Oligonucleotide compositions and methods thereof for exon skipping

Abstract

The present disclosure provides, among other things, various techniques, including chirally controlled oligonucleotide compositions and techniques for making and using such oligonucleotide compositions. In some embodiments, the disclosure provides techniques useful for the prevention or treatment of various conditions, disorders, or diseases, such as Duchenne muscular dystrophy.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 315,952 filed on March 2, 2022, U.S. Provisional Application No. 63 / 397,221 filed on August 11, 2022, U.S. Provisional Application No. 63 / 424,418 filed on November 10, 2022, and U.S. Provisional Application No. 63 / 433,733 filed on December 19, 2022, the entire contents of each of which are incorporated herein by reference. Background Art

[0002] Muscular dystrophy (MD) is a group of muscle conditions, diseases, or disorders that reportedly cause (increased) weakness and breakdown of skeletal muscle over time. The condition, disease, or disorder primarily affects the muscles, the degree of weakness when symptoms begin, and how quickly symptoms worsen. Many MD subjects will eventually be unable to walk. In many cases, muscular dystrophy is fatal. Some types are also associated with problems with other organs, including the central nervous system. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD). In some embodiments, the muscular dystrophy is Becker muscular dystrophy (BMD) Summary of the invention

[0003] Although the U.S. Food and Drug Administration (FDA) has approved treatments for DMD, including the oligonucleotides golodirsen and viltolarsen, there remains an unmet need for more technologies to treat muscular dystrophies, including DMD. Among other things, at the time of the present invention, the clinical benefit of the approved oligonucleotide drugs has not yet been established.

[0004] WVE-N531 is an oligonucleotide compound that has demonstrated exon skipping activity and functional restoration of DMD in vitro and in mouse models. Structurally, WVE-N531 belongs to a different type of oligonucleotide than grodisin and vitolin. For example, grodisin and vitolin are both phosphorodiamidate morpholino oligomers (PMOs), while WVE-N531 is a 2'-F and 2'-OMe modified oligonucleotide in which the internucleotide linkages are independently phosphorothioate linkages, n001 linkages, and natural phosphate linkages. In addition, the chiral-linked phosphorus centers are not stereo-defined, and both grodisin and vitolin are provided and administered as stereo-random compositions in which their chiral-linked phosphorus centers are randomly present in two possible configurations (Rp or Sp): the compositions are more than 33 million (2 25 ) and 1 million (2 20) random mixture of diastereomers. In contrast, the chiral bonded phosphorus in WVE-N531 is independently stereo defined: the bonded phosphorus of each phosphorothioate bond is Sp, and the bonded phosphorus of each n001 bond is Rp. As demonstrated herein, the WVE-N531 composition is a chiral controlled composition in which WVE-N531 or its salt is highly enriched compared to other diastereomers or their salts. In addition, to the applicant's knowledge prior to this disclosure, oligonucleotides containing guanidine phosphate linkages (such as n001) have not been systematically administered to human subjects and evaluated for therapeutic use. The oligonucleotide suvodirsen contains certain chemical moieties, such as 2'-F, 2'-OMe, phosphorothioate linkages, etc. Suvodirsen is more similar to WVE-N531 than grodisen and vitrolsen, and is also manufactured and administered as a chirality-controlled oligonucleotide composition, but failed in human clinical trials evaluating doses (including about 4.5 mg / kg).

[0005] Among other things, the present disclosure provides technologies for treating muscular dystrophy (e.g., DMD). In some embodiments, the present disclosure provides doses and dosage regimens that are sufficiently safe for clinical administration to subjects and are effective in providing DMD exon 53 skipping, production of truncated DMD polypeptides (which can perform one or more functions of an increased level of a wild-type DMD protein compared to the absence of WVE-N531), restoration or improvement of DMD function, and / or clinical benefit. In some embodiments, as demonstrated herein, WVE-N531 can provide improved properties and / or activities, such as AUC, C max , tissue distribution and / or plasma half-life. The various results presented herein support that WVE-N531 can be safely and effectively used to treat conditions, disorders or diseases associated with DMD mutations that are amenable to exon 53 skipping, including in human subjects.

[0006] In some embodiments, the present disclosure provides a method for treating muscular dystrophy (e.g., DMD), comprising administering WVE-N531 to a subject having the disease 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 / kg body weight) of WVE-N531 free acid form, wherein the subject has a DMD gene mutation suitable for exon 53 skipping. In some embodiments, WVE-N531 is administered as a pharmaceutically acceptable salt form. In some embodiments, WVE-N531 is administered as a hexasodium salt of WVE-N531. In some embodiments, WVE-N531 is administered as one or more forms, including one or more pharmaceutically acceptable salt forms, and the total amount of all WVE-N531 forms is equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of free acid form. In some embodiments, WVE-N531 is administered in a pharmaceutical composition.

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

[0008] In some embodiments, the present disclosure provides a method for providing DMD exon 53 skipping in a subject, the method comprising administering to the subject WVE-N531 or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for restoring the DMD RNA reading frame in a subject, the method comprising administering to the subject WVE-N531 or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for providing a truncated DMD polypeptide in a subject, the method comprising administering WVE-N531 or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier to the subject, the dosage administered being equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the truncated (compared to the wild-type DMD protein) DMD polypeptide is encoded by a DMD mRNA that skips exon 53. In some embodiments, such an mRNA may lack one or more additional exons in addition to exon 53. In some embodiments, such a truncated DMD polypeptide provides one or more functions (partial or complete) of a wild-type DMD protein. In some embodiments, the present disclosure provides a method for providing an increased level of DMD function in a subject, the method comprising administering to the subject WVE-N531 or a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, the dose administered being equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the subject has a DMD gene mutation suitable for exon 53 skipping. In some embodiments, the subject suffers from or is susceptible to muscular dystrophy, such as DMD.

[0009] In some embodiments, two or more doses are administered, each dose being independent of the amounts described herein, e.g., equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, about 2-10 doses are administered. In some embodiments, 10 or more doses are administered. In some embodiments, each dose is approximately the same.

[0010] In some embodiments, two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) doses are administered approximately weekly. In some embodiments, two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) doses are administered approximately every two weeks. In some embodiments, two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) doses are administered approximately every 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, the dose is administered approximately weekly. In some embodiments, the dose is administered approximately every two weeks. In some embodiments, the dose is administered approximately every three weeks. In some embodiments, the dose is administered approximately every four weeks. In some embodiments, the dose is administered approximately monthly. In some embodiments, the dose is administered approximately every 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, three or more doses are administered approximately every two weeks. In some embodiments, the dose is administered first at shorter intervals (e.g., about every two weeks) and then at longer intervals (e.g., about every 3, 4, 5, 6, 7, 8, 9, or 10 weeks). In some embodiments, two or more doses, such as about 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses are administered approximately every two weeks, followed by one or more doses, each dose being administered independently about 3, 4, 5, 6, 7, 8, 9 or 10 weeks after its previous dose. In some embodiments, about 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses are administered approximately every two weeks, and one or more doses are administered approximately every 3, 4, 5, 6, 7, 8, 9 or 10 weeks or approximately every month. In some embodiments, from the first dose, a dose is administered approximately every two weeks for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 weeks or more, and then a dose is administered approximately every 3, 4, 5, 6, 7, 8, 9 or 10 weeks. In some embodiments, from the first dose, a dose is administered every two weeks for about 4, 8, 12 or 16 weeks or more, and then a dose is administered approximately every 4 weeks. In some embodiments, starting from the first dose, the dose is administered every two weeks for about 4, 8, 12, or 16 weeks or more, and then the dose is administered about monthly. In some embodiments, the dose is first administered about every two weeks, and then about 4 weeks, 8 weeks, 12 weeks, or 16 weeks from the first dose, the dose is administered about every four weeks or about monthly. In some embodiments, each dose is independently about the same, for example, about 10 mg / kg.

[0011] In some embodiments, WVE-N531 or a composition thereof is administered intravenously. In some embodiments, WVE-N531 or a composition thereof is administered intramuscularly. In some embodiments, the dose is administered by intravenous infusion over about 20-60, e.g., about 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the infusion is administered over 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 boy subject. In some embodiments, the subject is not less than 5 years old. In some embodiments, the subject is not more than 18 years old. In some embodiments, the subject meets one or more or all inclusion criteria described in the examples. In some embodiments, the subject is selected according to one or more or all inclusion and exclusion criteria described in the examples.

