nm. -1 cm -1 (determined in water). Unless otherwise stated, when UV is used to measure the concentration / amount of WVE-N531, 260 nm is used and this number is used in the calculations. Using a molecular weight of 6918.72 g / mol (free acid form), this equates to an extinction coefficient of 27.0 OD / mg.

[0090] In some embodiments, WVE-N531 is provided as an isotonic solution for dilution for IV infusion. In some embodiments, the total volume of infusion is 100-500 mL based on the subject's weight. In some embodiments, the infusion is administered over approximately 1 hour. In some embodiments, the infusion is extended to about 3 hours, for example, to enhance tolerability. In some embodiments, the diluted infusion solution of WVE-N531 does not contain a preservative and is administered without a delay or within 4 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.

[0091] In some embodiments, WVE-N531 is provided and administered as a chiral controlled composition in which WVE-N531 is enriched relative to other diastereomers.

[0092] In particular, the present disclosure provides highly pure WVE-N531. In some embodiments, WVE-N531 has a purity level 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, the purity level is determined by IP-RP-UPLC as described herein. In some embodiments, it is about 70% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 71% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 72% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 73% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 74% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 75% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 76% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 77% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 78% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 79% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 80% as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 81% or greater as determined by IP-RP-UPLC as described herein. In some embodiments, it is about 82% as determined by IP-RP-UPLC as 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 greater as determined by IP-RP-UPLC described herein. In some embodiments, the IP-RP-UPLC utilizes Set A parameters. In some embodiments, the purity is the % area around a given wavelength, e.g., 260 nm.

[0093] In some embodiments, the level of impurities is about 10%-30% or less. In some embodiments, it is 10%-30%. In some embodiments, it is 15%-30%. In some embodiments, it is 20%-30%. In some embodiments, it is about 30% or less. In some embodiments, it is about 29% or less. In some embodiments, it is about 28% or less. In some embodiments, it is about 27% or less. In some embodiments, it is about 26% or less. In some embodiments, it is about 25% or less. In some embodiments, it is about 24% or less. In some embodiments, it is about 23% or less. In some embodiments, it is about 22% or less. In some embodiments, it is about 21% or less. In some embodiments, it is about 20% or less. In some embodiments, it is about 19% or less. In some embodiments, it is about 18% or less. In some embodiments, it is about 17% or less. In some embodiments, it is about 16% or less. In some embodiments, it is about 15% or less. In some embodiments, it is about 14% or less. In some embodiments, it is about 13% or less. In some embodiments, it is about 12% or less. In some embodiments, it is about 11% or less. In some embodiments, it is about 10% or less. In some embodiments, the level of impurities is assessed using IP-RP-UPLC. In some embodiments, the IP-RP-UPLC utilizes Set A parameters. In some embodiments, the purity is the % area around a given wavelength, for example 260 nm.

[0094] In some embodiments, the disclosure provides various techniques for characterizing WVE-N531 preparations, hi some embodiments, the disclosure provides techniques for assessing the purity of WVE-N531 or compositions thereof.

[0095] In some embodiments, the disclosure provides techniques for producing WVE-N531 or compositions thereof, hi some embodiments, the disclosure provides techniques for releasing WVE-N531 preparations.

[0096] manufacturing In some embodiments, the disclosure provides techniques for producing WVE-N531 or a salt thereof, such as WVE-N531 hexadeca sodium salt. In some embodiments, the disclosure provides techniques for producing WVE-N531 drug substance. In some embodiments, the disclosure provides techniques for producing a pharmaceutical composition comprising WVE-N531 or a pharma- ceutically acceptable salt form thereof and a pharma- ceutically acceptable carrier. In some embodiments, the disclosure provides WVE-N531 formulations.

[0097] In some embodiments, WVE-N531 or a salt thereof, such as WVE-N531 hexadeca sodium salt, is chemically produced in accordance with Good Manufacturing Practice (cGMP) regulations.

[0098] In some embodiments, the provided techniques involve 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.

[0099] In some embodiments, the stereochemistry of WVE-N531 or a salt thereof is established through control of the synthetic starting materials and the synthetic process. For example, in some embodiments, the techniques provided include the use of phosphoramidites prepared from the chiral auxiliaries (L)-DPSE and (L)-PSM during the coupling step to ensure the intended stereorestricted Sp phosphorothioate and Rp n001 linkages, respectively.

[0100] Useful manufacturing processes and specific results for WVE-N531 hexadeca sodium salt as a drug substance are described below as examples.

[0101] Useful manufacturing processes In some embodiments, the manufacturing process consists of synthesizing oligonucleotides containing a combination of three types of internucleotide linkages (phosphodiester, sterically restricted phosphorothioate and sterically restricted n001) on a solid support using phosphoramidite chemistry as shown in the following synthesis cycles. Each cycle for introducing sterically restricted phosphorothioate or sterically restricted n001 consists of 5'-detritylation, coupling, capping of the exposed chiral auxiliary secondary amine (capping-1), thiolation or imidization and capping of the unreacted 5'-hydroxyl group (capping-2), respectively. Each cycle for introducing phosphodiester consists of detritylation, coupling, oxidation and capping-2. Each coupling reaction is carried out by activation of the appropriate phosphoramidite and reaction with the free 5'-hydroxyl group of a protected nucleotide or oligonucleotide immobilized on a support. After an appropriate number of cycles and final detritylation, the (L)-PSM chiral auxiliary and the cyanoethyl phosphate protecting group are removed from the crude oligonucleotide by on-column treatment with diethylamine (DEA) in acetonitrile (ACN). The (L)-DPSE chiral auxiliary is then reacted with NEt 3 The base protecting groups are removed by treatment with a solution of triethylamine hydrogen fluoride (HF). The crude oligonucleotide is then cleaved from the solid support and removed from the column by treatment with ammonia. The base protecting groups are removed by continued treatment with ammonia 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 obtain, in some embodiments, WVE-N531 or a salt thereof, such as WVE-N531 hexadeca sodium salt, as the drug substance.

[0102] Useful cycles for phosphorothioate construction [ka]

[0103] Useful cycles for building n001 [ka]

[0104] A useful cycle for building phosphate [ka]

[0105] [Table 1]

[0106] The process for manufacturing the WVE-N531 drug substance is described in further detail below.

[0107] Stage 1: Synthesis The synthesis of oligonucleotides is carried out on a controlled pore glass (CPG) solid support functionalized with 5'-ODMTr-2'-F-dC(N4-Ac) in an automated oligonucleotide synthesizer. All reactions are carried out on the solid support packed in a column.

[0108] 1. Detritylation [ka] To start the synthesis, the CPG-5'-ODMTr-2'-F-dC(N4-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 in-line UV monitoring based on the watch command in the synthesis program. DMTr removal is carried out in the same way at the beginning of each synthesis cycle and after the final cycle. In all cases, after detritylation, the support-bound material is washed with acetonitrile in preparation for the next step of the synthesis.

[0109] 2. Coupling [ka] Elongation of the developing 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 either the activator, 5-(ethylthio)-1H-tetrazole (ETT) or 1-cyanomethylimidazolium triflate salt (CMIMT), dissolved in acetonitrile. The phosphoramidite is dissolved in acetonitrile or an 80:20 v / v acetonitrile:isobutyronitrile mixture. The phosphoramidite required for each coupling step is determined by the oligonucleotide sequence. For the introduction of a phosphate bond, a phosphoramidite and ETT are used. For the introduction of sterically restricted phosphorothioates or n001 bonds, a phosphoramidite derived from (L)-DPSE or (L)-PSM and CMIMT are used. In both cases, the phosphoramidite / activator solutions are mixed in series and pushed onto the synthesis column and then cycled through the column for the appropriate time. Excess reagent is then removed by flushing the synthesis column with acetonitrile.

[0110] 3. Cap addition-1 [ka] Capping 1 allows the secondary amine resulting from the oxazaphospholidine ring opening with the auxiliary group during the coupling step to be protected and rendered unreactive for the remainder of the synthesis. The secondary amine is capped by running capping reagent B ([Cap B]: acetic anhydride / 2,6-lutidine / acetonitrile (20:30:50, v:v:v) through the synthesis column. Excess reagent is flushed from the synthesis column with acetonitrile.

[0111] 4. Oxidation, thiolation or imidization The newly generated P(III) phosphite triester bond is processed in one of three ways: 1. Oxidation by treatment with iodine in pyridine:water (90:10, v:v) results in the formation of a P(V) phosphodiester bond. [ka] 2. Thiolation by treatment with xanthan hydride (XH) in pyridine:acetonitrile (1:1, v:v) leads to the generation of sterically restricted P(V) phosphorothioate triester linkages. [ka] 3. Imidization by treatment with 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (ADIH) in acetonitrile leads to the formation of sterically restricted P(V) phosphoroimidate triester linkages. [ka]

[0112] In each case, after the step, excess reagent is flushed from the synthesis column with acetonitrile.

[0113] 5. Cap addition-2 Usually, the coupling reaction proceeds in very high yield, but it is not quantitative. A small portion of the 5'-hydroxy groups available in any given cycle may not be able to couple with the activated phosphoramidite. To prevent reaction during subsequent cycles, these sites are blocked by capping with a mixture of reagents (1:1, v:v) (capping reagent A [Cap A]:N-methylimidazole / acetonitrile (20:80, v:v) and Cap B). As a result, a 5'-O-acetylated ("capped") support-bound oligonucleotide sequence is formed. Excess capping reagent is washed from the column with acetonitrile. [ka]

[0114] Oligonucleotide construction and final steps Three independent repetitions of the cycles shown above using appropriate protected phosphoramidites construct a sequence that is globally protected with a DMTr protecting group present at the 5'-terminal position. After addition of the last nucleotide in the sequence, the 5'-terminal DMTr group is removed during a final detritylation step.

[0115] Step 2: Cleavage and deprotection Removal of the phosphate protecting groups and (L)-PSM and (L)-DPSE chiral auxiliaries from the sterically restricted phosphoromidate and phosphorothioate triesters, cleavage of the crude oligonucleotides from the solid support, and removal of the exocyclic nucleobase protecting groups is accomplished in a three-step process.

[0116] Step 1: Removal of (L)-PSM chiral auxiliary and cyanoethyl by treatment with diethylamine [ka] The crude oligonucleotide on the solid support can be treated with a solution of diethylamine in ACN, which simultaneously removes the cyanoethyl protecting group from the phosphotriester linkage and the (L)-PSM chiral auxiliary from the phosphoroimidate triester to generate the phosphodiester and phosphoramidate diester linkages, respectively.

[0117] Step 2: Removal of the (L)-DPSE chiral auxiliary by fluoride treatment [ka] The crude oligonucleotides on the solid support were precipitated in dimethyl sulfoxide (DMSO), NEt 3 and H 2 NEt in a mixture of O 3 Treatment with a temperature controlled solution of 3HF results in removal of the chiral auxiliary. This process converts the stereorestricted phosphorothioate triester to the stereorestricted phosphorothioate diester as shown above.

[0118] Step 3: Cleavage and global nucleobase deprotection [ka] [ka] 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 result in the cyanoethyl phosphate group (which may have been deprotected in an earlier step) and in the global deprotection of the exocyclic amino group (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 + (a compound such as an "acid of formula P"). In some embodiments, each M +are independently cationic. In some embodiments, the crude oligonucleotide is a salt of an acid of formula P. In some embodiments, the crude oligonucleotide composition comprises a salt of an acid of formula P. In some embodiments, the crude oligonucleotide composition comprises two or more salts of an acid of formula P.

[0119] Typically, after deprotection, one or more WVE-N531 salts are provided. In some embodiments, the salts are ammonium salts. In some embodiments, the deprotection product composition comprises one or more WVE-N531 salts. In some embodiments, the deprotection product composition comprises a WVE-N531 ammonium salt.

[0120] Step 3: Purification by anion exchange chromatography Purification of the crude oligonucleotide solution is accomplished by AEX-HPLC. The crude oligonucleotide solution is loaded onto a purification column packed with TSK-GEL Super Q-5PW media. The purification run is performed using an eluent buffered with sodium hydroxide. A sodium chloride gradient is used to elute the oligonucleotide from the column. Purification is carried out at ambient temperature. The elution profile is monitored by ultraviolet (UV) spectrophotometry. Fractions are collected and the pseudopools are evaluated by IP-RP-UPLC. Pools containing oligonucleotides at or above the target purity level are subjected to the next step in the process.

[0121] Stage 4: Concentration and desalting (final UF / DF) 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 the pH is adjusted to 6.5-7.5 with hydrochloric acid or sodium hydroxide. The concentrated oligonucleotide is diafiltered against purified water to a target of 1200-1400 OD / mL, after which the desalted oligonucleotide is recovered. The system is flushed with purified water to maximize yield by combining the desalted concentrated oligonucleotide with the rinse water to a target final desalted concentration of 878 ± 68 OD / mL (32.5 ± 2.5 mg / mL).

[0122] Step 5: Filtration and packaging The oligonucleotide solution is filtered through a 0.2 micron filter before being filled into drug substance storage containers. The drug substance is packaged in sterile high density polyethylene (HDPE) bottles, each of which is labeled, sealed in a Mylar foil pouch, and stored at -20°C.

[0123] Starting materials Particular useful starting materials are listed below.

[0124] [Table 2]

[0125] [Table 3]

[0126] [Table 4]

[0127] [Table 5]

[0128] [Table 6]

[0129] Starting materials include nine phosphoramidites, a controlled pore glass (CPG) solid support, and 2-azido-1,3-dimethylimidazolinium hexafluorophosphate. Reactive exocyclic groups on the nucleobases are appropriately protected to render them unreactive during oligonucleotide synthesis, and 5'-hydroxyl functions are protected as 4,4'-dimethoxytrityl ethers (DMTr). The phosphoramidites are purified to about 90%, about 95%, or in some cases, greater than about 98% (by RP-HPLC at about 260 nm (area %) and / or integration). 31 P NMR), P(III) purity of about 97% or more ( 31 The controlled pore glass-5'-ODMTr-2'-F-dC(N4-Ac) succinic acid solid support has a particle size (analytical sieving) of about 120-200 mesh, a pore size (mercury intrusion) of about 540-660 Å, a density (tapped density) of about 0.20-0.24 g / cc, a pore volume (mercury intrusion) of ≥ about 0.8 cc / g, and a water content of ≥ about 75 m 2 / g surface area Hg intrusion (mercury intrusion) and 70-80 μmol / g (spectrophotometric at 498 nm) as assessed by DMT ligand assay. 2-Azido-1,3-dimethylimidazolinium hexafluorophosphate has a purity of greater than about 98.0% (HPLC) and a nitrogen content of about 23.70-24.80% (elemental analysis).

[0130] The stereochemistry can be established through control of the starting materials for synthesis and the synthetic process. Sterically restricted phosphoramidites are prepared from appropriately protected nucleosides and chiral auxiliaries, such as (L)-DPSE or (L)-PSM. The chemical purity of the stereoregulated phosphoramidites can be determined by: 31 The configuration can be determined by P NMR. 31 P NMR, 1 H NMR and 13The stereochemical configuration of the protected nucleoside and chiral auxiliary is fixed, so there are two possible diastereomers (trans and cis) that can result from the coupling reaction, of which the trans form predominates and the cis form exists as a minor impurity.

[0131] During the manufacturing process, various in-process controls are applied. Upon completion of the synthesis and cleavage and deprotection steps, the presence of WVE-N531 drug substance in the resulting crude oligonucleotide is identified by LC / MS and its purity is quantified by IP-RP-UPLC. During purification, individual fractions and selected pseudopools are assessed for purity and impurities by IP-RP-UPLC. Selected fractions are pooled and concentrated, and then desalting of the resulting solution is controlled via in-process measurements of conductivity, pH and concentration. In some embodiments, a drug substance or drug product is manufactured that meets one or more or all of the following criteria: In some embodiments, the drug substance, e.g., WVE-N531 or a salt form thereof, has a particular level of purity as described herein. In some embodiments, WVE-N531 hexadeca sodium salt has a particular level of purity as described herein. In some embodiments, WVE-N531 or a salt thereof has a particular level of purity in a composition, e.g., a pharmaceutical composition. In some embodiments, WVE-N531 or a salt thereof has a particular level of purity in the formulation. In some embodiments, WVE-N531 or a salt thereof is WVE-N531 hexadeca sodium salt. In some embodiments, WVE-N531 has a purity level 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 greater as determined by IP-RP-UPLC as described herein.

[0132] Characterization Among other things, the present disclosure provides techniques for evaluating WVE-N531 or compositions thereof.