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

[0014] In some embodiments, the subject is administered steroids at least about six months (or 24 weeks) before the first dose of WVE-N531 or its composition. In some embodiments, the subject is administered corticosteroids at least about six months (or 24 weeks) before the first dose of WVE-N531 or its composition. In some embodiments, the subject receives a stable corticosteroid treatment regimen. In some embodiments, the corticosteroid is deflazacort. In some embodiments, the subject is administered steroids at least about one month before the first dose of WVE-N531 or its composition.

[0015] In some embodiments, the technology provided (oligonucleotides, compositions, methods, dosages, dosage regimens, etc.) provides one or more desired biological effects. In some embodiments, the technology provided provides exon 53 skipping. In some embodiments, the technology provided provides clinically significant levels of exon 53 skipping. In some embodiments, the technology provided increases the level of internally truncated but functional DMD polypeptides (e.g., encoded by DMD mRNA skipped by exon 53). In some embodiments, the technology provided provides one or more restored DMD functions, for example, by truncated but functional DMD polypeptides. In some embodiments, the technology provided provides clinical benefits. In some embodiments, disease progression is delayed, slowed or prevented. In some embodiments, disease progression is delayed, slowed or prevented, as assessed by a 10-meter walk test. In some embodiments, the technology provided reduces loss of lung function (relative to baseline). In some embodiments, muscle weakness in a subject is delayed, slowed or prevented. In some embodiments, muscle mass loss in a subject is delayed, slowed or prevented. In some embodiments, the subject is improved in a muscle dystrophin assessment. In some embodiments, the subject is improved in a DMD assessment. In some embodiments, improvement is achieved in one or more functional assessments, including North Star Mobile Assessment (NSAA) 2.0, upper limb performance (PUL) 2.0, four-ladder climbing, hand-held muscle measurement and lung function test. In some embodiments, such assessments are performed in such order. In some embodiments, assessment is or includes lower limb motor function of timing functional test (including 10 meters walking / running time, four-ladder climbing time and time to get up from the ground). In some embodiments, assessment is or includes upper limb proximal strength assessed using a handheld muscle tester. In some embodiments, assessment is or includes lung function test (peak flow rate [PFR], cough peak flow [CPF] and FVC). In some embodiments, improvement is achieved independently in one or more or all of these assessments. In some embodiments, assessment is the assessment disclosed in the example. In some embodiments, improvement is compared with baseline. In some embodiments, improvement is compared with WVE-N531 before application. In some embodiments, improvement is compared with predicted WVE-N531 after application, severity and / or function. In some embodiments, improvement is compared with not applying WVE-N531. In some embodiments, improvement is compared with application of reference composition. In some embodiments, the reference composition is comparable to the administered WVE-N531 composition, but does not comprise WVE-N531 or comprises a smaller amount of WVE-N531.

[0016] In some embodiments, provided techniques reduce walking loss (relative to baseline).

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

[0018] In some embodiments, the technology provided does not cause adverse events or low levels of adverse events, or does not cause severe and / or serious adverse events or low levels of severe and / or serious adverse events. In some embodiments, the adverse events associated with the technology provided are acceptable or controllable.

[0019] In some embodiments, the present disclosure provides techniques for manufacturing WVE-N531. In some embodiments, the present disclosure provides techniques for manufacturing WVE-N531 drug substances. In some embodiments, the present disclosure provides techniques for manufacturing WVE-N531 drug products. In some embodiments, the present disclosure provides techniques for characterizing WVE-N531 drug substances and / or products. In some embodiments, the present disclosure provides release specifications for characterizing WVE-N531 drug substances and / or products. In some embodiments, the present disclosure provides WVE-N531, compositions thereof, drug substances thereof, and / or products manufactured by the provided methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 .Example flow chart of the WVE-N531 drug product manufacturing method.

[0021] Figure 2. WVE-N531 provides robust exon 53 skipping in non-human primates. (A): Example regimen. Monkey animals received 6 weekly IV infusions of PBS or 3, 7, or 25 mg / kg WVE-N531 (n=2 per dose). W: week. Necropsy at day 38. (B): Exon 53 skipping assessed by RT-PCR. Dystrophin levels are normal in healthy animals, but exon skipping can be assessed by detecting skipped transcripts.

[0022] Figure 3 . Schematic diagram of an example of an experimental design. Abbreviations: DSMB = Data Safety Monitoring Board; N = number of patients.

[0023] Figure 4 .WVE-N531 demonstrated increased plasma concentrations in human subjects compared to suvodisan. Human subjects received IV infusions of 1, 3, or 6 mg / kg WVE-N531 or 5 mg / kg suvodisan. Following infusion, plasma concentrations (μg / ml) of WVE-N531 or suvodisan were measured over a period of at least 42 days.

[0024] Figure 5 .WVE-N531 demonstrated increased plasma concentrations in human subjects. Human subjects received IV infusions of 1, 3, 6, or 10 mg / kg WVE-N531. After infusion, plasma concentrations of WVE-N531 (μg / ml) were measured over a period of at least 105 days.

[0025] Figure 6.WVE-N531 demonstrated increased plasma concentrations in human subjects compared to suvodisan. Human subjects received IV infusions of 1, 3, 6, or 10 mg / kg WVE-N531 or 5 mg / kg suvodisan. Following infusion, plasma concentrations (μg / ml) of WVE-N531 or suvodisan were measured over a period of at least 21 days.

[0026] Figure 7 .Schematic representation of an example study design. definition

[0027] As used herein, unless otherwise indicated, the following definitions shall apply. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and "March's Advanced Organic Chemistry", 5th edition, eds. Smith, MB and March, J., John Wiley & Sons, New York: 2001.

[0028] As used herein in the present disclosure, unless the context clearly indicates otherwise, (i) the term "a" or "an" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising," "comprise," "including" (whether or not used with "not limited to") and "include" (whether or not used with "not limited to") may be understood to cover the itemized components or steps, whether shown alone or with one or more other components or steps; (iv) the term "another" may be understood to mean at least one additional or second one or more; and (v) where a range is provided, the endpoints are included.

[0029] Unless otherwise indicated, oligonucleotides and elements thereof (e.g., base sequence, sugar modification, internucleotide linkage, linkage phosphorus stereochemistry, etc.) are described from 5' to 3'. Unless otherwise indicated, oligonucleotides as described herein can be provided and / or utilized in various forms (including salt forms, particularly pharmaceutically acceptable salt forms). As will be understood by those skilled in the art after reading this disclosure, in some embodiments, oligonucleotides can be provided in the form of salts, such as sodium salts or potassium salts. As will be understood by those skilled in the art, in some embodiments, a single oligonucleotide in a composition can be considered to have the same configuration and / or structure, even in such a composition (e.g., liquid composition), in particular, such an oligonucleotide may be in different one or more forms (including one or more salt forms) at a specific time (and, for example, in a liquid composition, it can be dissolved and the oligonucleotide chain can exist in anionic form). For example, one skilled in the art will recognize that, at a given pH, a single internucleotide linkage along an oligonucleotide chain may be in the acid (H) form, or in one of a variety of possible salt forms (e.g., sodium salts or salts of different cations, depending on which ions may be present in the formulation or composition), and will understand that so long as their acid form (e.g., with all cations, if any, replaced with H) has the same conformation and / or structure, such individual oligonucleotides may appropriately be considered to have the same conformation and / or structure.

[0030] Approximately: As used herein, the terms "about" and "approximately" may be understood to allow for standard deviations, as would be understood by one of ordinary skill in the art. In some embodiments, the terms "about" or "approximately" in relation to numbers are generally used to include numbers that fall within a range of 5%, 10%, 15%, 20%, 25%, or 30% of that number in either direction (greater than or less than), unless otherwise indicated or otherwise apparent from the context. In some embodiments, the range is ±5%. In some embodiments, the range is ±10%.

[0031] Dosing regimen: As used herein, a "dosing regimen" or "treatment regimen" refers to a group of unit doses (usually more than one) that are administered individually to a subject and are usually administered at intervals of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some embodiments, the dosing regimen comprises multiple doses, each of which is separated from each other by a time period of the same length; in some embodiments, the dosing regimen comprises multiple doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen have the same unit dosage amount. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dosage amount, followed by one or more additional doses in a second dosage amount that is different from the first dosage amount. In some embodiments, a dosing regimen comprises a first dose in a first dosage amount, followed by one or more additional doses in a second dosage amount that is the same as the first dosage amount.

[0032] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that shows a statistically significant probability of achieving a controlled therapeutic effect when administered to a relevant population.

[0033] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0034] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which is involved in carrying or transporting the subject compound from one organ (or part of the body) to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

[0035] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of such compounds that are suitable for use in a pharmaceutical setting, i.e., salts that are suitable for use in contact with tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc., and commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, the provided compounds contain more than one acidic group, for example, the provided oligonucleotides may contain two or more acidic groups (e.g., natural phosphate linkages and / or modified internucleotide linkages). In some embodiments, the pharmaceutically acceptable salts (or generally, salts) of such compounds contain two or more cations, which may be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or generally, a salt), each acidic group having sufficient acidity exists 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 exists independently in its salt form). In some embodiments, the pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide. In some embodiments, the pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide, wherein each acidic linkage (e.g., each natural phosphate linkage and phosphorothioate internucleotide linkage) exists in the form of a sodium salt (all sodium salts).

[0036] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a provided compound or composition according to the present disclosure is administered, e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes. 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.

[0037] Substantially: As used herein, the term "substantially" refers to a qualitative state of exhibiting an overall or nearly overall range or degree of a characteristic or property of interest. It should be understood by those of ordinary skill in the biological field that biological and chemical phenomena, if any, rarely reach completion and / or proceed to completion or achieve or avoid an absolute result. Therefore, the term "substantially" is used herein to capture the inherent completeness that is potentially lacking in many biological and / or chemical phenomena.

[0038] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with the disease, disorder, and / or condition and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0039] Susceptible to: An individual who is "susceptible" to a disease, disorder, and / or condition is an individual who has a higher risk of developing the disease, disorder, and / or condition than members of the general public. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0040] Systemic: As used herein, the phrases "systemic administration," "administering systemically," "peripheral administration," and "peripherally administering" have their art-understood meanings and refer to systemic administration of a compound or composition so that it enters a receptor.

[0041] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, mitigate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0042] Therapeutically effective amount: In some embodiments, the term "therapeutically effective amount" means the amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a 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 a disease, disorder, and / or condition. As will be appreciated by one of ordinary skill in the art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, 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 alleviates, ameliorates, mitigates, inhibits, prevents, delays the onset of one or more symptoms or features of a disease, disorder, and / or condition, reduces the severity of one or more symptoms or features of a disease, disorder, and / or condition, and / or reduces the incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. In some embodiments, a single dose is an infusion, which may take up to one or more hours.

[0043] Treatment: As used herein, the term "treat, treatment, or treating" refers to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of one or more symptoms or features of a disease, disorder, and / or condition, reducing the severity of one or more symptoms or features of a disease, disorder, and / or condition, and / or reducing the incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment can be administered to subjects who do not show signs of a disease, disorder, and / or condition. In some embodiments, treatment can be administered to subjects who only show early signs of a disease, disorder, and / or condition, for example, for the purpose of reducing the risk of developing pathology associated with a disease, disorder, and / or condition.

[0044] Chirality-controlled oligonucleotide composition: As used herein, the terms "chirality-controlled (stereo-controlled or stereo-defined) oligonucleotide composition", "chirality-controlled (stereo-controlled or stereo-defined) nucleic acid composition", etc. refer to a composition comprising a plurality of oligonucleotides (or nucleic acids, chirality-controlled oligonucleotides or chirality-controlled nucleic acids) that share 1) a common base sequence, 2) a common backbone bonding pattern; 3) a common pattern of backbone chiral centers, and 4) a common backbone phosphorus modification pattern (a specific type of oligonucleotide), wherein the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotide linkages (chirality-controlled internucleotide linkages whose chiral bonding phosphorus is Rp or Sp, rather than a random Rp and Sp mixture as a non-chiral controlled internucleotide linkage). The level of the plurality of oligonucleotides (or nucleic acids) in the chirality-controlled oligonucleotide composition is non-random (predetermined, controlled). Chirality-controlled oligonucleotide compositions are typically prepared by chirality-controlled oligonucleotide preparations to stereoselectively form one or more chiral internucleotide linkages (e.g., using chiral auxiliaries exemplified in the present disclosure to purposefully control stereoselectivity compared to non-chiral controlled (stereorandom, non-stereoselective, racemic) oligonucleotide synthesis (e.g., conventional phosphoramidite-based oligonucleotide synthesis using non-chiral auxiliaries or chiral catalysts). The chirality-controlled oligonucleotide composition is enriched for a plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides having a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern. In some embodiments, the chirality-controlled oligonucleotide composition comprises a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) backbone linkage pattern; 3) backbone chiral center pattern; and 4) backbone phosphorus modification pattern, wherein the chirality-controlled oligonucleotide composition is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence, backbone linkage pattern, and backbone phosphorus modification pattern. As those of ordinary skill in the art readily appreciate, the feature of this enrichment is that, compared with substantially racemic preparations, at the linkage between each chiral controlled nucleotide, the linkage phosphorus of higher level has a desired configuration. In certain embodiments, the linkage between each chiral controlled nucleotide independently has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% diastereomeric purity relative to its chiral linkage phosphorus. In certain embodiments, each independently has at least 90% diastereomeric purity. In certain embodiments, each independently has at least 95% diastereomeric purity. In certain embodiments, each independently has at least 97% diastereomeric purity. In certain embodiments, each independently has at least 98% diastereomeric purity. In certain embodiments, the oligonucleotides in a variety have identical structures.In some embodiments, the oligonucleotides in a plurality have the same architecture and stereochemistry, and are structurally identical.

[0045] In some embodiments, the multiple oligonucleotides in the chiral controlled oligonucleotide composition share the same base sequence, (if any) the same nucleobase, sugar, and internucleotide linkage modification, and independently the same stereochemistry (Rp or Sp) at the linkage phosphorus chiral center of one or more chiral controlled internucleotide linkages, although the stereochemistry of some linkage phosphorus chiral centers may be different. In some embodiments, about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 5%, 10%-10% of all oligonucleotides in the chiral controlled oligonucleotide composition. , 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 the plurality of oligonucleotides. In some embodiments, about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 50%-100%) of all oligonucleotides in a chiral controlled oligonucleotide composition that share a common base sequence. %, 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%) are the plurality of oligonucleotides.In some embodiments, about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 100%-100% or more) of all oligonucleotides in a chiral controlled oligonucleotide composition that share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern. 94%, 95%, 96%, 97%, 98%, or 99%) are the plurality of oligonucleotides. In some embodiments, about 0.1%-100% (e.g., about 1%-100%, 5%-10% of all oligonucleotides in a chiral controlled oligonucleotide composition, or about 0.1%-100%, 5%-10% of all oligonucleotides in a composition that share a common base sequence, or about 0.1%-100%, 5%-10% of all oligonucleotides in a composition that share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern, or about 0.1%-100% (e.g., about 1%-100%, 5%-10% of all oligonucleotides in a composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linkage type pattern, and / or a common internucleotide linkage modification pattern) of all oligonucleotides in a biological composition. %, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 90%, 95%, 100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 90%, 95%-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 90%, 95%-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 90%, 95%-100%, 50%-90%, or about In some embodiments, the percentage is at least (DP). NCI, wherein DP is a percentage selected from 85%-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 linkage of the linkage phosphorus chiral center. In some embodiments, the diastereomeric purity of the linkage phosphorus chiral center of the internucleotide linkage can be assessed using a suitable dimer (comprising such an internucleotide linkage and two nucleoside units connected 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 present in an amount ranging from 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%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 50%, ...70%, 80%, 90%, 95%, 50%, 90%, 95%, 50%, 90%, about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 40%, 50%, 60%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, 70%, In some embodiments, the chiral internucleotide linkages are at least 0%, 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% (or more) share the same stereochemistry at the chiral internucleotide linkages. 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 certain embodiments, the oligonucleotide composition of chirality control comprises independent oligonucleotide or nucleic acid type of predetermined level.For example, in certain embodiments, the oligonucleotide composition of chirality control is included in a kind of oligonucleotide type of (for example, as described above) at predetermined level.In certain embodiments, the oligonucleotide composition of chirality control comprises more than a kind of oligonucleotide type, independently of each other at predetermined level.In certain embodiments, the oligonucleotide composition of chirality control comprises multiple oligonucleotide types, independently of each other at predetermined level.In certain embodiments, the oligonucleotide composition of chirality control is the composition of the oligonucleotide of oligonucleotide type, and said composition comprises the multiple oligonucleotide of the oligonucleotide type of predetermined level. DETAILED DESCRIPTION