[0133] The product may be characterized by various techniques. For example, WVE-N531 preparations may be characterized by mass spectrometry. For some preparations, electrospray ionization mass spectrometry (ESI-MS) analysis was performed, and the experimentally determined value of 6722 Da is consistent with the theoretical average mass (free acid form) of WVE-N531 drug substance (6723 Da). In some embodiments, the sequence of WVE-N531 or its salts was confirmed by ESI-MS / MS sequencing. For example, in one evaluation, for 19 nucleobases in the sequence, at least one sequence-related fragment ion was observed within 5 ppm error of the respective calculated (predicted) mass. The monoisotopic mass of the full-length oligonucleotide was also verified by ESI-MS / MS analysis, thus verifying the identity and position of all 20 nucleobases and confirming the sequence.

[0134] Stereochemical identity includes the combination of the absolute stereochemical configuration of each chiral linking phosphorus (Rp or Sp) in the oligonucleotide (each linking phosphorus in a phosphorothioate or phosphoramidate group for WVE-N531 or a salt thereof). n The diastereomers (n=number of chiral phosphorus bonds) may give rise to a unique single diastereomer. In the case of WVE-N531 or a salt thereof, this combination may be represented by 5'-SSRSSRSSOSSSOSSSRSS-3' (where S, P and O represent Sp phosphorothioate, Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate and phosphate bonds, respectively). WVE-N531 or a salt thereof also includes 2'-fluoro (F) and 2'-O-methyl (OMe) modifications.

[0135] Establishing the stereochemical identity involves controlling the starting materials for synthesis. Synthetic coupling of phosphoramidites prepared from (L)-DPSE or (L)-PSM produces phosphorothioate linkages of Sp chirality and phosphoramidate linkages of Rp chirality, respectively, and those prepared from (D)-DPSE or (D)-PSM produces phosphorothioate linkages of Rp chirality and phosphoramidate linkages of Sp chirality. Consistent use of stereorestricted phosphoramidites for each coupling reaction allows control of the stereochemical identity of each phosphorothioate or phosphoramidate linkage.

[0136] Stereochemical identity may be confirmed using a variety of techniques in accordance with the present disclosure. For example, it may be confirmed by measurement using several different techniques, which, in combination and comparison with appropriate standards, provide precise information and confirmation of this attribute. Useful analytical techniques include NMR ( 1 H, 19 F, 31 In some instances, NMR is performed in a phosphate buffer (e.g., pH 7.0). 1 H, 19 F and 31 Results including P NMR as well as enzymatic digestion are consistent with the product structure. In some embodiments, NMR is referenced to water based on DSS standards. Data observed from specific experiments are described below.

[0137] Regarding the preparation of WVE-N531 drug substance (hexadeca sodium salt) 31 P NMR data acquired and recorded at 161.98 MHz.

[0138] [Table 7]

[0139] Regarding the preparation of WVE-N531 drug substance (hexadeca sodium salt) 19F NMR data acquired and recorded at 348K.

[0140] [Table 8]

[0141] In some embodiments, standards, e.g., characterized by NMR, enzymatic digestion, etc., are used to determine stereochemical identity by UPLC as part of a batch release. In some embodiments, the techniques provided provide standards.

[0142] The FTIR spectrum of solid WVE-N531 hexadeca sodium salt as drug substance was recorded. -1 and 1682 cm -1 The major absorbance bands at 3200-3300 cm are consistent with the C=O, C=N and C=C stretching vibrations of amides. -1 The broad peak in the region is also consistent with an oligonucleotide functional group.

[0143] Counterions may be analyzed according to the present disclosure. For example, a sodium content value of 5.1% has been determined by ICP-OES for a preparation of WVE-N531 drug substance lot, consistent with a theoretical sodium content value of 5.1% (w / w) (hexadeca sodium salt).

[0144] The prepared solution of WVE-N531 drug substance is a clear (no visible material and essentially free of particulate matter) and colorless solution. The pH of WVE-N531 drug substance in purified water was found to be about 7.3.

[0145] The molar extinction coefficient of WVE-N531 drug substance was 186486M using one lot. -1 cm -1 It was experimentally determined in water to be. Using a molecular weight of 6918.72 g / mol (free acid form), this equates to an extinction coefficient of 27.0 OD / mg.

[0146] In various embodiments, impurities are controlled at low levels as described herein. For example, in some embodiments, impurity levels are less than about 30% as described herein.

[0147] The WVE-N531 drug substance preparation (including relevant impurities) was evaluated under GLP in vitro genotoxicity studies and in vivo micronucleus assays. No genotoxicity was observed.

[0148] Various preparations of WVE-N531 drug substance met the specified constraints for residual solvents including acetonitrile, toluene, and pyridine (ICH class 2) and elemental impurities including various elements in ICH classes 1, 2A, 3, etc.

[0149] The fidelity of the transfer of chiral information (diastereoselectivity) from each stereorestricted phosphoramidite in WVE-N531 or its salts to its stereorestricted Sp phosphorothioate or Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate (PN) linkages during oligonucleotide synthesis is very high. The overall stereochemical purity of the oligonucleotide can be expressed by the product of the diastereoselectivities of the incorporation of each of the 17 stereorestricted internucleotide linkages.

[0150] In some embodiments, stereopurity is assessed using dimer modeling. Fourteen dimer units (fGn001RfU, fG*SfG and fU*SfC dimers each represented twice) representing the composition of stereorestricted phosphorothioate or PN internucleotide linkages within the WVE-N531 drug substance were independently synthesized and assessed for stereochemical purity. The stereochemical purity of WVE-N531 or its salts can be estimated as the product of the measured diastereoselectivities of the 17 individual dimers. The synthetic conditions used to generate each dimer were identical to those applied to the WVE-N531 drug substance. Compared to the full-length WVE-N531 drug substance, these model dimer units are more readily analyzed by standard analytical methods such as IP-RP-UPLC, and both the Rp and Sp diastereoisomers are easily separated and quantified by this technique. The IP-RP-UPLC data for the model dimers are shown below.

[0151] [Table 9]

[0152] This model study demonstrates excellent stereochemical fidelity at each position, with stereochemical purity values ​​of ≧98% at each position in the sequence. The overall stereochemical purity, based on the product of the stereochemical purities of each individual constituent dimer, is 85% (84.9%=product of the 17 diastereoselectivity fidelity values ​​above).

[0153] In some embodiments, the present disclosure provides WVE-N531 with high stereopurity, for example as assessed using dimer modeling as described herein. In some embodiments, the stereopurity is about 80% or greater. In some embodiments, the stereopurity is about 81% or greater. In some embodiments, the stereopurity is about 82% or greater. In some embodiments, the stereopurity is about 83% or greater. In some embodiments, the stereopurity is about 84% or greater. In some embodiments, the stereopurity is about 85% or greater. In some embodiments, the stereopurity is about 86% or greater. In some embodiments, the stereopurity is about 87% or greater. In some embodiments, the stereopurity is about 88% or greater. In some embodiments, the stereopurity is about 89% or greater. In some embodiments, the stereopurity is about 90% or greater. In some embodiments, the stereopurity is about 80%-90%. In some embodiments, the stereopurity is about 83%-87%.

[0154] The release specifications for the WVE-N531 drug substance may be determined based on one or more specifications described herein, such as 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 a solution in purified water), residual solvents (e.g., by gas chromatography), elemental impurities (e.g., by ICP-MS), stereochemical identity (e.g., by UPLC), endotoxins (e.g., USP <85> , Ph.Eur 2.6.14), total aerobic microbial count according to bioburden (USP<61), total yeast and mould count etc.) and / or Ph.Eur 2.6.12).

[0155] Useful parameters for identity (molecular weight) by LC MS are described below.

[0156] [Table 10]

[0157] In some embodiments, system suitability is confirmed when the molecular weight (deconvoluted mass) for the full length product (FLP) for the first three injections of analytical reference material should be 6923±3 Da for WVE-N531, and in addition, the blank chromatogram has no interfering peaks other than the solvent front and gradient shift.

[0158] The identity of WVE-N531 or its salts can be determined by liquid chromatography mass spectrometry (LC-MS). Samples are prepared in water and injected onto a Waters Acquity BEH C18 column. The analysis utilizes a gradient of mobile phase A (hexafluoroisopropanol [HFIP] and triethylamine [TEA] in water) and mobile phase B (acetonitrile). A summary of the method parameters is provided below as an example.

[0159] [Table 11]

[0160] In some embodiments, the disclosure provides techniques for assessing WVE-N531 purity and / or impurities. In some embodiments, the purity and impurities of WVE-N531 can be determined by ion-pair reversed-phase UPLC (IP-RP-UPLC), for example, using a Waters BEH C18 column. A useful procedure is described below by way of example (Protocol A). Separation is achieved using a gradient of mobile phase A (hexafluoroisopropanol [HFIP] and diisopropylethylamine [DIPEA] in water) and mobile phase B (30% acetonitrile in water). A useful set of parameters is provided below (Set A) and has been utilized to determine the purity / impurity levels for the various preparations described herein. A variety of impurities can be separated using this method, including diastereomeric impurities (of the 17 WVE-N531 diastereomers (DS1-DS17), each differing from WVE-N531 only at a single bond phosphorus, six diastereomers are fully resolved and nine are partially resolved). In some embodiments, the disclosure provides methods 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, mobile phase comprising one or more or all of the solvents in mobile phase A, mobile phase comprising one or more or all of the solvents in mobile phase B, mobile phase A, mobile phase B, flow rate, run time, gradient, etc.).

[0161] [Table 12]

[0162] [Table 13]

[0163] In some embodiments, >0.10 area % of all individual impurities and >0.10 area % of the sum of all impurities as a function of relative retention time to the main peak are included in the analysis. In some embodiments, WVE-N531 identity is confirmed by comparing the relative retention time to a system suitability standard.

[0164] In some embodiments, the present disclosure provides techniques for confirming the stereochemical identity of WVE-N531 or its diastereomers with respect to one or more bound phosphorus. In some embodiments, the stereochemical identity of WVE-N531 is determined by IP-RP-UPLC using a Waters BEH C18 column. A useful procedure is described below as an example (Protocol B). Separation is achieved using a gradient of mobile phase A (hexafluoroisopropanol [HFIP] and DIPEA in water) and mobile phase B (30% acetonitrile in water). A summary of the method parameters (Set B) is provided below. In some embodiments, the disclosure provides methods 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, mobile phase comprising one or more or all of the solvents in mobile phase A, mobile phase comprising one or more or all of the solvents in mobile phase B, mobile phase A, mobile phase B, flow rate, run time, gradient, etc.).

[0165] [Table 14]

[0166] In particular, the present disclosure provides high purity WVE-N531 as described herein. In some embodiments, the present disclosure provides high stereochemical purity WVE-N531, for example, as determined using IP-RP-UPLC with Set A and / or Set B parameters. In some embodiments, the disclosure provides compositions comprising WVE-N531 and one or more of DS1-DS17 (the "first group"), none of WVE-N531 and one or more of DS1-DS17 (the "second group"), and optionally reduced levels of WVE-N531 and one or more of DS1-DS17 (the "third group"), each reduced level being independently lower than one or more or each of the levels of the members of the first group (e.g., about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% or less, in some embodiments about 50% or less). In some embodiments, the disclosure provides a composition comprising WVE-N531 and one of DS1-DS17, wherein the composition does not include one or more of WVE-N531 and the remaining of DS1-DS17 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17). In some embodiments, the composition does not include one of WVE-N531 and the remaining of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining seven of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining eight of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining nine of DS1-DS17.In some embodiments, the composition does not include WVE-N531 and the remaining 10 of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining 11 of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining 12 of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining 13 of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining 14 of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining 15 of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining 16 of DS1-DS17. In some embodiments, the composition does not include WVE-N531 and the remaining 17 of DS1-DS17. In some embodiments, the composition includes WVE-N531. In some embodiments, the composition includes DS1. In some embodiments, the composition includes DS2. In some embodiments, the composition comprises DS3. In some embodiments, the composition comprises DS4. In some embodiments, the composition comprises DS5. In some embodiments, the composition comprises DS6. In some embodiments, the composition comprises DS7. In some embodiments, the composition comprises DS8. In some embodiments, the composition comprises DS9. In some embodiments, the composition comprises DS10. In some embodiments, the composition comprises DS11. In some embodiments, the composition comprises DS12. In some embodiments, the composition comprises DS13. In some embodiments, the composition comprises DS14. In some embodiments, the composition comprises DS15. In some embodiments, the composition comprises DS16. In some embodiments, the composition comprises DS17. In some embodiments, at least WVE-N531 is absent in the composition. In some embodiments, at least DS1 is absent in the composition. In some embodiments, at least DS2 is absent in the composition. In some embodiments, at least DS3 is absent in the composition. In some embodiments, at least DS4 is absent in the composition.In some embodiments, at least DS5 is absent in the composition. In some embodiments, at least DS6 is absent in the composition. In some embodiments, at least DS7 is absent in the composition. In some embodiments, at least DS8 is absent in the composition. In some embodiments, at least DS9 is absent in the composition. In some embodiments, at least DS10 is absent in the composition. In some embodiments, at least DS11 is absent in the composition. In some embodiments, at least DS12 is absent in the composition. In some embodiments, at least DS13 is absent in the composition. In some embodiments, at least DS14 is absent in the composition. In some embodiments, at least DS15 is absent in the composition. In some embodiments, at least DS16 is absent in the composition. In some embodiments, at least DS17 is absent in the composition. In some embodiments, the present disclosure provides techniques for preparing such compositions, such as the IP-RP-UPLC techniques described herein (e.g., those that can fully or partially resolve diastereomers). For example, in some embodiments, when a peak for WVE-N531 and one of DS1-DS17 is completely resolved from one or more peaks of WVE-N531 and one or more of DS1-DS17, that peak represents a composition comprising WVE-N531 and one of DS1-DS17 that does not include one or more of WVE-N531 and DS1-DS17. In some embodiments, the presence, absence and / or level is assessed using techniques provided, such as IP-RP-UPLC as described herein.

[0167] In some embodiments, the disclosure provides a composition comprising WVE-N531, wherein the composition does not include one or more of DS1-DS17 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17). In some embodiments, the composition does not include one of DS1-DS17. In some embodiments, the composition does not include two of DS1-DS17. In some embodiments, the composition does not include three of DS1-DS17. In some embodiments, the composition does not include four of DS1-DS17. In some embodiments, the composition does not include five of DS1-DS17. In some embodiments, the composition does not include six of DS1-DS17. In some embodiments, the composition does not include seven of DS1-DS17. In some embodiments, the composition does not include eight of DS1-DS17. In some embodiments, the composition does not include nine of DS1-DS17. In some embodiments, the composition does not include ten of DS1-DS17. In some embodiments, the composition does not include eleven of DS1-DS17. In some embodiments, the composition does not include twelve of DS1-DS17. In some embodiments, the composition does not include thirteen of DS1-DS17. In some embodiments, the composition does not include fourteen of DS1-DS17. In some embodiments, the composition does not include fifteen of DS1-DS17. In some embodiments, the composition does not include sixteen of DS1-DS17. In some embodiments, the composition does not include seventeen of DS1-DS17. In some embodiments, at least DS1 is not present in the composition. In some embodiments, at least DS2 is not present in the composition. In some embodiments, at least DS3 is not present in the composition. In some embodiments, at least DS4 is not present in the composition. In some embodiments, at least DS5 is not present in the composition. In some embodiments, at least DS6 is not present in the composition. In some embodiments, at least DS7 is not present in the composition. In some embodiments, at least DS8 is not present in the composition. In some embodiments, at least DS9 is not present in the composition.In some embodiments, at least DS10 is absent in the composition. In some embodiments, at least DS11 is absent in the composition. In some embodiments, at least DS12 is absent in the composition. In some embodiments, at least DS13 is absent in the composition. In some embodiments, at least DS14 is absent in the composition. In some embodiments, at least DS15 is absent in the composition. In some embodiments, at least DS16 is absent in the composition. In some embodiments, at least DS17 is absent in the composition.

[0168] As with WVE-N531, each of DS1-DS17 may exist independently in various forms. In some embodiments, in the composition, it exists in one form, and in some embodiments, it exists in two or more forms. In some embodiments, the form is a salt form. In some embodiments, the form is a pharma- ceutically acceptable salt form. In some embodiments, each form is independently a salt form. In some embodiments, each form is independently a pharma- ceutically acceptable salt form. In some embodiments, the form is a hexadeca sodium salt. In some embodiments, WVE-N531 and / or D1-DS17 may be dissolved in a liquid composition, such as an optionally buffered solution, and dissociated to provide cations and anions.