[0046] Among other things, the present disclosure provides a method for treating muscular dystrophy by administering a certain amount of WVE-N531 as described herein to a subject, for example, treating a subject with a DMD gene mutation suitable for exon 53 skipping. Dystrophin plays an important role in muscle function, and a variety of muscle-related diseases are characterized by a mutant form of this gene. In certain embodiments, the methods described herein can be used to induce exon skipping in transcripts (e.g., mRNA) of mutant forms of human dystrophin genes (e.g., mutant dystrophin gene forms found in muscle dystrophin, such as DMD and BMD). In certain embodiments, the technology provided provides DMDmRNA skipped in exon 53. In certain embodiments, the present disclosure provides a DMD polypeptide encoded by this DMD mRNA. In certain embodiments, the generation of such DMD polypeptides partially recovers in some embodiments and fully recovers one or more DMD functions in some embodiments.

[0047] WVE-N531 can induce exon 53 skipping and dystrophin restoration. It is reported that approximately 8% of DMD subjects will have a DMD gene mutation suitable for exon 53 skipping. As will be appreciated by those skilled in the art, various DMD mutations suitable for exon 53 skipping have been reported, and various techniques can be used to determine whether a mutation is suitable for exon 53 skipping. In some embodiments, DMD mutations, such as Δ3-52, Δ4-52, Δ5-52, Δ6-52, Δ9-52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17 -52, Δ19-52, Δ21-52, Δ23-52, Δ24-52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32-52, Δ3 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 Δ51-52. In some embodiments, the mutation comprises Δ52. In some embodiments, the mutation is Δ52. In some embodiments, WVE-N531 mediates efficient exon 53 skipping of DMD to restore the reading frame, thereby producing a shorter but at least partially functional dystrophin protein - the resulting dystrophin protein is not necessarily a "wild type" form of dystrophin protein, but rather a truncated but functional form of dystrophin protein that provides an enhanced level of one or more dystrophin protein functions compared to the absence of WVE-N531 / exon 53 skipping.

[0048] In some embodiments, WVE-N531 provides high muscle concentrations, e.g., in various muscles, e.g., 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 of about 6 weeks after the initial dose and / or after about 3 doses, wherein each dose is about 10 mg / kg WVE-N531. In some embodiments, WVE-N531 provides exon 53 skipping, for example, at a level of about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, etc. In some embodiments, exon skipping is provided at a time point of about 6 weeks after the initial dose and / or after about 3 doses, wherein each dose is about 10 mg / kg WVE-N531 (which is a significantly lower dose than grodisen and / or vitrolaxan).

[0049] In some embodiments, the disclosure provides methods comprising using WVE-N531 to induce exon 53 skipping in muscle cells DMD. In some embodiments, the disclosure provides methods comprising using WVE-N531 to increase the level of functional dystrophin in muscle cells. This can be used to prevent and treat muscular dystrophy, especially those forms of muscular dystrophy that are suitable for exon 53 skipping. Among other things, the methods described herein provide improved treatment options for subjects with muscular dystrophy and have significant and practical advantages over alternative methods (e.g., using other oligonucleotide treatment methods).

[0050] In some embodiments, the disclosure relates to improved methods for treating muscular dystrophy by inducing exon skipping in a subject. In some embodiments, exon 53 skipping in a subject's DMD is induced by administering an effective amount of WVE-N531, as described herein. In some embodiments, the disclosure relates to improved methods for treating muscular dystrophy by restoring / increasing the level of a dystrophin polypeptide and / or its activity (e.g., restoring / increasing the level of a functional dystrophin) in a subject.

[0051] In some embodiments, the present disclosure provides a method for treating DMD in a subject having a DMD gene mutation suitable for exon 53 skipping, the method comprising administering an effective dose of WVE-N531 (optionally as part of a composition (e.g., a pharmaceutical formulation or dosage form)) at a suitable frequency (e.g., every week, every two weeks, every three weeks, every four weeks, every five weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks), e.g., equivalent to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg of WVE-N531 free acid form.

[0052] In some embodiments, the present disclosure provides a method for treating DMD in a subject having DMD, wherein the subject has a DMD gene mutation suitable for exon 53 skipping, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

[0053] In some embodiments, the present disclosure provides a method for providing DMD exon skipping in a subject, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for increasing the level of exon 53 skipped DMD transcripts in a subject, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the present disclosure provides a method for restoring the DMD RNA reading frame in a subject, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the disclosure provides a method for increasing the level of a truncated DMD polypeptide in a subject, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the disclosure provides a method for providing an increased level of DMD function in a subject, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose is independently equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. In some embodiments, the truncated DMD polypeptide is encoded by a DMD mRNA with exon 53 skipping.In some embodiments, such mRNA may lack one or more additional exons in addition to exon 53. In some embodiments, the subject has a DMD gene mutation suitable for exon 53 skipping. In some embodiments, the subject suffers from or is susceptible to muscular dystrophy, such as DMD. In some embodiments, each dose of WVE-N531 is independently administered in a pharmaceutical composition independently comprising WVE-N531 and a pharmaceutically acceptable carrier, wherein 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 WVE-N531 free acid form.

[0054] WVE-N531 can be administered in a variety of forms. For example, in many embodiments, WVE-N531 is administered as a salt form. In some embodiments, a single form is present in the composition. In some embodiments, a single form is present in the dosage. In some embodiments, the form is a pharmaceutically acceptable salt form. In some embodiments, the form is WVE-N531 hexasodium salt. In some embodiments, two or more forms are present in the composition. In some embodiments, two or more forms are present in the dosage. In some embodiments, each of the two or more forms is independently a pharmaceutically acceptable salt form. In some embodiments, one of them is WVE-N531 hexasodium salt. In some embodiments, the composition is a liquid composition comprising WVE-N531 dissolved, for example, in phosphate buffered saline. In some embodiments, one or more cation types are present in the solution. In some embodiments, each cation 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 + .

[0055] 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 dosage is about 14 mg / kg. In some embodiments, the dosage is about 15 mg / kg. In some embodiments, the dosage is about 16 mg / kg. In some embodiments, the dosage is about 17 mg / kg. In some embodiments, the dosage is about 18 mg / kg. In some embodiments, the dosage is about 19 mg / kg. In some embodiments, the dosage is about 20 mg / kg.

[0056] In some embodiments, the dose is administered as a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is a solution of WVE-N531 as described herein, for example in a buffer. In some embodiments, the pharmaceutically acceptable carrier is isotonic. In some embodiments, the pharmaceutical composition is a solution of WVE-N531 in phosphate buffered saline, as described herein.

[0057] 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 and a pharmaceutically acceptable carrier, as described herein. In some embodiments, two or more doses are approximately the same (mg / kg). In some embodiments, each dose is approximately the same (mg / kg). In some embodiments, the pharmaceutical composition administered with two or more doses is approximately the same in terms of components and their relative amounts. In some embodiments, the pharmaceutical composition administered with all doses is approximately the same in terms of components and their relative amounts. In some embodiments, one or more earlier doses are independently lower than one or more later doses. In some embodiments, one or more earlier doses are independently higher than one or more later doses.