[0169] Liquid WVE-N531 drug substance is packaged in sterile high density polyethylene (HDPE) bottles with polypropylene screw caps that are labeled and sealed in Mylar foil pouches that provide a gas / moisture barrier with a high level of abrasion and puncture resistance.

[0170] Various batches of WVE-N531 hexadeca sodium 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 have been utilized in non-clinical studies, including GLP toxicology studies. Certain preparations have been manufactured for clinical trials. The manufactured product is stable: no significant changes in attributed stability have been observed for multiple lots of GMP drug substance after 24 months of storage at extended storage conditions of -20°C ± 5°C and after 14 days of storage at accelerated conditions of 5°C ± 3°C.

[0171] formulation In some embodiments, the disclosure provides a pharmaceutical composition that includes or delivers WVE-N531 or a pharma- ceutically acceptable salt form thereof and a pharma- ceutically acceptable carrier. In some embodiments, the disclosure provides a pharmaceutical composition that includes WVE-N531 hexadeca sodium salt and a pharma- ceutically acceptable carrier. In some embodiments, the disclosure provides a WVE-N531 formulation. In some embodiments, the WVE-N531 formulation includes a WVE-N531 drug substance, such as WVE-N531 hexadeca sodium salt, manufactured using the processes described above.

[0172] In some embodiments, the pharma- ceutically acceptable carrier is or comprises a buffer solution. In some embodiments, the pharma- ceutically acceptable carrier is or comprises a phosphate buffer solution. In some embodiments, the pharma- ceutically acceptable carrier is or comprises an isotonic phosphate buffer solution. In some embodiments, the WVE-N531 formulation is or comprises WVE-N531 drug substance in an isotonic phosphate buffer solution. In some embodiments, it is a pharmaceutical composition or formulation that closely matches the physiological conditions in blood in terms of pH and osmolality. In some embodiments, it is for intravenous (IV) administration.

[0173] Specific WVE-N531 formulations and useful manufacturing techniques are described below by way of example.

[0174] In some embodiments, the WVE-N531 pharmaceutical composition, e.g., formulation, is packaged in a 10 mL USP type 1 clear glass vial capped with an aluminum overseal with a FluroTec® coated chlorobutyl rubber stopper and a white flip-off cap. In some embodiments, each formulation vial contains WVE-N531 drug substance equivalent to 36 mg of WVE-N531 free acid form (6 mg / mL, nominal content 36 mg, total extractable volume 6 mL). In some embodiments, the components of the WVE-N531 formulation include 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 to the required range.

[0175] The composition of an example WVE-N531 formulation is shown below.

[0176] [Table 15]

[0177] In some embodiments, the WVE-N531 formulation is a colorless solution with no visible material and essentially free of particulates. In some embodiments, the pH of a 6 mg / mL solution in phosphate buffered saline solution is 7.0-7.8 and has an osmolality in the range of 280-320 mOsm / kg. In some embodiments, the density of the WVE-N531 formulation is 1.0 g / mL.

[0178] WVE-N531 formulation lots were manufactured at a concentration of 36 mg / vial (free acid form). The 36 mg strength is achieved by filling 6.35 mL of the 6 mg / mL formulation, which contained 0.35 mL more than the nominal volume of 6 mL. All material contact components were sterilized prior to use. The container closure system for the WVE-N531 formulation comprises or consists of 10 mL USP type 1 clear glass vials sealed with an aluminum overseal with a FluroTec® coated rubber stopper and a white flip-off cap. This container closure can protect the sterilized product from microbial contamination.

[0179] WVE-N531 formulations are typically sterile solutions. In some embodiments, WVE-N531 formulations may be diluted with 0.9% sodium chloride or 0.45% sodium chloride as a sterile, preservative-free solution prior to intravenous (IV) administration. Formulation lots may be created by pooling multiple drug substance lots.

[0180] In some embodiments, the formulations have a purity as described herein, e.g., as described for WVE-N531 or a salt thereof (e.g., hexadeca sodium salt), WVE-N531 drug substance, etc. In some embodiments, purity is determined and assessed using the IP-RP-UPLC method described herein for purity.

[0181] A flow diagram for a WVE-N531 formulation manufacturing process is provided, by way of example, in FIG.

[0182] The WVE-N531 drug substance containers are allowed to thaw for approximately 48 hours at 2-8° C. While the drug substance thawing is occurring, phosphate buffered saline (PBS) solutions of 1× and 2× strength are prepared, with the 1× solution containing 10 mM sodium / potassium phosphate and 142 mM sodium chloride at pH 7.4±0.2 and an osmolality of 285-315 mOsm / kg. The thawed drug substance contents from each container are pooled into the formulation container and mixed thoroughly.

[0183] When a single lot of drug substance is used for the drug product, the release purity result is used as the purity correction factor. If multiple lots of drug substance are to be pooled, a purity correction factor based on the amount of active ingredient from each drug substance lot is determined.

[0184] In some embodiments, water for injection (WFI) is used to rinse each drug substance container and the rinse is added to the next drug substance container. The rinse from the final drug substance container is added to the formulation container. The total amount of drug substance is weighed and an equal amount of 2x PBS solution is added to the formulation container. In-process samples are taken to measure concentration and density by ultraviolet (UV) spectrophotometry.

[0185] Based on the results of the WVE-N531 drug substance concentration and solution mass, the required volume of 1×PBS is calculated to achieve a WVE-N531 drug substance concentration of 6.0 mg / mL (WVE-N531 free acid form). The required amount of 1×PBS is then added directly to the formulation container and the contents of the container are mixed to ensure solution homogeneity.

[0186] In some embodiments, final in-process samples are collected from the formulation container for concentration, density, osmolality, and pH by UV spectrophotometry. In some embodiments, the WVE-N531 concentration in the formulation is about 6 mg / mL. In some embodiments, it is about 5.5-6.5 mg / mL (free acid form, corrected purity). The final formulation undergoes clarification filtration.

[0187] In some embodiments, an in-process pre-filtration bioburden sample is collected before sterile filtration. In some embodiments, the final formulation is sterile filtered through two 0.2 μm filters in succession before filling. In some embodiments, the sterile filter is checked for filter integrity by bubble point method before and after filtration.

[0188] Based on the final density measurements, a target fill weight is determined along with warning limits and action limits (reaching a target fill volume of 6.35 mL / vial). Filling is performed with periodic fill weight checks. Vials are filled, stoppered and aseptically sealed in a Grade A environment. Finished vials are visually inspected prior to release testing, bulk packaging and / or stability testing.

[0189] In some embodiments, process and in-process control tests and acceptance limits applied during manufacturing of WVE-N531 formulations include WVE-N531 concentration, pH, osmolality, bioburden, filter integrity, fill weight, appearance, and the like.

[0190] In some embodiments, the manufacture of WVE-N531 formulations involves in-process concentration measurements via ultraviolet (UV) spectrophotometry. In some embodiments, the in-process concentration measurements are made upon initial dilution of the WVE-N531 drug substance with an equal volume of 2× phosphate buffered saline (PBS). The results are used to determine the amount of 1× PBS to add for the final concentration to be measured prior to sterile filtration and filling.

[0191] The release specifications for the WVE-N531 formulation may be based on one or more specifications described herein, such as appearance (e.g., visual), identity (e.g., by retention time by IP-RP-UPLC), purity (e.g., area % by IP-RP-UPLC), impurities (e.g., area % by IP-RP-UPLC), % labeling (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 endotoxins (e.g., USP <85> and / or Ph.Eur.2.6.14), sterility (e.g., USP <71> and / or Ph.Eur.2.6.1), uniformity of dose (e.g., USP <905> and / or Ph.Eur.2.9.40), particulate matter (e.g., USP <788> and / or Ph.Eur.2.9.19), container content (e.g. USP <697> ), container closure integrity testing (e.g., USP <1207> ), etc. In some embodiments, IP-RP-UPLC techniques utilizing one or more or all of Set A parameters are utilized to assess identity (retention time), purity, and / or impurities. In some embodiments, the 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 greater.In some embodiments, it is measured by IP-RP-UPLC area % at 260 nm using Set A parameters.

[0192] WVE-N531 formulations may contain impurities similar to the WVE-N531 drug substance at levels as described herein.

[0193] In some embodiments, the container closure system for the WVE-N531 formulation comprises or consists of a USP Type 1 glass vial (10 mL capacity, 20 mm opening) with a stopper molded from FluroTec® coated elastomeric compound 4432 / 50 gray with an aluminum overseal and a white flip-off cap.

[0194] In some embodiments, storage conditions for WVE-N531 formulations are −20±5° C. In some embodiments, conditions of 5° C.±3° C., for example for relatively short-term storage.