[0058] In some embodiments, the dose is administered about weekly or about every 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks or about every 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 months. In some embodiments, WVE-N531 is administered about every two weeks, wherein each dose is administered independently, as described herein. In some embodiments, WVE-N531 is administered about every four weeks, wherein each dose is administered independently, as described herein. In some embodiments, WVE-N531 is administered about every four weeks or more weeks, wherein each dose is administered independently, as described herein. In some embodiments, each dose interval (the time between a dose and its previous or subsequent dose) is approximately the same. In some embodiments, one or more earlier dose intervals are independently longer than one or more later dose intervals. In some embodiments, one or more earlier dose intervals are independently shorter than one or more later dose intervals.

[0059] In some embodiments, "about" is + / - 1% (99% to 101% of a value). In some embodiments, "about" is + / - 2%. In some embodiments, "about" is + / - 3%. In some embodiments, "about" is + / - 4%. In some embodiments, "about" is + / - 5%. In some embodiments, "about" is + / - 6%. In some embodiments, "about" is + / - 7%. In some embodiments, "about" is + / - 8%. In some embodiments, "about" is + / - 9%. In some embodiments, "about" is + / - 10%.

[0060] Unless otherwise stated, amounts, concentrations, doses, etc. of WVE-N531 are in the free acid form.

[0061] Quantities, concentrations, dosages, etc. are typically corrected for purity. In some embodiments, purity is measured based on area % by IP-RP-UPLC as described herein.

[0062] Vyondys 53 TM (grolodisan) is an oligonucleotide of the phosphorodiamidate morpholino oligomer (PMO) subclass designed to increase dystrophin production via exon skipping. Grolodisan was approved in the United States in 2019 for the treatment of patients with a confirmed DMD gene mutation 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 TM(Vitolasen) is another PMO subclass exon-skipping ASO that was approved in the United States in 2020 for the treatment of DMD patients who are suitable for exon 53 skipping. Approval was also based on the observation of increased production of dystrophin, 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 grodisen and vitolasen were approved under the US accelerated approval mechanism, which is based on changes in surrogate endpoints of dystrophin production, and at the time of approval, clinical benefit of both drugs had not been demonstrated in DMD patients. Continued approval in the United States is contingent upon demonstration of clinical benefit in confirmatory trials. At the time of this invention, neither drug has been approved in the UK or the European Union (EU).

[0063] Among other things, the provided technology offers a variety of advantages over grodisen and / or vitolase, such as lower single doses, lower total doses, less frequent administration, improved exon 53 skipping in DMD, higher levels of functional DMD polypeptides, improved clinical outcomes, etc. Dystrophin

[0064] In some embodiments, the dystrophin (DMD) gene or its product, or a variant or portion thereof, may be referred to as DMD, BMD, CMD3B, DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270, DXS272, MRX85, or dystrophin; External ID: OMIM: 300377MGI: 94909; Homologous gene: 20856; Gene Card: DMD; In humans: Entr ez:1756;Ensembl:ENSG00000198947;UniProt:P11532;RefSeq(mRNA):NM_000109;NM_004006;NM_004007;NM_004009;NM_004010;RefSeq(protein):NP_000100;NP_003997;NP_004000;NP_004001;NP_004002;Location(UCSC):Chr X: 31.1-33.34 Mb; in mouse: Entrez: 13405; Ensembl: ENSMUSG00000045103; UniProt: P11531; RefSeq (mRNA): NM_007868; NM_001314034; NM_001314035; NM_001314036; NM_001314037; RefSeq (protein): NP_001300963; NP_001300964; NP_001300965; NP_001300966; NP_001300967; Position (UCSC): ChrX: 82.95-85.21 Mb.

[0065] Dystrophin is reported to be present in muscle cells and is essential for strengthening and protecting muscle fibers. Normal dystrophin is reported to be part of a protein complex called the dystrophin-glycoprotein complex, which, according to various reports, provides structural stability to skeletal muscle and protects the muscle from damage during contraction and relaxation. In addition, dystrophin is reported to be essential for cell survival via transmembrane signaling functions and regulating vasomotion in response to physical activity.

[0066] It is reported that the DMD gene contains 79 exons, which are distributed in a genetic real estate of more than 2.3 million bp on the X chromosome; however, it is reported that only about 14,000 bp (<1%) are used to translate into protein (coding sequence). It is reported that about 99.5% of the genetic sequence (i.e., intron sequence) is spliced ​​from the initial heteronuclear RNA transcript of 2.3 million bp to provide mature 14,000 bp mRNA, which includes all the key information for the production of dystrophin. In some embodiments, the subject with DMD has one or more mutations in the DMD gene, which prevent the appropriate construction of wild-type DMD mRNA and / or the production of wild-type dystrophin, and the subject with DMD often shows significant dystrophin deficiency in their muscles.

[0067] In some embodiments, the dystrophin transcript (e.g., mRNA or protein) encompasses those associated with or produced by alternative splicing. For example, after analyzing the splicing pattern of the DMD gene in skeletal muscle, brain, and heart tissue, at least sixteen alternative transcripts of the dystrophin gene were reported.

[0068] It is reported that dystrophin has several isoforms. In certain embodiments, dystrophin refers to a specific isoform. At least three full-length dystrophin isoforms have been reported, each controlled by a tissue-specific promoter. It is reported that the muscle isoform is mainly expressed in skeletal muscle, but is also expressed in smooth muscle and cardiac muscle, it is reported that brain dystrophin has specificity to cortical neurons, but can also be detected in cardiac and cerebellar neurons, and it is reported that Purkinje cell types account for almost all cerebellar dystrophin. It is reported that alternative splicing provides a means of dystrophin diversification: it is reported that the 3' region of the gene has undergone alternative splicing, which produces tissue-specific transcripts in brain neurons, cardiac Purkinje fibers, and smooth muscle cells, and 12 alternative splicing patterns have been reported in the 5' region of the skeletal muscle gene.

[0069] In some embodiments, the dystrophin mRNA, gene or protein is a revertant version.

[0070] Various mutations in the DMD gene can and / or have been reported to cause muscular dystrophy. Mutations in the form of large deletions (1 or more exons) have been reported to account for approximately two-thirds of all DMD gene mutations; the remainder have been reported to be due to duplications and small deletions, insertions, point mutations, or splicing mutations. According to some reports, deletions are often clustered in a hotspot region between exons 45 and 55 and prevent translation of dystrophin. It has been reported that dystrophin loss or defects due to DMD gene mutations disrupt the dystrophin-glycoprotein complex, leading to increased muscle membrane fragility, chronic muscle damage, inflammation, replacement of muscle fibers with fat and fibrotic tissue, and then loss of muscle function. Muscular dystrophy

[0071] Muscular dystrophy (MD) is any of a group of muscle disorders, diseases or disorders that, over time, can lead to (increase) skeletal muscle weakness and breakdown. The disorder, disease or disorder that primarily affects the muscles, the degree of weakness when symptoms begin, and the speed at which symptoms worsen. Many MD subjects will eventually be unable to walk. In many cases, muscular dystrophy is fatal. Some types are also associated with problems with other organs (including the central nervous system). In some embodiments, muscular dystrophy is Duchenne muscular dystrophy (DMD). In some embodiments, muscular dystrophy Becker muscular dystrophy (BMD).

[0072] In some embodiments, symptoms of Duchenne muscular dystrophy are weakness associated with muscle wasting, with voluntary muscles being affected first, especially those in the buttocks, pelvic area, thighs, shoulders, and calves. Muscle weakness can also occur in the arms, neck, and other areas. The calves are often swollen. Symptoms usually appear before the age of six and may appear in early infancy. Other physical symptoms are: awkward way of walking, stepping or running (in some cases, subjects tend to walk on their front feet due to increased calf muscle tone), frequent falls, fatigue, difficulty with motor skills (e.g., running, hopping on one foot, hopping on both feet), excessive lumbar lordosis, which may lead to shortened hip flexors, unusual overall posture and / or way of walking, stepping or running, muscle contractures of the Achilles tendon and hamstrings that impair function, progressive difficulty walking, muscle fiber deformities, pseudohypertrophy (enlargement) of the tongue and calf muscles, high risk of neurobehavioral disorders (e.g., ADHD), learning disabilities (e.g., dyslexia), and non-progressive weaknesses in specific cognitive skills (e.g., short-term verbal memory), believed to be a result of a lack of dystrophin or dystrophin function in the brain, eventual loss of the ability to walk (usually by age 12), skeletal deformities (including scoliosis in some cases), and trouble getting up from a lying or sitting position. In some embodiments, the technology provided delays the onset of one or more symptoms, slows the progression of one or more symptoms, reduces the severity of one or more symptoms, and / or prevents one or more symptoms.

[0073] In some embodiments, Becker muscular dystrophy (BMD) is caused by mutations that produce shortened but in-frame transcripts, resulting in a truncated but partially functional protein or proteins. Such partially functional proteins or proteins reportedly retain key amino-terminal, cysteine-rich and C-terminal domains, but typically lack elements of the central rod domain, which is reported to be of less functional significance. England et al. 1990 Nature, 343, 180-182.

[0074] In some embodiments, the BMD phenotype ranges from mild DMD to virtually asymptomatic, depending on the precise mutation and the level of dystrophin produced. Yin et al. 2008 Hum. Mol. Genet. 17:3909-3918.

[0075] In some embodiments, dystrophic subjects with an out-of-frame mutation are generally diagnosed as having a more severe form of Duchenne muscular dystrophy, and dystrophic subjects with an in-frame mutation are generally diagnosed as having a less severe form of Becker muscular dystrophy. Exon skipping as a treatment for muscular dystrophy

[0076] Exon skipping can induce cellular machinery to skip one or more targeted exons and restore the reading frame, thereby generating an internally truncated but functional dystrophin polypeptide. In various exon skipping approaches, oligonucleotides can bind to target complementary sequences in messenger RNA (mRNA), which engage the splicing machinery to exclude the exon from the final transcript. In DMD subjects, this approach may slow disease progression by converting severe DMD symptoms to milder symptoms, such as seen in Becker muscular dystrophy (BMD) patients, where patients can walk longer and have a longer life expectancy.

[0077] Various DMD genotypes are suitable for exon skipping, including exon 53 skipping. Exon skipping can restore the DMD mRNA reading frame, thereby producing a shorter but functional dystrophin. Exon 53 skipping is applicable to, but not limited to, patients with DMD gene deletions with exons 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, 51-52 or exon 52 alone. According to some reports, approximately 8% to 10% of DMD patients can be treated by exon 53 skipping. In a patient cohort with deletions suitable for skipping exons 44, 45, 51 or 53, 36 months of longitudinal natural history data showed that, compared with other subgroups, patients who were particularly suitable for exon 53 skipping seemed to begin to decline earlier and had a higher risk of severe functional loss (e.g., loss of walking).

[0078] In some embodiments, exon skipping restores or maintains the correct reading frame and / or creates an artificial internally truncated DMD protein that provides at least partially improved or fully restored biological activity. In some embodiments, an internally truncated DMD protein produced by a dystrophin transcript with one or more skipped exons is more functional than a terminally truncated DMD protein (e.g., produced by a dystrophin transcript with an out-of-frame deletion).

[0079] In some embodiments, an internally truncated DMD protein produced from a dystrophin transcript with one or more skipped exons is more resistant to nonsense-mediated decay, which can degrade a terminally truncated DMD protein (e.g., produced from a dystrophin transcript with an out-of-frame deletion).

[0080] In some embodiments, the DMD genotype of the DMD subject is analyzed, and it is confirmed that the subject has a DMD gene mutation suitable for exon 53 skipping before WVE-N531 administration. In some embodiments, the DMD gene mutation suitable for exon 53 skipping is or includes one or more nucleobase changes (e.g., replaced by a different nucleobase), insertions and / or deletions, etc., wherein exon 53 skipping can produce a DMD polypeptide that can provide improved function compared to not having exon 53 skipping.

[0081] Prior to this disclosure, (Deflazacort) is a corticosteroid approved for the treatment of DMD in the United States. Emflaza showed improved muscle strength (as measured by the modified Medical Research Council scale, with an average change of 0.15 points) after a 12-week treatment period at a dose of 0.9 mg / kg / day. In a long-term placebo-controlled study in children with DMD aged 6 to 12 years, deflazacort had no significant effect on the average muscle strength score at 2 years. Although Emflaza is not approved for the treatment of DMD in the European Union (EU), in the EU, corticosteroids such as deflazacort are often used off-label in DMD patients to slow the decline in muscle strength and prolong walking and respiratory function. However, long-term use of corticosteroids is associated with serious side effects, such as increased risk of immunosuppression and infection, changes in endocrine function, hypertension, cataracts, bone demineralization, gastrointestinal perforation, and growth retardation in children. In addition, corticosteroids cannot correct the underlying genetic defects of DMD.

[0082] Vyondys 53 TM (grolodisan) is an oligonucleotide of the phosphorodiamidate morpholino oligomer (PMO) subclass designed to increase dystrophin production via exon 53 skipping. Grolodisan was approved in the United States in 2019 for the treatment of patients with a confirmed DMD gene mutation 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 TM(Vitolasen) is another PMO subclass exon skipping oligonucleotide that 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 the observation of increased production of dystrophin, 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 grodisen and vitolasen were approved under the US accelerated approval mechanism, which is based on an increase in the surrogate endpoint of dystrophin production, but neither drug has been shown to provide clinical benefit to patients with DMD. Continued approval in the United States is contingent upon demonstration of clinical benefit in confirmatory trials. Prior to this disclosure, neither drug had been approved in the United Kingdom (UK) or the EU.

[0083] Likewise, some patients may have mutations that make them suitable for exon 53 skipping oligonucleotides and translarna TM (ataluren) or Exondys (eteplirsen) treatment. Ataluren is a small molecule designed to enhance ribosome readthrough of nonsense mutations, which is not approved for use in the United States (US). It has received conditional marketing approval in the EU for the treatment of DMD caused by nonsense mutations in the dystrophin gene in ambulatory patients aged 5 years or older. The clinical efficacy of ataluren has not been established in non-ambulatory patients, and additional evidence is needed to support its continued approval in the EU. Given the low incidence of nonsense mutations and the limited indications of ataluren for ambulatory patients, it is expected that the use of ataluren will be limited to a small proportion of the DMD population. Eteplirsen is an oligonucleotide of the PMO subclass designed to increase the production of dystrophin via exon skipping. Eteplirsen is approved in the United States for the treatment of patients with confirmed DMD gene mutations suitable for exon 51 skipping. Approval was based on the detection of a median dystrophin level of 0.93% of that in normal muscle and a median increase in truncated dystrophin expression of 0.1% in the only study that could estimate dystrophin expression in response to eplastin treatment. Prior to this disclosure, the drug had not been approved in the EU.

[0084] Grodison's plasma C maxand the area under the plasma concentration-time curve (AUC) increased proportionally with increasing doses, and the half-life was approximately 3 hours, with no observed plasma accumulation. Based on Phase 1 studies of viltorazan, urinary excretion rates indicated that 70% to 80% of the total administered drug was cleared within 24 hours, indicating a short half-life (<24 hours). Renal function was within normal range in all patients. Epleren was rapidly cleared from plasma, with the majority of drug clearance occurring within 24 hours. The mean concentration of Epleren was the maximum (peak) drug concentration in plasma (C max ) is 0.07%. Renal clearance of iplesen accounts for more than 60% of the administered dose, and its terminal half-life (t 1 / 2 ) was 3 to 4 hours. No accumulation of epleren was observed during once-weekly dosing.

[0085] Among other things, the present disclosure provides techniques for treating DMD, inducing DMD exon 53 skipping, increasing the level of DMD transcripts that skip exon 53, producing or increasing the level of truncated DMD polypeptides that skip exon 53, increasing the level of one or more DMD functions, and / or restoring one or more DMD functions, etc., comprising administering WVE-N531 or a composition thereof according to the present disclosure to a subject, wherein the subject has a mutant DMD that is suitable for skipping exon 53. In some embodiments, the provided techniques provide various advantages over one or more currently approved drugs, such as improved safety, tolerability, clearance, accumulation, exon skipping, clinical effects, and / or one or more other pharmacokinetic or pharmacodynamic aspects. WVE-N531

[0086] WVE-N531 can be described as (5' to 3' unless otherwise stated, as understood by the skilled artisan): fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC m: 2'-OMe modified nucleoside; f: 2'-F modified nucleoside; *S: Sp phosphorothioate linkage; and n001R: Rp N-(1,3-dimethylimidazolidine-2-vinylidene)phosphoramide linkage.