[0195] Among other things, the present disclosure provides the following exemplary embodiments: 1. A method of treating muscular dystrophy comprising administering to a subject suffering therefrom WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form, wherein the subject has a mutation in the DMD gene amenable to exon 53 skipping. 2. A method of treating muscular dystrophy in a subject suffering from same, comprising administering to the subject one or more doses of WVE-N531, each dose being 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 of WVE-N531 free acid form, wherein the subject has a mutation in the DMD gene that is amenable to exon 53 skipping. 3. A method comprising administering to a subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. 4. A method comprising administering to a subject one or more doses of WVE-N531, each dose being 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 of WVE-N531 free acid form. 5. A method for effecting DMD exon 53 skipping in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. 6. A method for effecting DMD exon 53 skipping in a subject, comprising administering to the subject one or more doses of WVE-N531, each dose being 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 of WVE-N531 free acid form. 7. A method of restoring a DMD RNA reading frame in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. 8. A method of restoring a DMD RNA reading frame in a subject, comprising administering to the subject one or more doses of WVE-N531, each dose 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 of WVE-N531 free acid form. 9. A method of providing a DMD polypeptide in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein. 10. A method of providing a DMD polypeptide in a subject, comprising administering to the subject one or more doses of WVE-N531, each dose being 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 of WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein. 11. The method of any one of embodiments 9-10, wherein the DMD polypeptide is encoded by a DMD mRNA in which exon 53 has been skipped. 12. The method of any one of embodiments 9-11, wherein the DMD polypeptide provides one or more functions of a wild-type DMD protein. 13. A method for producing an increased level of DMD function in a subject, comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. 14. A method for producing an increased level of DMD function in a subject, comprising administering to the subject one or more doses of WVE-N531, each dose being 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 of WVE-N531 free acid form. 15. The method of any one of embodiments 3 to 14, wherein the subject is suffering from muscular dystrophy. 16. The method of any one of embodiments 3-15, wherein the subject is afflicted with DMD. 17. The method of any one of embodiments 3-16, wherein the subject has a mutation in the DMD gene that is amenable to exon 53 skipping. 18. The method of any one of the preceding embodiments, wherein WVE-N531 is administered in one or more forms. 19. The method of any one of the preceding embodiments, wherein WVE-N531 is administered in one or more pharma- ceutically acceptable salt forms. 20. The method of any one of embodiments 18-19, wherein one form is WVE-N531 hexadeca sodium salt. 21. The method of any one of embodiments 18-20, wherein each dose of WVE-N531 is administered independently in a pharmaceutical composition comprising WVE-N531 and a pharma- ceutically acceptable carrier. 22. A method of treating muscular dystrophy comprising administering to a subject suffering from same a pharmaceutical composition comprising WVE-N531 and a pharma- ceutical acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form, wherein the subject has a mutation in the DMD gene amenable to exon 53 skipping. 23. A method comprising administering to a subject a pharmaceutical composition comprising WVE-N531 and a pharma- ceutical acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. 24. A method for effecting DMD exon 53 skipping in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharma- ceutical acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. 25. A method of restoring a DMD RNA reading frame comprising administering to a subject a pharmaceutical composition comprising WVE-N531 and a pharma- ceutical acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. 26. A method of providing a DMD polypeptide in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharma- ceutical acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein. 27. The method of embodiment 26, wherein the DMD polypeptide is encoded by a DMD mRNA in which exon 53 has been skipped. 28. The method of any one of embodiments 26-27, wherein the DMD polypeptide provides one or more functions of a wild-type DMD protein. 29. A method for producing an increased level of DMD function in a subject, comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharma- ceutical acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. 30. The method of any one of embodiments 23-29, wherein the subject is suffering from muscular dystrophy. 31. The method of any one of embodiments 23-30, wherein the subject is suffering from DMD. 32. The method of any one of embodiments 23-31, wherein the subject has a mutation in the DMD gene that is amenable to exon 53 skipping. 33. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 1 mg / kg of WVE-N531 free acid form. 34. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 1 mg / kg of WVE-N531 free acid form. 35. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 2 mg / kg of WVE-N531 free acid form. 36. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 3 mg / kg of WVE-N531 free acid form. 37. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 4 mg / kg of WVE-N531 free acid form. 38. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 5 mg / kg of WVE-N531 free acid form. 39. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 6 mg / kg of WVE-N531 free acid form. 40. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 7 mg / kg of WVE-N531 free acid form. 41. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 8 mg / kg of WVE-N531 free acid form. 42. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 9 mg / kg of WVE-N531 free acid form. 43. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 10 mg / kg of WVE-N531 free acid form. 44. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 11 mg / kg of WVE-N531 free acid form. 45. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 12 mg / kg of WVE-N531 free acid form. 46. ​​The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 13 mg / kg of WVE-N531 free acid form. 47. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 14 mg / kg of WVE-N531 free acid form. 48. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 15 mg / kg of WVE-N531 free acid form. 49. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 16 mg / kg of WVE-N531 free acid form. 50. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 17 mg / kg of WVE-N531 free acid form. 51. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 18 mg / kg of WVE-N531 free acid form. 52. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 19 mg / kg of WVE-N531 free acid form. 53. The method of any one of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 20 mg / kg of WVE-N531 free acid form. 54. The method of any one of embodiments 33-53, wherein about is ±1%. 55. The method of any one of embodiments 33-53, wherein about is ±2%. 56. The method of any one of embodiments 33-53, wherein about is ±3%. 57. The method of any one of embodiments 33-53, wherein about is ±4%. 58. The method of any one of embodiments 33-53, wherein about is ±5%. 59. The method of any one of embodiments 33-53, wherein about is ±6%. 60. The method of any one of embodiments 33-53, wherein about is ±7%. 61. The method of any one of embodiments 33-53, wherein about is ±8%. 62. The method of any one of embodiments 33-53, wherein about is ±9%. 63. The method of any one of embodiments 33-53, wherein about is ±10%. 64. The method of any one of the preceding embodiments, wherein two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) doses are administered. 65. The method of any one of the preceding embodiments, wherein about 10 doses or more are administered. 66. The method of any one of embodiments 64-65, wherein each dose is administered independently in a pharmaceutical composition comprising WVE-N531 and a pharma- ceutically acceptable carrier. 67. The method of any one of embodiments 64-66, wherein each dose has approximately the same amount of WVE-N531. 68. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 1 mg / kg of WVE-N531 free acid form. 69. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 1 mg / kg of WVE-N531 free acid form. 70. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 2 mg / kg of WVE-N531 free acid form. 71. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 3 mg / kg of WVE-N531 free acid form. 72. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 4 mg / kg of WVE-N531 free acid form. 73. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 5 mg / kg of WVE-N531 free acid form. 74. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 6 mg / kg of WVE-N531 free acid form. 75. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 7 mg / kg of WVE-N531 free acid form. 76. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 8 mg / kg of WVE-N531 free acid form. 77. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 9 mg / kg of WVE-N531 free acid form. 78. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 10 mg / kg of WVE-N531 free acid form. 79. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 11 mg / kg of WVE-N531 free acid form. 80. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 12 mg / kg of WVE-N531 free acid form. 81. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 13 mg / kg of WVE-N531 free acid form. 82. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 14 mg / kg of WVE-N531 free acid form. 83. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 15 mg / kg of WVE-N531 free acid form. 84. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 16 mg / kg of WVE-N531 free acid form. 85. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 17 mg / kg of WVE-N531 free acid form. 86. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 18 mg / kg of WVE-N531 free acid form. 87. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 19 mg / kg of WVE-N531 free acid form. 88. The method of any one of embodiments 64-67, wherein the WVE-N531 in each dose is equivalent to about 20 mg / kg of WVE-N531 free acid form. 89. The method of any one of embodiments 67-88, wherein about is ±1%. 90. The method of any one of embodiments 67-88, wherein about is ±2%. 91. The method of any one of embodiments 67-88, wherein about is ±3%. 92. The method of any one of embodiments 67-88, wherein about is ±4%. 93. The method of any one of embodiments 67-88, wherein about is ±5%. 94. The method of any one of embodiments 67-88, wherein about is ±6%. 95. The method of any one of embodiments 67-88, wherein about is ±7%. 96. The method of any one of embodiments 67-88, wherein about is ±8%. 97. The method of any one of embodiments 67-88, wherein about is ±9%. 98. The method of any one of embodiments 67-88, wherein about is ±10%. 99. The method of any one of the preceding embodiments, wherein WVE-N531 is present in the pharmaceutical composition as one or more pharma- ceutically acceptable salt forms. 100. The method of embodiment 99, wherein the pharma- ceutically acceptable salt form is a hexadeca sodium salt. 101. The method of any one of the preceding embodiments, wherein the pharmaceutical composition is a liquid composition comprising dissolved WVE-N531. 102. The method of any one of the preceding embodiments, wherein the pharma- ceutically acceptable carrier is or comprises a phosphate buffer solution. 103. The method of any one of the preceding embodiments, wherein the components in the pharmaceutical composition are WVE-N531, potassium phosphate monobasic, sodium phosphate dibasic, sodium chloride and water, as well as hydrochloric acid and / or sodium hydroxide for pH adjustment. 104. The method of any one of the preceding embodiments, wherein the pharmaceutical composition is isotonic. 105. The method of any one of the preceding embodiments, wherein the pharmaceutical composition has a pH of about 7-8. 106. The method of any one of the preceding embodiments, wherein the pharmaceutical composition has a pH of about 7.3. 107. The method of embodiment 105, wherein the pharmaceutical composition has a pH of about 7.4. 108. The method of any one of the preceding embodiments, wherein the two or more consecutive doses are administered about once a week, 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. The method of any one of the preceding embodiments, wherein 109.10 or more successive doses are administered about every week 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. 110. The method of any one of the preceding embodiments, wherein all doses are administered about every week, 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. 111. The method of any one of the preceding embodiments, wherein the two or more successive doses are administered about every two weeks. 112. The method of any one of the preceding embodiments, wherein the three successive doses are administered about every two weeks. 113. The method of any one of the preceding embodiments, wherein three or more successive doses are administered about every two weeks. The method of any one of the preceding embodiments, wherein 114.10 or more successive doses are administered about every two weeks. 115. The method of any one of the preceding embodiments, wherein all doses are administered about every two weeks. 116. The method of any one of embodiments 1-114, wherein two or more successive doses are administered about every four weeks. 117. The method of any one of embodiments 1-114, wherein 10 or more successive doses are administered about every 4 weeks. 118. The method of any one of embodiments 108-117, wherein each of the successive doses is independently equivalent to about 10 mg / kg of WVE-N531 free acid form. 119. The method of any one of embodiments 1-110, wherein all doses are administered about every 4 weeks. 120. The method of any one of embodiments 1-110, wherein one or more doses are administered about every two weeks for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks from a first dose, followed by doses administered about every 3, 4, 5, 6, 7, 8, 9, or 10 weeks. 121. The method of any one of embodiments 1-110, wherein a dose is initially administered about every 2 weeks, followed by about 4 weeks, 8 weeks, 12 weeks, or 16 weeks after the initial dose, a dose is administered about every 4 weeks. 122. The method of any one of embodiments 1-110, wherein a dose is initially administered about every 2 weeks, followed by about 4 weeks, 8 weeks, 12 weeks, or 16 weeks after the initial dose, a dose is administered about every month. 123. The method of any one of embodiments 1-110, wherein two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every two weeks, and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every four weeks. 124. The method of any one of embodiments 1-110, wherein two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every two weeks, and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) doses are administered about every month. 125. The method of any one of embodiments 123-124, wherein the two or more doses administered about every two weeks are administered before the two or more doses administered about every four weeks or every month. 126. The method of any one of the preceding embodiments, wherein the composition has a purity of about 70%-85%. 127. The method of any one of the preceding embodiments, wherein the composition has a purity of about 70%-90%. 128. The method of any one of the preceding embodiments, wherein the composition has a purity of about 70%-80%. 129. The method of any one of the preceding embodiments, wherein the composition has a purity of about 70% or greater. 130. The method of any one of the preceding embodiments, wherein the composition has a purity of about 71% or greater. 131. The method of any one of the preceding embodiments, wherein the composition has a purity of about 72% or greater. 132. The method of any one of the preceding embodiments, wherein the composition has a purity of about 73% or greater. 133. The method of any one of the preceding embodiments, wherein the composition has a purity of about 74% or greater. 134. The method of any one of the preceding embodiments, wherein the composition has a purity of about 75% or greater. 135. The method of any one of the preceding embodiments, wherein the composition has a purity of about 76% or greater. 136. The method of any one of the preceding embodiments, wherein the composition has a purity of about 77% or greater. 137. The method of any one of the preceding embodiments, wherein the composition has a purity of about 78% or greater. 138. The method of any one of the preceding embodiments, wherein the composition has a purity of about 79% or greater. 139. The method of any one of the preceding embodiments, wherein the composition has a purity of about 80% or greater. 140. The method of any one of the preceding embodiments, wherein the composition has a purity of about 81% or greater. 141. The method of any one of the preceding embodiments, wherein the composition has a purity of about 82% or greater. 142. The method of any one of the preceding embodiments, wherein the composition has a purity of about 83% or greater. 143. The method of any one of the preceding embodiments, wherein the composition has a purity of about 84% or greater. 144. The method of any one of the preceding embodiments, wherein the composition has a purity of about 85% or greater. 145. The method of any one of the preceding embodiments, wherein the composition has a purity of about 86% or greater. 146. The method of any one of the preceding embodiments, wherein the composition has a purity of about 87% or greater. 147. The method of any one of the preceding embodiments, wherein the composition has a purity of about 88% or greater. 148. The method of any one of the preceding embodiments, wherein the composition has a purity of about 89% or greater. 149. The method of any one of the preceding embodiments, wherein the composition has a purity of about 90% or greater. 150. The method of any one of embodiments 126-149, wherein purity is measured by IP-RP-UPLC using area % at 260 nm. 151. The method of any one of embodiments 126-150, wherein purity is measured by IP-RP-UPLC using area % at 260 nm and set A parameters. 152. The method of any one of embodiments 126-150, wherein the purity is measured by an IP-RP-UPLC method for purity as described herein. 153. The method of any one of the preceding embodiments, wherein impurities in the composition are no more than about 10%-30%. 154. The method of any one of the preceding embodiments, wherein impurities in the composition are no more than about 15%-30%. 155. The method of any one of the preceding embodiments, wherein impurities in the composition are about 20%-30% or less. 156. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 30%. 157. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 29%. 158. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 28%. 159. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 27%. 160. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 26%. 161. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 25%. 162. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 24%. 163. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 23%. 164. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 22%. 165. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 21%. 166. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 20%. 167. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 19%. 168. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 18%. 169. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 17%. 170. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 16%. 171. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 15%. 172. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 14%. 173. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 13%. 174. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 12%. 175. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 11%. 176. The method of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 10%. 177. The method of any one of embodiments 153-176, wherein the impurities are measured by IP-RP-UPLC using area % at 260 nm. 178. The method of any one of embodiments 153-177, wherein the impurities are measured by IP-RP-UPLC using area % at 260 nm and set A parameters. 179. The method of any one of embodiments 153-178, wherein the impurities are measured by an IP-RP-UPLC method for purity as described herein. 180. The method of any one of the preceding embodiments, wherein the stereochemical purity of WVE-N531 is about 80% or greater. 181. The method of any one of the preceding embodiments, wherein the stereochemical purity of WVE-N531 is about 85% or greater. 182. The method of any one of the preceding embodiments, wherein the stereochemical purity of WVE-N531 is about 80%-90%. 183. The method of any one of the preceding embodiments, wherein stereochemical purity is assessed by dimer modeling. 184. The method of any one of the preceding embodiments, wherein the amount of WVE-N531 is measured by UV at 260 nm and 27 OD / mg. 185. The method of any one of the preceding embodiments, wherein the WVE-N531 drug substance is manufactured by a process described herein. 186. The method of any one of the preceding embodiments, wherein the WVE-N531 drug substance is characterized by one or more methods described herein. 187. The method of any one of the preceding embodiments, wherein the stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC. 188. The method of any one of the preceding embodiments, wherein the stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC according to Set B parameters. 189. The method of any one of the preceding embodiments, wherein the stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC method for stereochemical identity as described herein. 190. The method of any one of the preceding embodiments, wherein the WVE-N531 drug substance is shipped by one or more methods described herein. 191. The method of any one of the preceding embodiments, wherein the WVE-N531 drug substance is stored by one or more methods described herein. 192. The method of any one of embodiments 185-191, wherein the WVE-N531 drug substance is a hexadeca sodium salt. 193. The method of any one of the preceding embodiments, wherein the WVE-N531 formulation is manufactured by a process described herein. 194. The method of any one of the preceding embodiments, wherein the WVE-N531 formulation is characterized by one or more methods described herein. 195. The method of any one of the preceding embodiments, wherein the WVE-N531 formulation is shipped by one or more methods described herein. 196. The method of any one of the preceding embodiments, wherein the WVE-N531 formulation is stored by one or more methods described herein. 197. The method of any one of the preceding embodiments, wherein the pharmaceutical composition is manufactured by a process described herein. 198. The method of any one of the preceding embodiments, wherein the pharmaceutical composition is characterized by one or more methods described herein. 199. The method of any one of the preceding embodiments, wherein the pharmaceutical composition is delivered by one or more methods described herein. 200. The method of any one of the preceding embodiments, wherein the pharmaceutical composition is stored by one or more methods described herein. 201. The method of any one of the preceding embodiments, wherein WVE-N531 is administered intravenously. 202.DMD mutations are Δ3-52, Δ4-52, Δ5-52, Δ6-52, Δ9-52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17-52, Δ1 9-52, 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 36-52, 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 or Δ54-77. 203. The method of any one of the preceding embodiments, wherein the mutation comprises Δ52. 204. The method of any one of the preceding embodiments, wherein the mutation is Δ52. 205. The method of embodiment 203, wherein the mutation is Δ45-52. 206. The method of embodiment 203, wherein the mutation is Δ47-52. 207. The method of embodiment 203, wherein the mutation is Δ48-52. 208. The method of embodiment 203, wherein the mutation is Δ49-52. 209. The method of embodiment 203, wherein the mutation is Δ50-52. 210. The method of embodiment 203, wherein the mutation is Δ51-52. 211. The method of any one of the preceding embodiments, wherein the muscle dystrophin is DMD. 212. The method of any one of the preceding embodiments, wherein exon 53 is skipped in DMD mRNA. 213. The method of any one of the preceding embodiments, wherein the level of exon 53-skipped DMD mRNA is elevated. 214. 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 the DMD mRNA is exon 53 skipped DMD mRNA after administration over a particular period of time or after a particular number of doses. 215. The method of any one of the preceding embodiments, wherein the truncated DMD polypeptide is generated relative to a wild-type DMD protein. 216. The method of any one of the preceding embodiments, wherein the level of the truncated DMD polypeptide is elevated. 217. The method of any one of embodiments 215-216, wherein the truncated DMD polypeptide performs one or more functions of the wild-type DMD protein. 218. The method of any one of the preceding embodiments, which results in an increase from baseline in dystrophin levels of about 1% or more of normal levels after administration over a particular period of time or after a particular number of doses. 219. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 2% of normal levels. 220. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 5% of normal levels. 221. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 5.3% of normal levels. 222. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 6% of normal levels. 223. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 7% of normal levels. 224. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 8% of normal levels. 225. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 9% of normal levels. 226. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 10% of normal levels. 227. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 11% of normal levels. 228. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 12% of normal levels. 229. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 13% of normal levels. 230. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 14% of normal levels. 231. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 15% of normal levels. 232. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 16% of normal levels. 233. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 17% of normal levels. 234. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 18% of normal levels. 235. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 19% of normal levels. 236. The method of embodiment 218, wherein the increase from baseline in dystrophin levels is greater than or equal to about 20% of normal levels. 237. The method of any one of embodiments 218-236, wherein the increase is measured after about 12 weeks of administration from the first dose. 238. The method of any one of embodiments 218-236, wherein the increase is measured after about 13 weeks of administration from the first dose. 239. The method of any one of embodiments 218-236, wherein the increase is measured after about 14 weeks of administration from the first dose. 240. The method of any one of embodiments 218-236, wherein the increase is measured after about 24 weeks of administration from the first dose. 241. The method of any one of embodiments 218-236, wherein the increase is measured after about 25 weeks of administration from the first dose. 242. The method of any one of embodiments 218-236, wherein the increase is measured after about 26 weeks of administration from the first dose. 243. The method of any one of embodiments 218-236, wherein the increase is measured after about 36 weeks of administration from the first dose. 244. The method of any one of embodiments 218-236, wherein the increase is measured after about 37 weeks of administration from the first dose. 245. The method of any one of embodiments 218-236, wherein the increase is measured after about 38 weeks of administration from the first dose. 246. The method of any one of embodiments 218-236, wherein the increase is measured after about 48 weeks of administration from the first dose. 247. The method of any one of embodiments 218-236, wherein the increase is measured after about 49 weeks of administration from the first dose. 248. The method of any one of embodiments 218-236, wherein the increase is measured after about 50 weeks of administration from the first dose. 249. The method of any one of embodiments 218-236, wherein the increase is measured after about 72 weeks of administration from the first dose. 250. The method of any one of embodiments 218-236, wherein the increase is measured after about 73 weeks of administration from the first dose. 251. The method of any one of embodiments 218-236, wherein the increase is measured after about 74 weeks of administration from the first dose. 252. The method of any one of embodiments 218-236, wherein the increase is measured after about 96 weeks of administration from the first dose. 253. The method of any one of embodiments 218-236, wherein the increase is measured after about 97 weeks of administration from the first dose. 254. The method of any one of embodiments 218-236, wherein the increase is measured after about 98 weeks of administration from the first dose. 255. The method of any one of embodiments 218-254, wherein the increase is measured after 6 doses. 256. The method of any one of embodiments 218-254, wherein the increase is measured after 7 doses. 257. The method of any one of embodiments 218-254, wherein the increase is measured after 8 doses. 258. The method of any one of embodiments 218-254, wherein the increase is measured after 12 doses. 259. The method of any one of embodiments 218-254, wherein the increase is measured after 13 doses. 260. The method of any one of embodiments 218-254, wherein the increase is measured after 14 doses. 261. The method of any one of embodiments 218-254, wherein the increase is measured after 24 doses. 262. The method of any one of embodiments 218-254, wherein the increase is measured after 25 doses. 263. The method of any one of embodiments 218-254, wherein the increase is measured after 30 doses. 264. The method of any one of embodiments 218-254, wherein the increase is measured after 35 doses. 265. The method of any one of embodiments 218-254, wherein the increase is measured after 40 doses. 266. The method of any one of embodiments 218-254, wherein the increase is measured after 45 doses. 267. The method of any one of embodiments 218-254, wherein the increase is measured after 48 doses. 268. The method of any one of the preceding embodiments, wherein DMD function is elevated. 269. The method of any one of the preceding embodiments, wherein DMD function is restored. 270. The method of any one of the preceding embodiments, wherein loss of ambulation in the subject is reduced. 271. The method of any one of the preceding embodiments, wherein disease progression in the subject is delayed or slowed. 272. The method of any one of the preceding embodiments, wherein muscle weakness in the subject is delayed or slowed. 273. The method of any one of the preceding embodiments, wherein loss of muscle mass in the subject is delayed or slowed. 274. The method of any one of the preceding embodiments, wherein the decline in pulmonary function in the subject is delayed or slowed. 275. The method of any one of the preceding embodiments, wherein the subject improves in a muscular dystrophy assessment. 276. The method of any one of the preceding embodiments, wherein the subject improves in the 10 meter walk test. 277. The method of any one of the preceding embodiments, wherein the subject improves in one or more functional assessments. 278. The method of any one of the preceding embodiments, wherein the subject improves in the North Star Ambulatory Ability Assessment (NSAA) 2.0. 279. The method of any one of the preceding embodiments, wherein the subject improves in Upper Limb Performance (PUL) 2.0. 280. The method of any one of the preceding embodiments, wherein the subject improves in one or more lower extremity motor functions by timed test. 281. The method of any one of the preceding embodiments, wherein the subject improves in four rank progressions. 282. The method of any one of the preceding embodiments, wherein the subject improves in time to rise from bed. 283. The method of any one of the preceding embodiments, wherein the subject improves in upper extremity proximal muscle strength. 284. The method of any one of the preceding embodiments, wherein the subject improves in a handheld muscle strength measurement. 285. The method of any one of the preceding embodiments, wherein the subject improves in one or more pulmonary function tests. 286. The method of any one of the preceding embodiments, wherein the subject improves in one or more of peak flow rate [PFR], peak expiratory flow rate [CPF] and FVC. 287. The method of any one of the preceding embodiments, wherein the improvement is compared to a baseline. 288. The method of any one of the preceding embodiments, wherein the improvement is compared to the absence of WVE-N531 administration. 289. The method of any one of the preceding embodiments, wherein the improvement is compared to administration of a reference composition. 290. The method of any one of the preceding embodiments, wherein the reference composition is equivalent to the administered WVE-N531 composition, but does not contain WVE-N531. 291. The method of any of the preceding embodiments, wherein the subject is a pediatric subject. 292. The method of any of the preceding embodiments, wherein the subject is administered a steroid at least about 1 month prior to the first dose of WVE-N531. 293. The method of any of the preceding embodiments, wherein the subject is administered a steroid at least about 6 months prior to the first dose of WVE-N531. 294. The method of any of the preceding embodiments, wherein the subject is administered a corticosteroid at least about 1 month prior to the first dose of WVE-N531. 295. The method of any of the preceding embodiments, wherein the subject is administered a corticosteroid at least about 6 months prior to the first dose of WVE-N531. 296. The method of any one of embodiments 294-295, wherein the corticosteroid is deflazacort. 297. A composition comprising WVE-N531. 298. The composition of embodiment 297, wherein the form of WVE-N531 in the composition is a pharma- ceutically acceptable salt form. 299. The composition of any one of embodiments 297-298, wherein each form of WVE-N531 in the composition is independently a salt form. 300. The composition of any one of embodiments 297-299, wherein each form of WVE-N531 in the composition is independently a pharma- ceutically acceptable salt form. 301. The composition of any one of embodiments 297-300, wherein the form of WVE-N531 in the composition is WVE-N531 hexadeca sodium salt. 302. The composition of any one of the preceding embodiments, which is an active pharmaceutical ingredient. 303. The composition of any one of embodiments 297-301, which is a formulation. 304. The compound of any one of the preceding embodiments, wherein the composition is a liquid and WVE-N531 is dissolved. 305. The composition of any one of the preceding embodiments, which is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier. 306. The composition of embodiment 305, wherein the pharma- ceutically acceptable carrier is or comprises a phosphate buffer solution. 307. The composition of any one of the preceding embodiments, wherein the components in the composition are WVE-N531, potassium phosphate monobasic, sodium phosphate dibasic, sodium chloride and water, as well as hydrochloric acid and / or sodium hydroxide for pH adjustment. 308. The composition of any one of the preceding embodiments, which is isotonic. 309. The composition of any one of the preceding embodiments, having a pH of about 7-8. 310. The composition of any one of the preceding embodiments, having a pH of about 7.3. 311. The composition of embodiment 309, wherein the pharmaceutical composition has a pH of about 7.4. 312. The composition of any one of the preceding embodiments, wherein the concentration of WVE-N531 is equivalent to about 5-45 mg / mL of WVE-N531 free acid form. 313. The composition of embodiment 312, wherein the concentration of WVE-N531 is equivalent to about 25-40 mg / mL of WVE-N531 free acid form. 314. The composition of embodiment 312, wherein the concentration of WVE-N531 is equivalent to about 5-7 mg / mL of WVE-N531 free acid form. 315. The composition of embodiment 312, wherein the concentration of WVE-N531 is equivalent to about 6 mg / mL of WVE-N531 free acid form. 316. The composition of embodiment 315, packaged in a vial, wherein the volume of the composition in the vial is at least 6 mL. 317. The composition of any one of embodiments 315-316, packaged in a vial, wherein the volume of the composition in the vial is at least 6-7 mL. 318. The composition of any one of embodiments 315-316, packaged in a vial, wherein the volume of the composition in the vial is at least 6-6.5 mL. 319. The composition of any one of the preceding embodiments, having a purity of about 70%-90%. 320. The composition of any one of the preceding embodiments, having a purity of about 70%-85%. 321. The composition of any one of the preceding embodiments, having a purity of about 70%-80%. 322. The composition of any one of the preceding embodiments, having a purity of about 70% or greater. 323. The composition of any one of the preceding embodiments, having a purity of about 71% or greater. 324. The composition of any one of the preceding embodiments, having a purity of about 72% or greater. 325. The composition of any one of the preceding embodiments, having a purity of about 73% or greater. 326. The composition of any one of the preceding embodiments, having a purity of about 74% or greater. 327. The composition of any one of the preceding embodiments, having a purity of about 75% or greater. 328. The composition of any one of the preceding embodiments, having a purity of about 76% or greater. 329. The composition of any one of the preceding embodiments, having a purity of about 77% or greater. 330. The composition of any one of the preceding embodiments, having a purity of about 78% or greater. 331. The composition of any one of the preceding embodiments, having a purity of about 79% or greater. 332. The composition of any one of the preceding embodiments, having a purity of about 80% or greater. 333. The composition of any one of the preceding embodiments, having a purity of about 81% or greater. 334. The composition of any one of the preceding embodiments, having a purity of about 82% or greater. 335. The composition of any one of the preceding embodiments, having a purity of about 83% or greater. 336. The composition of any one of the preceding embodiments, having a purity of about 84% or greater. 337. The composition of any one of the preceding embodiments, having a purity of about 85% or greater. 338. The composition of any one of the preceding embodiments, having a purity of about 86% or greater. 339. The composition of any one of the preceding embodiments, having a purity of about 87% or greater. 340. The composition of any one of the preceding embodiments, having a purity of about 88% or greater. 341. The composition of any one of the preceding embodiments, having a purity of about 89% or greater. 342. The composition of any one of the preceding embodiments, having a purity of about 90% or greater. 343. The composition of any one of embodiments 319-342, wherein purity is measured by IP-RP-UPLC using area % at 260 nm. 344. The composition of any one of embodiments 319-342, wherein purity is measured by IP-RP-UPLC using area % at 260 nm and set A parameters. 345. The composition of any one of embodiments 319-342, wherein the purity is measured by an IP-RP-UPLC method for purity as described herein. 346. The composition of any one of the preceding embodiments, wherein impurities in the composition are no more than about 10%-30%. 347. The composition of any one of the preceding embodiments, wherein impurities in the composition are no more than about 15%-30%. 348. The composition of any one of the preceding embodiments, wherein impurities in the composition are about 20%-30% or less. 349. The composition of any one of the preceding embodiments, wherein impurities in the composition are less than or equal to about 30%. 350. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 29%. 351. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 28%. 352. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 27%. 353. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 26%. 354. The composition of any one of the preceding embodiments, wherein impurities in the composition are less than or equal to about 25%. 355. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 24%. 356. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 23%. 357. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 22%. 358. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 21%, or about 21%. 359. The composition of any one of the preceding embodiments, wherein impurities in the composition are less than or equal to about 20%. 360. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 19%. 361. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 18%. 362. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 17%. 363. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 16%. 364. The composition of any one of the preceding embodiments, wherein impurities in the composition are less than or equal to about 15%. 365. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 14%, or about 14%. 366. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 13%. 367. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 12%. 368. The composition of any one of the preceding embodiments, wherein the impurities in the composition are less than or equal to about 11%, or about 11%. 369. The composition of any one of the preceding embodiments, wherein impurities in the composition are less than or equal to about 10%. 370. The composition of any one of embodiments 346-369, wherein the impurities are measured by IP-RP-UPLC using area % at 260 nm. 371. The composition of any one of embodiments 346-370, wherein the impurities are measured by IP-RP-UPLC using area % at 260 nm and set A parameters. 372. The composition of any one of embodiments 346-371, wherein the impurities are measured by an IP-RP-UPLC method for purity as described herein. 373. The composition of any one of the preceding embodiments, wherein the stereochemical purity of WVE-N531 is about 80% or greater. 374. The composition of any one of the preceding embodiments, wherein the stereochemical purity of WVE-N531 is about 85% or greater. 375. The composition of any one of the preceding embodiments, wherein the stereochemical purity of WVE-N531 is about 80%-90%. 376. The composition of any one of the preceding embodiments, wherein stereochemical purity is assessed by dimer modeling. 377. The composition of any one of the preceding embodiments, wherein the amount of WVE-N531 is measured by UV at 260 nm. 378. The composition of any one of the preceding embodiments, wherein the amount of WVE-N531 is measured by UV at 260 nm and 27 OD / mg. 379. The composition of any one of the preceding embodiments, wherein the WVE-N531 drug substance in the composition is manufactured by a process described herein. 380. The composition of any one of the preceding embodiments, wherein the WVE-N531 drug substance in the composition is characterized by one or more methods described herein. 381. The composition of any one of the preceding embodiments, wherein the WVE-N531 drug substance in the composition is shipped by one or more methods described herein. 382. The composition of any one of the preceding embodiments, wherein the WVE-N531 drug substance is stored by one or more methods described herein. 383. The composition of any one of embodiments 379-382, wherein the WVE-N531 drug substance is a hexadeca sodium salt. 384. The composition of any one of the preceding embodiments, which is a WVE-N531 formulation. 385. The composition of any one of the preceding embodiments, wherein the WVE-N531 formulation is manufactured by a process described herein. 386. The composition of any one of the preceding embodiments, wherein the WVE-N531 formulation is characterized by one or more methods described herein. 387. The composition of any one of the preceding embodiments, wherein the WVE-N531 formulation is delivered by one or more methods described herein. 388. The composition of any one of the preceding embodiments, wherein the WVE-N531 formulation is stored by one or more methods described herein. 389. The composition of any one of the preceding embodiments, produced by the process described herein. 390. The composition of any one of the preceding embodiments, wherein the pharmaceutical composition is characterized by one or more of the methods described herein. 391. The composition of any one of the preceding embodiments, wherein the pharmaceutical composition is delivered by one or more methods described herein. 392. The composition of any one of the preceding embodiments, wherein the pharmaceutical composition is stored by one or more methods described herein. 393. The composition of any one of the preceding embodiments, which does not contain DS1. 394. The composition of any one of the preceding embodiments, which does not contain DS2. 395. The composition of any one of the preceding embodiments, which does not contain DS3. 396. The composition of any one of the preceding embodiments, which does not contain DS4. 397. The composition of any one of the preceding embodiments, which does not contain DS5. 398. The composition of any one of the preceding embodiments, which does not contain DS6. 399. The composition of any one of the preceding embodiments, which does not contain DS7. 400. The composition of any one of the preceding embodiments, which does not contain DS8. 401. The composition of any one of the preceding embodiments, which does not contain DS9. 402. The composition of any one of the preceding embodiments, which does not contain DS10. 403. The composition of any one of the preceding embodiments, which does not contain DS11. 404. The composition of any one of the preceding embodiments, which does not contain DS12. 405. The composition of any one of the preceding embodiments, which does not contain DS13. 406. The composition of any one of the preceding embodiments, which does not contain DS14. 407. The composition of any one of the preceding embodiments, which does not contain DS15. 408. The composition of any one of the preceding embodiments, which does not contain DS16. 409. The composition of any one of the preceding embodiments, which does not contain DS17. 410. A composition comprising WVE-N531 and one of DS1-DS17, wherein the composition does not include one or more of the remaining WVE-N531 and DS1-DS17 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17). 411. The composition of embodiment 410, comprising WVE-N531. 412. The composition of embodiment 410, comprising DS1. 413. The composition of embodiment 410, comprising DS2. 414. The composition of embodiment 410, comprising DS3. 415. The composition of embodiment 410, comprising DS4. 416. The composition of embodiment 410, comprising DS5. 417. The composition of embodiment 410, comprising DS6. 418. The composition of embodiment 410, comprising DS7. 419. The composition of embodiment 410, comprising DS8. 420. The composition of embodiment 410, comprising DS9. 421. The composition of embodiment 410, comprising DS10. 422. The composition of embodiment 410, comprising DS11. 423. The composition of embodiment 410, comprising DS12. 424. The composition of embodiment 410, comprising DS13. 425. The composition of embodiment 410, comprising DS14. 426. The composition of embodiment 410, comprising DS15. 427. The composition of embodiment 410, comprising DS16. 428. The composition of embodiment 410, comprising DS17. 429. The composition of any one of the preceding embodiments, wherein about is ±1%. 430. The composition of any one of the preceding embodiments, wherein about is ±2%. 431. The composition of any one of the preceding embodiments, wherein about is ±3%. 432. The composition of any one of the preceding embodiments, wherein about is ±4%. 433. The composition of any one of the preceding embodiments, wherein about is ±5%. 434. The composition of any one of the preceding embodiments, wherein about is ±6%. 435. The composition of any one of the preceding embodiments, wherein about is ±7%. 436. The composition of any one of the preceding embodiments, wherein about is ±8%. 437. The composition of any one of the preceding embodiments, wherein about is ±9%. 438. The composition of any one of the preceding embodiments, wherein about is ±10%. 439. A method of producing a WVE-N531 composition according to the methods described herein. 440. The method of embodiment 439, comprising utilizing IP-RP-UPLC to assess purity and / or impurities in the manufactured WVE-N531 composition, and shipping the preparation if the purity and / or impurities meet specified criteria. 441. The method of any one of embodiments 439-440, wherein the composition is an active pharmaceutical ingredient. 442. The method of any one of embodiments 439-440, wherein the composition is a formulation. 443. A method of shipping a WVE-N531 preparation, comprising utilizing IP-RP-UPLC to assess purity and / or impurities in a WVE-N531 preparation, and shipping the preparation if the purity and / or impurities meet specified criteria. 444. Method for assessing purity of WVE-N531 utilizing IP-RP-UPLC. 445. The method of any one of embodiments 440-444, wherein the IP-RP-UPLC utilizes one or more parameters described herein. 446. The method of any one of embodiments 440-444, wherein the IP-RP-UPLC utilizes one or more parameters of set A. 447. The method of any one of embodiments 440 to 444, wherein the IP-RP-UPLC utilizes set A parameters. 448. The method of any one of embodiments 439-447, wherein the stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC. 449. A method to confirm the stereochemical identity of WVE-N531 using IP-RP-UPLC. 450. The method of any one of embodiments 439-449, wherein the stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC according to Set B parameters. 451. The method of any one of embodiments 439-449, wherein the stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC method for stereochemical identity as described herein. 452. The method of any one of embodiments 439-451, wherein the composition is any one of the preceding embodiments. 453. A compound, oligonucleotide, composition, method, process, use, dose or administration regimen described herein. EXAMPLES