[0087] WVE-N531 can be named using generally accepted oligonucleotide nomenclature: (Sp)-2'-Fluoro-2'-deoxy-P-thio-cytidylyl-(O 3 '→O 5')-(Sp)-2'-fluoro-2'-deoxy-P-thio-uridyl-(O 3 '→O 5 ')-(Rp)-2'-fluoro-2'-deoxy-P-((1,3-dimethylimidazolidine-2-vinylene)amino)-cytidine-(O 3 '→O 5 ')-(Sp)-2'-fluoro-2'-deoxy-P-thio-cytidylyl-(O 3 '→O 5 ')-(Sp)-2'-fluoro-2'-deoxy-P-thio-guanylate-(O 3 '→O 5 ')-(Rp)-2'-fluoro-2'-deoxy-P-((1,3-dimethylimidazolidine-2-vinylene)amino-guanylyl-(O 3 '→O 5 ')-(Sp)-2'-fluoro-2'-deoxy-P-thio-uridyl-(O 3 '→O 5 ')-(Sp)-2'-fluoro-2'-deoxy-P-thio-uridyl-(O 3 '→O 5 ')-2'-O-methylcytidyl-(O 3 '→O 5 ')-(Sp)-2'-fluoro-2'-deoxy-P-thio-uridyl-(O 3 '→O 5 ')-(Sp)-2'-O-methyl-P-thio-guanylate-(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-guanylate-(O 3 '→O 5 ')-(Sp)-2'-fluoro-2'-deoxy-P-thio-guanylate-(O 3 '→O 5 ')-(Sp)-2'-fluoro-2'-deoxy-P-thio-uridyl-(O 3 '→O 5 ')-(Rp)-2'-fluoro-2'-deoxy-P-((1,3-dimethylimidazolidine-2-vinylene)amino)-guanylyl-(O 3 '→O 5')-(Sp)-2'-fluoro-2'-deoxy-P-thio-uridyl-(O 3 '→O 5 ')-(Sp)-2'-fluoro-2'-deoxy-P-thio-uridyl-(O 3 '→O 5 ')-2'-fluoro-2'-deoxycytidine.

[0088] In some embodiments, WVE-N531 is provided in a composition (e.g., a pharmaceutical composition) and / or is administered as one or more pharmaceutically acceptable salts. In some embodiments, the disclosure provides a pharmaceutically acceptable salt of WVE-N531. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the disclosure provides WVE-N531 hexasodium salt. In some embodiments, the WVE-N531 drug substance is WVE-N531 hexasodium salt.

[0089] The molecular formula and molecular weight of WVE-N531 and its hexadecasodium salt are as follows: 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 (hexadecanoic acid salt form): C 206 H 234 F 17 N 78 O 109 P 19 S 14 Na 16 Molecular weight (hexadecanoic acid salt form): 7274.73 g / mol

[0090] WVE-N531 has 17 stereo-defined internucleotide linkages, 14 of which were identified as Sp phosphorothioate linkages and 3 as Rp N-(1,3-dimethylimidazolidine-2-vinylidene)phosphoramide linkages. Its internucleotide linkages can be represented as: 5'-SSRSSRSSOSSSOSSSRSS-3', where "S", "R" and "O" represent Sp phosphorothioate linkages, Rp N-(1,3-dimethylimidazolidine-2-vinylidene)phosphoramide linkages and phosphate linkages, respectively. The N-(1,3-dimethylimidazolidine-2-vinylidene)phosphoramide linkage can be referred to as n001 linkage.

[0091] The structure of WVE-N531 can be described as Formula I:

[0092] WVE-N531 hexadecasodium salt can be described as Formula Ia:

[0093] In Formulas I and Ia, due to the size of the structure, the structure is presented as 4 rows of five nucleoside units each. As shown in these formulas, each linkage in WVE-N531 is independently a Sp phosphorothioate linkage, an Rp n001 linkage, or a natural phosphate linkage, while in the WVE-N531 salt, the acidic hydrogen atoms of the phosphorothioate and phosphate linkages can be independently substituted with cations, for example, in the hexadecanoic acid salt, each is substituted with sodium.

[0094] Aqueous solutions of WVE-N531 hexadecasodium salt are typically clear, colorless to pale yellow solutions, and may have a concentration of about 150 mg / mL or higher, as determined by UV. In some embodiments, the pH of WVE-N531 hexadecasodium salt in purified water ranges from 6.0-8.0.

[0095] The extinction coefficient of WVE-N531 at 260nm is 186,486M -1 cm -1 (Measured in water). Unless otherwise stated, when measuring the concentration / amount of WVE-N531 using UV, 260 nm will be used and this value will be used in calculations. A molecular weight of 6918.72 g / mol (free acid form) was used, which corresponds to an absorption coefficient of 27.0 OD / mg.

[0096] In some embodiments, WVE-N531 is provided as a diluted isotonic solution 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 a period of about 1 hour. In some embodiments, the infusion is extended to about 3 hours, for example, to improve tolerability. In some embodiments, the diluted infusion solution of WVE-N531 is preservative-free and is administered immediately or within four hours after dilution. In some embodiments, 0.45% sodium chloride injection is used for dilution. In some embodiments, 0.9% sodium chloride injection is used for dilution.

[0097] In some embodiments, WVE-N531 is provided and administered as a chirality-controlled composition, wherein WVE-N531 is enriched compared to the other diastereomer.

[0098] Among other things, the present disclosure provides a high purity WVE-N531. In some embodiments, the purity level of WVE-N531 is about 70%-90%, such as 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, the purity level is about 70% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 71% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 72% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 73% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 74% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 75% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 76% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 77% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 78% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 79% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 80% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 81% or more as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 82% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 83% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 84% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 85% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 86% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 87% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 88% as determined by IP-RP-UPLC as described herein.In some embodiments, the purity level is about 89% as determined by IP-RP-UPLC as described herein. In some embodiments, the purity level is about 90% or more as determined by IP-RP-UPLC as described herein. In some embodiments, IP-RP-UPLC utilizes Group A parameters. In some embodiments, purity is the area % at a given wavelength (e.g., about 260 nm).

[0099] In some embodiments, the impurity level is about 10%-30% or less. In some embodiments, the impurity level is about 10%-30%. In some embodiments, the impurity level is about 15%-30%. In some embodiments, the impurity level is about 20%-30%. In some embodiments, the impurity level is about 30% or less. In some embodiments, the impurity level is about 29% or less. In some embodiments, the impurity level is about 28% or less. In some embodiments, the impurity level is about 27% or less. In some embodiments, the impurity level is about 26% or less. In some embodiments, the impurity level is about 25% or less. In some embodiments, the impurity level is about 24% or less. In some embodiments, the impurity level is about 23% or less. In some embodiments, the impurity level is about 22% or less. In some embodiments, the impurity level is about 21% or less. In some embodiments, the impurity level is about 20% or less. In some embodiments, the impurity level is about 19% or less. In some embodiments, the impurity level is about 18% or less. In some embodiments, the impurity level is about 17% or less. In some embodiments, the impurity level is about 16% or less. In some embodiments, the impurity level is about 15% or less. In some embodiments, the impurity level is about 14% or less. In some embodiments, the impurity level is about 13% or less. In some embodiments, the impurity level is about 12% or less. In some embodiments, the impurity level is about 11% or less. In some embodiments, the impurity level is about 10% or less. In some embodiments, the impurity level is assessed using IP-RP-UPLC. In some embodiments, IP-RP-UPLC utilizes Group A parameters. In some embodiments, purity is the area % at a given wavelength (e.g., about 260nm).

[0100] In some embodiments, the present disclosure provides various techniques for characterizing WVE-N531 preparations. In some embodiments, the present disclosure provides techniques for assessing the purity of WVE-N531 or compositions thereof.