[0196] The foregoing has been a description of certain non-limiting embodiments of the present disclosure. It is therefore to be understood that the embodiments of the present disclosure described herein are merely illustrative of the application of the principles of the present disclosure. Reference herein to details of the illustrated embodiments is not intended to limit the scope of any claims.

[0197] The function and advantages of certain embodiments of the present disclosure may be more fully understood from the following examples, which are intended to illustrate certain advantages of such embodiments.

[0198] Example 1. WVE-N531 is sufficiently safe for human administration based on in vitro and animal studies Among other things, the present disclosure provides data, including in primates, demonstrating that WVE-N531 can be administered to human subjects according to various doses and administration regimens as described herein.

[0199] WVE-N531 is an oligonucleotide that can target human Duchenne muscular dystrophy (DMD) messenger ribonucleic acid (mRNA) to induce exon 53 skipping and restoration of functional dystrophin protein in patients with DMD.

[0200] Notably, WVE-N531 induced exon skipping and protein recovery in patient-derived cell lines. In vivo, exon skipping, dystrophin protein recovery, and improved survival were achieved in mdx23 and / or double knockout (dKO) mouse models using surrogate molecules. These models have point mutations in exon 23, and therefore mouse surrogate oligonucleotides with sequences intended to specifically target mouse exon 23 with phosphoramidate (PN) backbone that mimics the chemical reaction of WVE-N531 were used in these experiments. In vivo target engagement was also demonstrated in healthy cynomolgus monkeys, a pharmacologically relevant species, using WVE-N531. In dystrophin-deficient muscles, biodistribution and exon skipping may be greater than that observed in healthy normal muscles with other oligonucleotides.

[0201] WVE-N531 has been evaluated in non-clinical toxicity studies and no risks have been identified that would preclude its administration in humans. After 14 weeks of repeated dosing in monkeys at 45 mg / kg, no adverse effects were deemed to be present due to the mild severity of findings, rare incidence and / or lack of impact on the health and well-being of the animals. The No Observed Adverse Effect Level (NOAEL) was deemed to be at least 45 mg / kg / dose, the highest dose evaluated.

[0202] Other findings observed in both mice and monkeys were consistent with those commonly observed with oligonucleotides, including non-detrimental and reversible to partially reversible renal and hepatic findings, proinflammatory effects, and transient effects on clotting factors [prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen]. These risks can be easily monitored in the clinic.

[0203] WVE-N531 was observed to be free of genotoxicity in vitro or in vivo or off-target effects via hybridization to the human genome. In vitro, WVE-N531 did not activate Toll-like receptor 9 (TLR9) in human reporter cells or increase inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) ex vivo.

[0204] WVE-N531 had no effect on cardiovascular or respiratory function in monkeys up to the highest dose evaluated (45 mg / kg). No adverse effects on CNS function were observed in mice up to the highest dose evaluated (180 mg / kg).

[0205] The various findings observed in various non-clinical studies are consistent with class effects observed with other oligonucleotides.

[0206] WVE-N531 has been demonstrated to result in DMD exon skipping and restore dystrophin protein in DMD patient-derived myoblast cell lines (e.g., DMD Δ45-52 and DMD Δ52). Additionally, WVE-N531, a mouse exon 23-specific oligonucleotide with equivalent chemistry to WV-21218, demonstrated a dose-response for exon 23 skipping and dystrophin restoration in the mdx23 mouse model of DMD. This oligonucleotide also demonstrated other biological effects, including exon skipping, dystrophin protein restoration, and lifespan extension, in a dKO mouse model of DMD that lacks both utrophin and dystrophin and develops a severe muscular dystrophy phenotype equivalent to that observed in patients with DMD. See, e.g., WO 2021 / 237223.

[0207] Bioinformatics tools were used to determine potential off-target effects based on sequence homology to WVE-N531. Analysis suggested that sequence-dependent off-target effects were minimal.

[0208] WVE-N531 was analyzed for its ability to activate the innate immune protein TLR9 in a cell-based reporter assay. WVE-N531 exposure to the cells tested (human embryonic kidney cells overexpressing human TLR9) did not increase TLR9 reporter activity at concentrations up to 30 μM compared to cells treated with water (negative control). The potential of WVE-N531 to activate human TLR9 is negligible based on these test data.

[0209] The immune stimulatory potential of WVE-N531 was also evaluated by assessment of cytokine release following incubation of WVE-N531 with PBMCs from healthy human donors. The cytokine response to WVE-N531 was not statistically significantly different from the cytokine response to water for any of the seven analytes: IFNα2, IL12p40, IL-1β, IL-6, MIP-1α, MIP-1β, TNFα. A statistically significant reduction in MCP-1 levels was observed compared to both the negative (HO) and positive controls (TLR7 / TLR8 agonist R848). These data indicated that WVE-N531 has negligible immune stimulatory potential as measured in this ex vivo PBMC assay.

[0210] The effects of WVE-N531 on cardiovascular and respiratory function in cynomolgus monkeys and CNS function in CD-1 mice after IV administration were evaluated in GLP studies. The study design is outlined in the table below (IV = intravenous).

[0211] [Table 16]

[0212] In studies in mice, the effects of WVE-N531 on CNS function were evaluated in male and female CD-1 mice following IV (bolus) injections of vehicle (dPBS without calcium or magnesium) or 15, 60, or 180 mg / kg / dose of WVE-N531 every other week on days 1, 15, 29, 43, 57, 71, 85, and 99. Functional Observational Board (FOB) data showed that no adverse CNS effects were observed at doses up to 180 mg / kg / dose.

[0213] In one monkey study, the effects of WVE-N531 on cardiovascular and respiratory function were evaluated in conscious, radiotelemetry-instrumented male cynomolgus monkeys after a single dose of WVE-N531. Animals received WVE-N531 (3, 15 or 45 mg / kg) or dPBS (without calcium or magnesium) via 60-minute IV infusion. No WVE-N531-related effects on cardiovascular or respiratory function were observed after administration of up to 45 mg / kg of WVE-N531. Dose-dependent increases in complement factor Bb and C3a plasma concentrations were observed at 2 minutes after EOI with 15 and 45 mg / kg WVE-N531. Compared to controls, at 2 min after EOI, increases in factor Bb were 3.7-fold and 34.6-fold at the 15 and 45 mg / kg dose levels, respectively, and increases in C3a were 2.0-fold and 32.4-fold at the 15 and 45 mg / kg dose levels, respectively, returning to baseline by 24 hours.

[0214] In another study in monkeys, the effects of WVE-N531 on cardiovascular function were evaluated in anesthetized male and female cynomolgus monkeys following repeated administration of WVE-N531. Animals received vehicle (dPBS without calcium or magnesium) or doses of 3, 15, or 45 mg / kg WVE-N531 via 1-hour IV infusion every other week. Electrocardiograms (ECGs) were collected once during the pre-dose phase and 0.5 hours (±0.25 hours) post-dose on days 15 and 71 of the dosing phase. No effects were observed following 1-hour IV infusion administration of WVE-N531 to male or female cynomolgus monkeys at doses up to 45 mg / kg / dose.

[0215] WVE-N531 demonstrated a favorable pharmacokinetic profile, including plasma protein binding, tissue distribution, metabolism, etc., for administration to human subjects in pharmacokinetic studies performed in animals, including mice and monkeys.

[0216] Non-GLP and GLP repeat-dose toxicity studies, including using the IV route of administration, have been performed in CD-1 mice (up to 14 weeks) and cynomolgus monkeys (up to 14 weeks). WVE-N531 has also been evaluated for potential toxicity in two in vitro studies and an in vivo study in mice. All GLP studies met the Organization for Economic Cooperation and Development (OECD) data mutual acceptance criteria [OECD Decision Reference C(89) 87 (Final)].

[0217] One non-clinical program evaluating WVE-N531 includes non-GLP repeat-dose toxicity studies in both mice and monkeys, a 14-week repeat-dose GLP toxicity study with 13-week recovery in both species, in vitro and in vivo genotoxicity, and in vivo safety pharmacology.

[0218] Mice: In male mice, some inflammatory effects were observed after 6 weeks of WVE-N531 administration via weekly IV bolus injection (0, 30, 90 or 180 mg / kg / dose). Overall, WVE-N531 was deemed well tolerated in mice at doses up to 180 mg / kg. Repeated doses of WVE-N531 (0, 15, 60 or 180 mg / kg / dose) administered every other week for 14 weeks via IV bolus injection (8 doses total) were evaluated in male and female CD-1 mice. The NOAEL for this study was considered to be 60 mg / kg / dose. This dose level resulted in gender-combined mean C of 1002 ± 135 μg / mL and 1330 ± 109 μg·h / mL, respectively, on day 99 of the dosing phase. max and AUC last Corresponded to the value.

[0219] Monkeys: In male monkeys, WVE-N531 was considered well tolerated up to dose levels of 25 mg / kg administered once weekly and 45 mg / kg administered every other week. Given the mild severity of findings, low incidence and / or lack of effects on health and well-being in animals administered the 45 mg / kg / dose, effects associated with this dose were considered not adverse. The NOAEL for this study was considered to be 45 mg / kg / dose. The 45 mg / kg / dose dose level (administered every other week) resulted in Cs of 529 μg / mL and 4380 μg hr / mL, respectively, on day 29 of the dosing period. max and AUC last Corresponded to the value.

[0220] Results from various preclinical studies indicate that the primary findings associated with WVE-N531 in mice and monkeys are consistent with oligonucleotide class effects, including some potential for systemic inflammatory and coagulation effects, reduced thymic cellularity (mice only), and effects in the liver and kidney. No effects were observed on cardiovascular or respiratory function following a 60-minute IV infusion of WVE-N531 administered to male cynomolgus monkeys at doses up to 45 mg / kg / dose. WVE-N531 was not associated with genotoxicity as assessed by standard in vitro and in vivo assays.

[0221] These findings establish, inter alia, that WVE-N531 is sufficiently safe for administration to human subjects according to a variety of methods, including the various doses and administration regimens described herein.

[0222] Example 2. WVE-N531 provides safe and effective exon skipping in primates Among other things, the present disclosure demonstrates that WVE-N531 can provide effective exon skipping in non-human primates at various doses. Monkeys were necropsied 2 days after the end of six weekly doses. cDNA was prepared from RNA samples to detect exon 53 skipping in DMD transcripts. Using a non-quantitative RT-PCR assay, exon 53 skipping was assessed in monkeys receiving WVE-N531. As shown in FIG. 2, "non-skipped" transcripts were detected in all samples (Panel A, arrows indicating bands), while "skipped" transcripts were detected only in samples from animals treated with WVE-N531 (Animals #3-#8, Panel B), demonstrating that exon skipping was achieved at all doses of 3 mg / kg or higher.

[0223] Compared with suvodirsen (25 mg / kg twice weekly), WVE-N531 (45 mg / kg every other week) increased plasma C max (in some instances, about 2-3 fold), AUC (in some instances, about 8-13 fold) and Ctrough. WVE-N531 concentrations in muscles, including the heart and diaphragm, were substantially higher, and in various instances multiple-fold higher, than suvodirsen in non-human primates.

[0224] Example 3. Clinical trials of WVE-N531. The safety and efficacy of WVE-N531 will be evaluated in clinical trials.

[0225] The WVE-N531 clinical trial is a Phase 1b / 2a, open-label study to evaluate the safety, tolerability, PK, PD, and clinical activity of IV WVE-N531 in patients with DMD harboring mutations amenable to exon 53 skipping intervention.

[0226] A dose of 1 mg / kg (unless otherwise specified, dose is equivalent to WVE-N531 free acid form) has been shown to be safe in human subjects.

[0227] Various higher doses, for example, one or more of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg, are administered via a dose escalation study to evaluate the safety profile and efficacy (e.g., DMD exon 53 skipping, DMD protein restoration, restoration of biological function, etc.) of various doses and administration regimens. In some embodiments, the dose or each dose is independently about 3 mg / kg. In some embodiments, the dose or each dose is independently about 4 mg / kg. In some embodiments, the dose or each dose is independently about 5 mg / kg. In some embodiments, the dose or each dose is independently about 6 mg / kg. In some embodiments, the dose or each dose is independently about 7 mg / kg. In some embodiments, the dose or each dose is independently about 8 mg / kg. In some embodiments, the dose or each dose is independently about 9 mg / kg. In some embodiments, the or each dose is independently about 10 mg / kg. In some embodiments, the or each dose is independently about 11 mg / kg. In some embodiments, the or each dose is independently about 12 mg / kg. In some embodiments, the or each dose is independently about 13 mg / kg. In some embodiments, the or each dose is independently about 14 mg / kg. In some embodiments, the or each dose is independently about 15 mg / kg. In some embodiments, the or each dose is independently about 16 mg / kg. In some embodiments, the or each dose is independently about 17 mg / kg. In some embodiments, the or each dose is independently about 18 mg / kg. In some embodiments, the or each dose is independently about 19 mg / kg. In some embodiments, the or each dose is independently about 20 mg / kg. In some embodiments, about 1 mg / kg is administered. In some embodiments, about 3 mg / kg is administered. In some embodiments, about 5 mg / kg is administered. In some embodiments, about 8 mg / kg is administered. In some embodiments, about 10 mg / kg is administered.In some embodiments, about 15 mg / kg is administered. In some embodiments, about 20 mg / kg is administered. In some embodiments, about 3-5 mg / kg is administered. In some embodiments, about 5-10 mg / kg is administered. In some embodiments, about 10-15 mg / kg is administered. In some embodiments, about 15-20 mg / kg is administered. In some embodiments, the dosing regimen comprises or consists of one or more doses, each independently about 3-20 mg / kg, e.g., about 3-5, 5-10, 10-15, 15-20 or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg / kg, each independently administered about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after the immediately preceding dose (if more than one dose is administered). In some embodiments, each dose in the dosing regimen is approximately the same. In some embodiments, two or more or all intervals between two consecutive doses are the same. In some embodiments, one or more subsequent doses may be administered at longer intervals compared to the first or initial dose.

[0228] In some embodiments, the clinical trial is or includes one or more conditions, protocols, methods, criteria, off-scales, designs, etc. of NCT04906460. In some embodiments, the clinical trial is NCT04906460.

[0229] Useful clinical trial protocols are shown below as examples. In some embodiments, the clinical trial is or includes one or more of the conditions, protocols, methods, criteria, off-scale, designs, etc. of the following studies. In some embodiments, the clinical trial is carried out according to the following studies. Those skilled in the art will understand that the studies can be adjusted.

[0230] An Open-Label Phase 1b / 2a Study of WVE-N531 in Patients with Duchenne Muscular Dystrophy In particular, the study will evaluate one or more of the safety and tolerability of WVE-N531 in patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping intervention, e.g., by assessing changes in dystrophin levels in deltoid muscle tissue, the pharmacokinetics (PK) of WVE-N531, and the clinical effects of WVE-N531, e.g., by assessing changes in muscle function, muscle strength, and lung function, concentrations of WVE-N531 in muscle tissue, and pharmacodynamic (PD) effects of WVE-N531.

[0231] To participate in the trial, patients must have a documented mutation in the DMD gene that is amenable to exon 53 skipping intervention.

[0232] This study has two parts. Patients in Part A (up to 5 patients) will receive escalating doses of WVE-N531 followed by a safety monitoring period of ≥ 28 days between doses, beginning at the lowest dose level. The sponsor plans to evaluate 4 dose levels to carefully assess acute tolerability with escalating doses in an attempt to titrate to a dose level optimized for PD response. The number of dose levels will be determined based on ongoing review of safety and PK data, as well as dose levels for the multiple dose treatment in Part B of the study. Approximately 12 patients in Part B will receive 7 doses administered every other week at the selected dose level. Patients in Part A may receive up to 3 additional doses of WVE-N531 every other week at the fixed dose level, provided they have not experienced any safety events that would prevent further participation in the study. Patients in Part A will receive up to 7 doses total. All dose recommendations will be reviewed and endorsed by an independent Data Safety Monitoring Board (DSMB) with input from site investigators.

[0233] Patients in Part A will undergo a biopsy (from the deltoid muscle) at the follow-up period after receiving 7 doses. Patients in Part B will undergo a baseline biopsy and a biopsy at the follow-up period after completion of dosing.

[0234] The criteria for dose selection in Part A and the decision to initiate Part B are detailed below.

[0235] Part A. Patients will begin screening no later than 6 weeks prior to the start of Part A. Part A will have an intrapatient dose escalation portion and a multiple dose portion. A minimum of 3 patients will be enrolled and will receive a maximum of 7 doses of WVE-N531. A maximum of 5 patients may be enrolled. Four ascending doses of WVE-N531 will be administered with a safety monitoring period of ≥28 days between each dose. Up to 3 additional doses every other week at the fixed dose level.

[0236] The first dose level evaluated will be 1 mg / kg. Dosing of Part A at all dose levels will be performed in a staggered fashion. No more than one patient will be dosed during Part A per 48 hour period. All patients will remain in the clinic through a 24 hour post-dose monitoring period for safety follow-up and will return on Day 3 (approximately 48 hours after dosing). The decision to proceed to dosing the next patient at the same dose level will be based on review of the safety data from the 48 hour observation period of the previous patient dosed, and the decision to proceed to the next patient will take into account the investigator's assessment. All available safety and PK data will be reviewed after at least three patients have completed the 2 week post-dose follow-up visit at a dose level. The recommended dose level for subsequent dose dosing will be reviewed and supported by the DSMB with input from the site investigator. Patients will continue to be evaluated for safety, PK, and functional assessments for a minimum of 28 days post-dose for each dose level evaluated before receiving the next dose level. Any additional patients enrolled after the DSMB review will receive subsequent dose levels selected for evaluation. Details regarding the DSMB review are provided below.

[0237] Once the maximum tolerated dose (MTD) is achieved, patients in Part A will receive up to three additional doses of WVE-N531 at a fixed dose level (e.g., 10 mg / kg) every other week, provided they have not experienced any safety events that would prevent further participation in the study. Patients will receive up to seven doses in Part A of this study. Patients will undergo safety and PK assessments throughout the treatment period. Patients will undergo an incisional muscle biopsy two weeks after completion of multi-dose treatment. Patients will continue to be monitored for safety for 10 weeks after the final dose. In some embodiments, the escalating intrapatient single doses include or are about 1 mg / kg, about 3 mg / kg, about 6 mg / kg, and about 10 mg / kg. In some embodiments, three additional doses of about 10 mg / kg every other week are administered.

[0238] The decision to initiate Part B will be based on a review of the safety, PK, and muscle tissue concentrations of WVE-N531.

[0239] Part B. Approximately 12 new patients will be enrolled in Part B. Patients will begin screening up to 6 weeks prior to dosing. Once patients are deemed eligible, they will undergo a baseline incisional muscle biopsy. The baseline biopsy should be performed at least 2 weeks prior to the first dose thereafter. Patients in Part B will receive 7 doses of WVE-N531 every other week. All patients will be evaluated for safety, PK, PD, and clinical efficacy of WVE-N531 in Part B. In addition, patients will undergo routine functional assessments including North Star Ambulatory Assessment (NSAA) 2.0, Performance of the Upper Limb (PUL) 2.0, 4-step ramp, handheld strength test, and pulmonary function testing. These assessments should be performed in the order specified below. Functional assessments may be performed up to 24 hours prior to dosing and should be performed prior to any other assessments that day, particularly blood draws. Patients will undergo a second incisional muscle biopsy 2 weeks after administration of the final dose. Patients will continue to be monitored for safety for 10 weeks after the final dose.

[0240] Dose selection will be primarily guided by clinical safety, tolerability, and PK data. In addition, the planned exposure at the highest dose to be tested will be within the quantifiable concentration (AUC) from time zero to the end of the study of 1330 μg h / mL, observed in a 14-week Good Laboratory Practice (GLP) toxicity study in mice (60 mg / kg / dose). last ) value or the lowest maximum observed concentration (C ) of 852 μg / mL observed at the NOAEL in a 14-week GLP toxicity study in monkeys (45 mg / kg / dose). max It is suggested that the value not be exceeded.

[0241] The dose increase for each dose level is no more than 3-fold from the previous dose level.

[0242] The same dose levels will be evaluated in Part B as the multiple-dose portion of Part A, but different dose levels may be selected based on findings from Part A. The doses used in Part B will not exceed the MTD determined in Part A.

[0243] Number of patients: Approximately 15 patients.

[0244] Study Population: Patients will be enrolled if they qualify according to all of the following inclusion and exclusion criteria.

[0245] Inclusion Criteria: 1. Patient and / or parent or legal guardian must be able and willing to provide written informed consent prior to any study-related procedures. 2. Diagnosis of DMD based on clinical phenotype with increased serum creatine kinase. 3. Demonstrated mutations in the DMD gene associated with DMD susceptible to exon 53 intervention. 4. A score of ≥ 1 on items 1 or 2 of the shoulder component of the PUL. 5. Ambulatory or non-ambulatory males. 6. Age ≥ 5 years and ≤ 18 years at screening. 7. Willing and able to comply with all study procedures, including scheduled clinic visits, drug regimens, clinical tests, study restrictions, and undergoing muscle biopsy procedures. 8. Stable pulmonary and cardiac function as measured by: A reproducible percent predicted forced vital capacity (FVC) of ≥ 50%. b. 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 echocardiogram (ECHO) or cardiac magnetic resonance imaging (MRI). 9. Sufficient deltoid muscle at screening to perform an incisional muscle biopsy. 10. Currently receiving a stable corticosteroid therapy regimen, defined as systemic corticosteroid therapy initiated ≥6 months prior to screening, with no change in dose ≤3 months prior to the screening visit. 11. Sexually mature males must be willing to use contraception for the duration of the study and for 10 weeks (i.e., 5 half-lives) after the last dose of study drug if the patient is sexually active. 12. Patients and caregivers must agree not to post any study-related information on social media.

[0246] Exclusion criteria: 1. Clinically significant medical findings on physical examination other than DMD that, in the opinion of the investigator, make the patient unsuitable to participate in and / or complete study procedures. 2. Other previous or ongoing medical conditions, including: Acute illness within 4 weeks of the initial screening visit, b. Abnormal physical findings other than those associated with musculoskeletal findings attributable to DMD. 3. Laboratory abnormalities that, in the opinion of the investigator, may adversely affect patient safety, preclude completion of the treatment or follow-up period, or impair the assessment of study results. These include, but are not limited to: Abnormal renal function as defined by elevated serum cystatin C levels >1.5x the upper limit of normal (ULN) at the screening visit, b. Impaired liver function with glutamate dehydrogenase (GLDH) of ≥ 2.5 x ULN and bilirubin of ≥ 2 x ULN (or international normalized ratio [INR] ≥ 1.5 x ULN); c. Activated partial thromboplastin time (aPTT) value of ≥ 1.5 × ULN; d. Platelet count below the lower limit of normal (LLN). 4. Documented positive Hepatitis B surface antigen or Hepatitis C antibody test. 5. Known to be positive for Human Immunodeficiency Virus (HIV). 6. Severe cognitive impairment and / or behavioral problems that, in the opinion of the investigator, may prohibit participation in this study. 7. Heart failure: a. Severe cardiomyopathy that, in the investigator's judgment, prohibits participation in this study; however, cardiomyopathy controlled with angiotensin-converting enzyme (ACE) inhibitors or beta-blockers will be permitted, provided the patient meets the LVEF inclusion criteria. b. Any other evidence of clinically significant structural or functional cardiac abnormalities. 8. In the opinion of the investigator, mechanical or non-invasive ventilation is required during the day or is predicted to be required during the day within the next year. Nocturnal non-invasive ventilation is permitted. 9.Has changed or will change (dose or regimen) any dietary or herbal supplements or concomitant medications within 1 month prior to the screening visit during the study. 10. Currently using anticoagulants or medications known to significantly increase the risk of bleeding, such as chronic nonsteroidal anti-inflammatory drugs (NSAIDs) and heparin. 11. Previous treatment with an investigational peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) or drisapersen. 12. Previously treated with gene therapy for DMD. 13. Received treatment with ataluren, viltolarsen, eteplirsen, or golodirsen within 14 weeks prior to screening. 14. Received any investigational drug within 3 months or 5 half-lives prior to screening, whichever is longer. 15. Known hypersensitivity to any oligonucleotide as demonstrated by a generalized allergic reaction, including changes in pulse, blood pressure, and respiratory function. 16.A parent or legal guardian is directly or indirectly involved in the conduct and management of this study as an investigator, co-investigator, study coordinator or other study staff, or the patient is a first-degree family member, significant other, or cohabiting relative of any of the above persons who is directly or indirectly involved in this study.

[0247] Investigational Drug, Dose, Route, Regimen: WVE-N531 will be provided as an isotonic solution for dilution for infusion. Four dose levels of WVE-N531 are planned. The route of administration will be IV. Infusions should be administered over a minimum of 60 minutes; however, infusion times may be extended up to 3 hours if necessary to enhance tolerability.

[0248] Study Duration: Part A will contain dose escalation with a safety monitoring period of ≥ 28 days between each dose. After selection of multiple dose levels, patients in Part A will receive up to 3 additional doses every other week followed by 10 weeks of follow-up after the final dose. Patients will receive a total of 7 doses. Part B will consist of 7 doses every other week and 10 weeks of follow-up after the final dose.

[0249] Evaluation items: safety · AEs: Vital signs, safety laboratory tests (hematology, chemistry, coagulation, urinalysis) and physical examination findings including electrocardiogram (ECG) will be monitored / collected to assess the safety and tolerability of WVE-N531. Pharmacokinetics and Pharmacodynamics Dystrophin levels (% normal dystrophin) as assessed by Western blot in muscle tissue following multiple doses of WVE-N531. Concentration of WVE-N531 in muscle tissue. ·PK for WVE-N531. Clinical Efficacy NSAA (Version 2.0) PUL (Version 2.0) Lower limb motor function by timed tests (including 10m walking / running time, time to ascend 4 steps, and time to rise from the floor) Proximal upper limb strength assessed by handheld dynamometer Pulmonary function tests (peak flow rate [PFR], peak expiratory flow rate [CPF] and FVC)

[0250] Statistical method: No formal sample size determination was performed for Part A. Up to 5 patients were considered sufficient for a preliminary evaluation of the safety, tolerability, and PK of escalating doses of WVE-N531.

[0251] Part B was designed to demonstrate the effect of WVE-N531 on dystrophin (% normal) compared to baseline. Sample size determination was based on the following assumptions: · One-sample t-test testing that the change from baseline in dystrophin is greater than zero. A mean increase in change from baseline in dystrophin of 5 (% of normal) or greater. One-sided significance level of 2.5%. · Standard deviation (SD) of change from baseline in dystrophin levels of 4.5%. · 10% dropout or non-assessment rate.

[0252] Analysis population The safety population will include all treated patients.

[0253] The dystrophin analysis population will be a subset of the safety population and will include patients with post-baseline dystrophin values.

[0254] The PK population will be a subset of the safety population and will include subjects with evaluable PK data.

[0255] Safety Analysis The safety evaluation of WVE-N531 will include a summary of TEAEs, TEAEs leading to treatment discontinuation, TEAEs by severity, TEAEs by relatedness, and serious TEAEs. Clinical evaluation changes will also be summarized.

[0256] Analysis of changes in dystrophin levels (% of normal) Changes in dystrophin levels (% of normal) from baseline will be analyzed using a one-sample t-test.

[0257] Specific Study Design This is an open-label study to evaluate the safety, tolerability, PK, PD, and clinical efficacy of intravenous (IV) WVE-N531 in patients with DMD. To participate in the study, patients must have a documented mutation in the DMD gene amenable to exon 53 skipping intervention.

[0258] screening The screening period allows for the determination of the patient's eligibility for the study. It will begin when the informed consent for the study is signed by the patient and / or parent or legal guardian (as appropriate). In addition, the patient may be required to provide assent (if applicable). Patients will begin screening up to 6 weeks prior to dosing. Screening assessments can be performed on multiple dates as long as they fall within the screening period. The investigator will determine whether the patient meets the eligibility criteria and will collect demographic and medical data that will allow for a sufficient characterization of the patient.

[0259] To participate in the study, patients must have a documented mutation in the DMD gene that is amenable to exon 53 skipping. This evaluation had to be performed using an accepted test method for mutation analysis.

[0260] treatment Part A Part A will consist of an intrapatient dose escalation portion and a multiple-dose portion. Three patients will be enrolled and will receive up to seven doses of WVE-N531.

[0261] Dose Escalation Portion In the intrapatient dose escalation portion, patients will receive ascending doses of WVE-N531 with a ≥ 28 day safety monitoring period between each dose, beginning at the lowest dose level. The first dose level evaluated will be 1 mg / kg. Dosing of Part A at all dose levels will be conducted in a staggered fashion. No more than one patient will be dosed during Part A per 48 hour period. All patients will remain in the clinic throughout the 24 hour post-dose monitoring period for safety follow-up and will return at Day 3 / 4 visit (approximately 48 hours after dosing). The decision to proceed to dosing the next patient at the same dose level will be based on review of the safety data from the 48 hour observation period of the previous patient dosed, and the decision to proceed to the next patient will take into account the investigator's assessment. Patients will return to the clinic for weekly visits until Day 22 / 7 visit. Subsequent weekly safety monitoring will be completed by the investigator via telephone / telehealth visits until the patient returns for administration of the next dose. A minimum of 28 days must elapse between doses.

[0262] After a minimum of three patients have completed the 2-week post-dose follow-up visit at a dose level, all available safety and PK data will be reviewed. Recommended dose levels for subsequent dose administration will be reviewed and endorsed by the DSMB, with input from the investigators. Patients will continue to be evaluated for safety, PK, and functional assessments for a minimum of 28 days post-dose at each dose level evaluated before receiving the next dose level. Any additional patients enrolled after DSMB review will receive subsequent dose levels selected for evaluation.

[0263] Multiple dose portion Once the maximum tolerated dose (MTD) is achieved, patients in Part A will receive up to three additional doses of WVE-N531 every other week at the fixed dose level, provided they have not experienced any safety events that would prevent further participation in the study. All patients will receive up to seven doses in total. Patients will undergo safety and PK assessments throughout the treatment period. Patients will undergo an open muscle biopsy from the deltoid muscle two weeks after completion of multi-dose treatment. Patients will continue to be monitored for safety for up to 10 weeks after the final dose. A decision to initiate Part B will be made based on review of safety, PK, and muscle tissue concentrations of WVE-N531.

[0264] Part B Approximately 12 new patients will be enrolled in Part B. Part B patients will receive 7 doses of WVE-N531 every other week at a fixed dose level and will be evaluated for safety, PK, PD and clinical efficacy. Part B patients will undergo a baseline incisional muscle biopsy. This incisional muscle biopsy will be taken from the deltoid muscle and will be taken at least 2 weeks prior to the first subsequent dose (Part B Day 1 / Visit 2) to allow recovery from surgery before obtaining a baseline functional assessment. Patients will undergo a second incisional muscle biopsy 2 weeks after completion of the last dose. All patients will undergo routine evaluations of WVE-N531 safety, PK and PD throughout Part B. In addition, patients will undergo routine functional assessments including NSAA 2.0, PUL 2.0, 4-step ascending, handheld muscle strength testing and pulmonary function testing. Functional assessments may be performed up to 24 hours prior to dosing and should be performed prior to any other assessments of the day, particularly blood draws.

[0265] At Week 14 / Visit 10 in Part B (minimum 2 weeks after the final dose), all patients will undergo a second muscle biopsy to evaluate the PD effect of WVE-N531 on muscle tissue. In addition, the concentration of WVE-N531 in muscle tissue will be evaluated. All patients in Part B will be followed for safety until Week 22 / Visit 13 (10 weeks after the final dose).

[0266] If a patient leaves the study early, they must complete an Early Discontinuation (ET) visit.

[0267] safety Adverse events (AEs) and vital signs, safety laboratory tests (hematology, chemistry, coagulation, urinalysis) and physical examination findings including electrocardiogram (ECG) will be monitored / collected to assess the safety and tolerability of WVE-N531.

[0268] Pharmacokinetics and Pharmacodynamics Dystrophin levels as assessed by Western blot in muscle tissue following multiple doses of WVE-N531 (% normal dystrophin) Concentration of WVE-N531 in muscle tissue ·PK for WVE-N531

[0269] Clinical Efficacy NSAA (Version 2.0) PUL (Version 2.0) Lower limb motor function by timed tests (including 10m walking / running time, time to ascend 4 steps, and time to rise from the floor) Proximal upper limb strength assessed by handheld dynamometer Pulmonary function tests (peak flow rate [PFR], peak expiratory flow rate [CPF] and FVC)

[0270] WVE-N531 will be provided as an isotonic solution for dilution for IV infusion. WVE-N531 will be supplied as a solution in single-use vials. The study medication should be diluted in 0.45% Sodium Chloride Injection or 0.9% Sodium Chloride Injection prior to infusion as per instructions. Study medication will be administered at the study site by trained personnel. Route of administration will be IV and total volume of infusion will be 100mL-500mL based on patient weight. Infusion should be administered over a minimum of 60 minutes. However, infusion time may be extended up to 3 hours if necessary to potentially enhance tolerability. Prepared infusion solution does not contain preservatives and should be administered within 4 hours. WVE-N531 will be reconstituted / diluted in a saline infusion bag / container. Patients should remain in place during infusion.

[0271] Treatment Adherence: Because this is an IV administered drug that will be administered in the clinic, non-adherence is not expected to be an issue.

[0272] Evaluation method and evaluation items Safety Assessment. The safety assessment will include: ·AE Medical History and Demographics Previous and concurrent medications Physical examination (including neurological and psychiatric) ·Life signs Height and weight 12-lead ECG ECHO or cardiac MRI · Clinical laboratory evaluation (including clinical chemistry, hematology and urinalysis)

[0273] Any abnormal clinical laboratory result (hematology, clinical chemistry, or urinalysis) or other safety assessment (e.g., vital sign measurements) that, in the medical and scientific judgment of the investigator, is clinically significant will be recorded as an AE or SAE.

[0274] Pharmacokinetic evaluation Plasma Pharmacokinetics: Sample collection for all patients will be collected according to the schedule above. Blood samples will be collected for analysis to be done by a sponsor approved CRO. Samples will be used by the sponsor and / or contracted vendors only for research related to the development of treatment for DMD and will be stored for a maximum of 15 years. All biological materials will be stored and secured to prevent unauthorized access and to ensure that samples are not accidentally or unlawfully lost, deteriorated or destroyed. Detailed instructions for sample collection, storage, processing and shipping will be provided in a study specific manual.

[0275] WVE-N531 Muscle Concentration: WVE-N531 concentration in muscle tissue may be assessed by hybridization-ligation enzyme-linked immunosorbent assay (HL-ELISA, if sufficient tissue sample is available). Samples will be used by the sponsor and / or contracted vendors only for research related to the development of treatments for DMD and will be stored for a maximum of 15 years. All biological materials will be stored and secured to ensure that unauthorized access is prohibited and samples are not accidentally or unlawfully lost, deteriorated or destroyed. Detailed instructions for sample collection, storage, processing and shipping will be provided in a study-specific manual.

[0276] Immunogenicity: Immunogenicity samples will be collected to assess anti-drug and anti-dystrophin antibodies in serum according to the assessment schedule. Samples will be used by the sponsor and / or contracted vendors only for research related to the development of treatments for DMD and will be stored for a maximum of 15 years. All biological materials will be stored and secured to prevent unauthorized access and to ensure that samples are not accidentally or unlawfully lost, deteriorated or destroyed. Detailed instructions for sample collection, storage, processing and shipping will be provided in a study-specific manual.

[0277] Pharmacodynamic Evaluation: Tissue samples taken from muscle biopsies will be evaluated for changes in dystrophin protein levels. In addition, exploratory immunohistochemistry evaluation for the presence of drugs or biomarkers may be performed. Serum samples for potential emerging biomarkers will also be collected at the time points described in the evaluation plan. Samples will be used by the sponsor and / or contracted vendors only for research related to the development of treatments for DMD and will be stored for a maximum of 15 years. All biological materials will be stored and secured to ensure that unauthorized access is prohibited and that samples are not accidentally or unlawfully lost, deteriorated or destroyed.

[0278] Dystrophin quantification by Western blot: Dystrophin levels will be quantified by Western blot in muscle tissue collected at baseline (Part B) and at the end of treatment (Parts A and B). The amount of restored dystrophin protein from muscle tissue will be assessed using a validated Western blot method.

[0279] Clinical Effects: All functional assessments must be performed pre-dose (on the day of dosing or up to 24 hours prior to each dose). Functional assessments included in both the NSAA and timed tests will be administered only once per time point as part of the NSAA and will be scored according to both assessments. Functional assessments should be performed in the order specified below. NSAA 2.0 PUL 2.0 · 4th rank promotion Handheld muscle strength test Pulmonary function tests (PFR, CPF and FVC)

[0280] Measurements of motor function will be performed according to the plan of evaluation. Patients should perform motor function and strength testing only if possible. If a patient is unable to complete the testing, this should be documented on the case report form.

[0281] North Star Ambulatory Ability Assessment-Functional Assessment: The NSAA is a validated unidimensional scale specifically designed to measure motor function in ambulatory children with DMD. The scale is suitable for multi-center global studies and is widely used internationally as an appropriate functional assessment for patients with DMD. The NSAA includes 17 items necessary to maintain functional ambulation from standing (item 1) to running (item 17). The scale includes items assessing abilities that may be present in the early stages of the disease, such as head lifting and standing on heels, as well as other activities such as hopping or running that are generally not fully accomplished in children with untreated DMD. Each item is scored on a 3-point scale using the following simple criteria: 2 - normal, achieves the goal without any assistance; 1 - modified manner, achieves the goal without relying on physical assistance from another person; and 0 - unable to achieve independently. The total score is the sum of the individual item scores. Scores can range from 0, unable to perform the activity independently, to 34, able to achieve all activities without any assistance. The scale is generally completed in a maximum of 15 minutes. The NSAA also includes the following timed assessments of lower extremity motor function: supine-to-stand time and time to walk 10 meters. An additional timed function test that is not part of the NSAA is the time to climb four standard-sized steps.

[0282] Performance of the Upper Limb (PUL 2.0) Test: The PUL test is an assessment tool to evaluate upper limb function in ambulatory and nonambulatory patients with DMD, developed by an international consortium of boys with DMD and their families. The PUL test was designed with a conceptual framework that reflects the natural history of progression of weakness and functional decline in DMD.

[0283] Climbing four standard-sized stairs: The time to climb four standard-sized stairs is a measure of lower extremity motor function in ambulatory patients.

[0284] Handheld dynamometry: Proximal upper limb muscle strength will be assessed by handheld dynamometer.

[0285] Pulmonary function tests: Pulmonary function tests (PFR, CPF and FVC) will be performed using a Microlab spirometer. PFR and CPF: Muscle weakness caused by neuromuscular disorders results in reduced values ​​of PFR. This test is effort dependent. The use of CPF may minimize effort-related variability. Therefore, for patients with neuromuscular weakness, CPF measured by a peak flow meter is a reliable measure of expiratory muscle strength. FVC: During FVC assessment, the patient takes a maximal breath, filling the lungs to total lung capacity (TLC), followed by a maximal exhalation. The total volume of gas expelled during a forced exhalation after a maximal inspiration is the FVC. Values ​​are reduced in patients with neuromuscular disorders. FVC measurements will be collected while the patient is in a sitting position. If the patient is wearing a thoracic-lumbar device, measurements will be taken with the patient wearing the device and in a sitting position.

[0286] statistical methods Study Drug Exposure and Compliance: The extent of study drug exposure and compliance will be summarized for the safety population.

[0287] Safety Data Analysis The analyses will include Part A and Part B analyses. All safety analyses will be performed on the safety population using the following common rules. Baseline values ​​are generally defined as the last available value before the first dose of study drug. The observation period for adverse events is defined as follows: A treatment-pretreated AE is an AE that developed or worsened before the first dose of study drug. Treatment-emergent AEs are those that occurred or worsened from the first dose of study drug to study completion / discontinuation. The TEAE summary will include all treatment-emergent AEs. Quantitative safety parameters based on descriptive statistical readout of central laboratories / measurements will be used to summarize outcomes and changes from baseline to visit.

[0288] The amount of WVE-N531 present in plasma and muscle will be assessed. Pharmacokinetic parameters include C max (maximum observed concentration), AUC last (area under the plasma concentration-time curve from time zero to the last quantifiable concentration), AUC inf (area under the plasma concentration-time curve from zero to infinity), t 1 / 2 (terminal half-life), CL (total clearance) and Vd ss (Volume of distribution at steady state). In some embodiments, the provided technology provides improved pharmacokinetic properties compared to existing oligonucleotide therapeutics.

[0289] The immunogenicity of WVE-N531, including antibodies to WVE-N531 and / or dystrophin, is evaluated. In some embodiments, the techniques provided do not induce antibodies to WVE-N531 and / or dystrophin in serum, or the levels of such antibodies are acceptable such that WVE-N531 can be administered to a subject.

[0290] In some embodiments, the change in dystrophin levels (% of normal) from baseline is assessed, which in some embodiments is analyzed using a one-sample t-test.

[0291] In some embodiments, an interim analysis is performed. In some embodiments, an interim analysis is not performed.

[0292] In some embodiments, the study will be conducted in accordance with a clinical trial protocol and SOPs that meet the guidelines provided by the International Conference on Harmonization (ICH) for Good Clinical Practice (GCP) in clinical trials and any other applicable local regulatory requirements.

[0293] In a Phase 1b / 2a open-label clinical trial, patients received a single dose of WVE-N531 and suvodirsen, both at 1 mg / kg. The plasma AUC for WVE-N531 was approximately 4-fold that of suvodirsen, and the C max was approximately 2.5-fold that of suvodirsen. WVE-N531 demonstrated a much longer plasma half-life than suvodirsen: whereas the plasma half-life for suvodirsen was less than 24 hours, the half-life of WVE-N531 was estimated to be greater than 1 week.

[0294] Additional data confirmed that WVE-N531 can provide a variety of benefits supporting its therapeutic use as described herein. In a Phase 1b / 2b, open-label clinical trial, patients received a single dose of 1 mg / kg, 3 mg / kg, or 6 mg / kg WVE-N531 or a single dose of 1 mg / kg, 5 mg / kg, 7 mg / kg, or 10 mg / kg suvodirsen. As shown in Figures 4 and 6 and Table 1 below, the plasma AUC last was approximately 4.6-fold higher than that of 5 mg / kg suvodirsen, and the plasma C max was about 2.1-fold that of 5 mg / kg suvodirsen. The plasma concentration for 6 mg / kg WVE-N531 was about 43-fold that of 5 mg / kg suvodirsen on day 7. As shown in FIG. 5, a single dose of 6 mg / kg WVE-N531 can result in quantifiable plasma concentrations (e.g., >0.002 μg / mL) for at least 105 days post-infusion. Furthermore, as seen in FIG. 6 and Table 1 below, the plasma AUC for 10 mg / kg WVE-N531 was about 2.1-fold that of 5 mg / kg suvodirsen on day 7. lastwas about 11-fold that of 5 mg / kg suvodirsen, and plasma concentrations for 10 mg / kg WVE-N531 were about 58-fold that of 5 mg / kg suvodirsen on day 7. In some embodiments, plasma concentrations and other pharmacokinetic parameters for a single dose of 10 mg / kg WVE-N531 demonstrate a half-life of approximately 25 days, which may support monthly dosing. Even at lower doses, WVE-N531 can produce higher plasma concentrations compared to suvodirsen (see, e.g., data for 3 mg / kg and 1 mg / kg WVE-N531 compared to that for 5 mg / kg or even 10 mg / kg suvodirsen).

[0295] [Table 17]

[0296] In a Phase 1b / 2a open-label clinical trial, patients received single ascending doses of WVE-N531 at 1, 3, 6, and 10 mg / kg, followed approximately 1-2 months later by three doses of 10 mg / kg WVE-N531 given every other week (weeks 0, 2, and 4). Two weeks after the final dose (week 6), muscle biopsies were performed. Muscle biopsies were analyzed for WVE-N531 concentration, WVE-N531 localization, % exon 53 skipping, and dystrophin expression. Specific data for individual patients are presented below in Table 2. Mean WVE-N531 muscle concentration was approximately 42 μg / g (approximately 6.1 μM). High levels of exon 53 skipping were observed: mean exon 53 skipping by RT-PCR was approximately 53%. To the applicant's knowledge, as of December 19, 2022, it was the earliest time point at which exon skipping has been reported in a clinical trial in boys with DMD. Mean dystrophin expression by Western blot was approximately 0.27% of normal (below the quantification limit of 1%). Without intending to be limited by theory, the applicant acknowledges that protein production may lag behind splicing of the RNA transcript. In some embodiments, WVE-N531 was visualized in myofiber nuclei using RNA in situ hybridization, confirming that WVE-N531 can reach the nucleus in muscle cells. Notably, these data confirm that WVE-N531 can produce very high levels of exon 53 skipping, even at 10 mg / kg every two weeks, which is significantly lower than golodirsen and / or viltolarsen.

[0297] [Table 18]

[0298] WVE-N531 appears to be safe and well tolerated. Treatment-emergent adverse events (TEAEs) were mild, except for a single COVID-19 infection of moderate intensity. All adverse events (AEs) related to WVE-N531 (headache, pruritic rash) were mild, transient, and resolved without sequelae. There were no serious adverse events (SAEs), events meeting discontinuation criteria, trends for an increase in TEAEs, and no evidence of oligonucleotide class-related safety events (e.g., thrombocytopenia, coagulation, complement activation, cytokine activation).

[0299] Additional clinical trials may also be designed and performed by one of skill in the art following the present disclosure to evaluate the safety and efficacy of WVE-N531, for example, longer time periods may be utilized to evaluate and confirm the production of dystrophin.

Claims

1. 1. A pharmaceutical composition for use in a method for treating muscular dystrophy, comprising: an oligonucleotide and a pharmaceutically acceptable carrier, fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC or a salt thereof, where m represents a 2'-OMe modified nucleoside; f represents a 2'-F modified nucleoside; *S represents an Sp phosphorothioate bond; n001R represents an Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate bond; The method comprises: The method comprises administering to a subject suffering therefrom an oligonucleotide at a dose equivalent to 1-20 mg / kg of the free acid form of the oligonucleotide, wherein the subject has a mutation in the DMD gene that is susceptible to exon 53 skipping.

2. The pharmaceutical composition of claim 1, wherein the oligonucleotide is a pharmaceutically acceptable salt.

3. The pharmaceutical composition described in claim 2, wherein the oligonucleotide is a hexadeca sodium salt.

4. 10. The pharmaceutical composition of claim 1, wherein the dose is equivalent to 10 mg / kg of the free acid form of the oligonucleotide.

5. 10. The pharmaceutical composition of claim 1, wherein two or more doses are administered.

6. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is a liquid composition in which the oligonucleotide is dissolved.

7. 7. The pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable carrier is or comprises a phosphate buffer solution.

8. The pharmaceutical composition according to claim 7, wherein the components in the pharmaceutical composition are oligonucleotide, potassium phosphate monobasic, sodium phosphate dibasic, sodium chloride, water, and hydrochloric acid and / or sodium hydroxide for pH adjustment.

9. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is isotonic.

10. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition has a pH of 7 to 8.

11. The pharmaceutical composition described in claim 9, wherein the pH of the pharmaceutical composition is 7.3 or 7.

4.

12. The pharmaceutical composition of claim 1, wherein three or more consecutive doses are administered every two weeks.

13. The pharmaceutical composition of claim 1, wherein the oligonucleotide is administered intravenously.

14. DMD gene mutations are Δ3-52, Δ4-52, Δ5-52, Δ6-52, Δ9-52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17-52, Δ 19-52, Δ21-52, Δ23-52, Δ24-52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32-52, Δ33-52, Δ 34-52, 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 or Δ54-77.

15. The pharmaceutical composition of claim 1 , wherein the subject is a pediatric subject.

16. 10. The pharmaceutical composition of claim 1, wherein the subject has been administered a corticosteroid at least six months prior to the first dose of the pharmaceutical composition.

17. A pharmaceutical composition for use in a method for effecting DMD exon 53 skipping in a subject, comprising: an oligonucleotide and a pharmaceutically acceptable carrier, fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC or a salt thereof, where m represents a 2'-OMe modified nucleoside; f represents a 2'-F modified nucleoside; *S represents an Sp phosphorothioate bond; n001R represents an Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate bond; The method comprises administering to the subject an oligonucleotide at a dose equivalent to 1-20 mg / kg of the free acid form of the oligonucleotide.

18. A pharmaceutical composition for use in a method for restoring a DMD RNA reading frame in a subject, comprising: an oligonucleotide and a pharmaceutically acceptable carrier, fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC or a salt thereof, where m represents a 2'-OMe modified nucleoside; f represents a 2'-F modified nucleoside; *S represents an Sp phosphorothioate bond; n001R represents an Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate bond; The method comprises administering to the subject an oligonucleotide at a dose equivalent to 1-20 mg / kg of the free acid form of the oligonucleotide.

19. A pharmaceutical composition for use in a method of providing a DMD polypeptide in a subject, comprising: the DMD polypeptide is truncated relative to a wild-type DMD protein; an oligonucleotide and a pharmaceutically acceptable carrier, fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC or a salt thereof, where m represents a 2'-OMe modified nucleoside; f represents a 2'-F modified nucleoside; *S represents an Sp phosphorothioate bond; n001R represents an Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate bond; The method comprises administering to the subject an oligonucleotide at a dose equivalent to 1-20 mg / kg of the free acid form of the oligonucleotide.

20. A pharmaceutical composition for use in a method for producing an increased level of DMD function in a subject, comprising: an oligonucleotide and a pharmaceutically acceptable carrier, fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC or a salt thereof, where m represents a 2'-OMe modified nucleoside; f represents a 2'-F modified nucleoside; *S represents an Sp phosphorothioate bond; n001R represents an Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate bond; The method comprises administering to the subject an oligonucleotide at a dose equivalent to 1-20 mg / kg of the free acid form of the oligonucleotide.

21. A composition comprising an oligonucleotide, an oligonucleotide and a pharmaceutically acceptable carrier, fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC or a salt thereof, where m represents a 2'-OMe modified nucleoside; f represents a 2'-F modified nucleoside; *S represents an Sp phosphorothioate bond; n001R represents an Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate bond; The composition has a purity of 70% or greater as measured by IP-RP-UPLC using area % at 260 nm and set A parameters.

22. a method comprising utilizing IP-RP-UPLC to assess purity and / or impurities in a preparation of oligonucleotides, and releasing the preparation if the purity and / or impurities meet specified criteria; or 1. A method comprising confirming the stereochemical identity of an oligonucleotide using IP-RP-UPLC, The oligonucleotide fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC or a salt thereof, where m represents a 2'-OMe modified nucleoside; f represents a 2'-F modified nucleoside; *S represents an Sp phosphorothioate bond; n001R represents an Rp N-(1,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate bond.

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