[0101] In some embodiments, the disclosure provides techniques for making WVE-N531 or compositions thereof. In some embodiments, the disclosure provides techniques for releasing WVE-N531 formulations. manufacture

[0102] In some embodiments, the disclosure provides techniques for making WVE-N531 or a salt thereof (e.g., WVE-N531 hexasodium salt). In some embodiments, the disclosure provides techniques for making WVE-N531 drug substances. In some embodiments, the disclosure provides techniques for making pharmaceutical compositions comprising WVE-N531 or a pharmaceutically acceptable salt form thereof and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides WVE-N531 drug products.

[0103] In some embodiments, WVE-N531 or a salt thereof, such as WVE-N531 hexadecasodium salt, is chemically manufactured in accordance with cGMP regulations.

[0104] In some embodiments, provided techniques include multi-step methods that include solid phase oligonucleotide synthesis, cleavage of crude protected oligonucleotides from a solid support, removal of protecting groups (deprotection), preparative anion exchange (AEX) chromatography purification, concentration and / or desalting.

[0105] In some embodiments, the stereochemistry of WVE-N531 or a salt thereof is established by controlling the starting materials and the synthetic process of the synthesis. For example, in some embodiments, the provided techniques include using phosphoramidites prepared from chiral auxiliaries (L)-DPSE and (L)-PSM during the coupling step to ensure that the desired stereo-defined Sp phosphorothioate linkage and Rp n001 linkage are obtained, respectively.

[0106] The following describes by way of example a method and certain results that can be used to make the hexadecasodium salt of WVE-N531 as a drug substance. Available manufacturing methods

[0107] In some embodiments, the manufacturing method consists of: synthesizing oligonucleotides on a solid support using phosphoramidite chemistry, the oligonucleotides containing a combination of three internucleotide linkages (phosphodiester, stereo-defined phosphorothioate and stereo-defined n001), as shown in the synthesis cycle below. Each cycle of introducing stereo-defined phosphorothioate or stereo-defined n001 consists of: 5'-detritylization, coupling, end-capping of exposed chiral auxiliary secondary amines (end-capping-1), correspondingly thiolation or imidization and end-capping of unreacted 5'-hydroxyl groups (end-capping-2). Each cycle of introducing phosphodiester consists of: detritylization, coupling, oxidation and end-capping-2. Each coupling reaction is carried out by activating the appropriate phosphoramidite and reacting with the free 5'-hydroxyl group of the protected nucleotide or oligonucleotide fixed to the support. After an appropriate number of cycles and final detritylation, the (L)-PSM chiral auxiliary and cyanoethyl phosphate protecting groups are removed from the crude oligonucleotides by on-column treatment with diethylamine (DEA) in acetonitrile (ACN). The (L)-DPSE chiral auxiliary is then removed by treatment with a NEt3·HF (triethylamine hydrogen fluoride) solution. The crude oligonucleotides are then cleaved from the solid support and removed from the column by ammonia treatment. The base protecting groups are removed by continuous ammonia treatment in a reaction vessel of appropriate size. The resulting crude oligonucleotides are purified using preparative anion exchange (AEX) high performance liquid chromatography (HPLC), followed by concentration and desalting to produce WVE-N531 or a salt thereof, such as WVE-N531 hexasodium salt, as a drug substance in some embodiments. Available cycles for phosphorothioate assembly Available cycles for n001 assembly Available cycles for phosphate assembly

[0108] The steps for manufacturing the WVE-N531 drug substance are described in further detail below. Stage 1: Synthesis

[0109] Oligonucleotide synthesis was performed on an automated oligonucleotide synthesizer on a controlled pore glass (CPG) solid support functionalized with 5'-ODMTr-2'-F-dC(N4-Ac). All reactions took place on the solid support of the column.

[0110] 1. Detritylation

[0111] To begin the synthesis, the CPG-5'-ODMTr-2'-F-dC(N4-Ac) solid support was subjected to acid-catalyzed removal of the DMTr protecting group from the 5'-hydroxyl group by treatment with 3% dichloroacetic acid (DCA) in toluene. Complete removal of DMTr was ensured by online UV monitoring based on the watch command in the synthesis program. DMTr removal was performed in the same manner at the beginning of each synthesis cycle and after the final cycle. In each case, after detritylation, the support-bound material was washed with acetonitrile in preparation for the next step of the synthesis.

[0112] 2. Coupling

[0113] Extension of the growing oligonucleotide chain is achieved by reacting the 5'-hydroxyl group of the support-bound oligonucleotide with an excess solution of protected phosphoramidites in the presence of an activator (5-(ethylthio)-1H-tetrazole (ETT) or 1-cyanomethylimidazolium trifluoromethanesulfonate (CMIMT) dissolved in acetonitrile). The phosphoramidites are dissolved in acetonitrile or an 80:20 v / v acetonitrile:isobutyronitrile mixture. The phosphoramidites required for each coupling step are determined by the oligonucleotide sequence. To introduce phosphate linkages, phosphoramidites and ETT are used. To introduce stereo-defined phosphorothioates or n001 linkages, (L)-DPSE or (L)-PSM derived phosphoramidites and CMIMT are used. In either case, the phosphoramidite / activator solutions are mixed online, pushed onto the synthesis column, and then circulated on the column for an appropriate amount of time. Subsequently, the excess reagents are removed by rinsing the synthesis column with acetonitrile.

[0114] 3. Capping-1

[0115] Capping 1 protects the secondary amine generated by the ring opening of the oxazophosphine on the auxiliary group during the coupling step from reacting in the remainder of the synthesis. The secondary amine was capped by passing a flowing capping reagent B ([Cap B]: acetic anhydride / 2,6-lutidine / acetonitrile (20:30:50, v:v:v)) through the synthesis column. Excess reagent was flushed from the synthesis column with acetonitrile.

[0116] 4. Oxidation, thiolation or imidization

[0117] The newly created P(III) phosphite triester linkage is processed in one of three ways: 1. Oxidation by treatment with iodine in pyridine:water (90:10, v:v) resulted in the formation of P(V) phosphodiester linkages. 2. Thiolation by treatment with xanthane hydride (XH) in pyridine:acetonitrile (1:1, v:v) resulted in a stereodefined P(V) phosphorothioate triester linkage. 3. Imidization by treatment with 2-azido-1,3-dimethylimidazolium hexafluorophosphate (ADIH) in acetonitrile results in the formation of stereodefined P(V)phosphoimidate triester linkages.

[0118] In each case, after the step, the synthesis column was flushed of excess reagents with acetonitrile.

[0119] 5. Capping-2

[0120] Typically, the coupling reaction will proceed with very high yields, but the yields are not quantitative. A small fraction of the 5'-hydroxyl groups available in any given cycle may not be coupled to the activated phosphoramidite. To prevent reaction in subsequent cycles, these sites are blocked by capping with a reagent mixture (1:1, v:v) (capping reagent A [Cap A]: N-methylimidazole / acetonitrile (20:80, v:v) and Cap B). Thus, a 5'-O-acetylated ("capped") support-bound oligonucleotide sequence is formed. Excess capping reagent is washed from the column with acetonitrile.

[0121] Oligonucleotide assembly and final steps

[0122] The above three cycles are repeated independently to assemble the complete protected sequence using the appropriate protected phosphoramidites, wherein the DMTr protecting group is present at the 5'-terminal position. After the last nucleotide in the sequence is added, the 5'-terminal DMTr group is removed during a final detritylation step. Stage 2: Fragmentation and deprotection

[0123] A three-step process was used to remove phosphate protecting groups and (L)-PSM and (L)-DPSE chiral auxiliaries from stereodefined phosphorimidates and phosphorothioate triesters, cleave crude oligonucleotides from solid supports, and remove exocyclic nucleobase protecting groups.

[0124] Step 1: Removal of the (L)-PSM chiral auxiliary and cyanoethyl groups using diethylamine treatment

[0125] The crude oligonucleotides on the solid support are treated with a solution of diethylamine in ACN, which can simultaneously remove the cyanoethyl protecting group from the phosphotriester linkage and the (L)-PSM chiral auxiliary from the phosphorimidate, generating phosphodiester linkages and phosphoramidate diester linkages, respectively.

[0126] Step 2: Removal of the (L)-DPSE chiral auxiliary using fluoride treatment

[0127] The crude oligonucleotides on the solid support are treated with a temperature-controlled solution of NEt3·3HF in a mixture of dimethyl sulfoxide (DMSO), NEt3, and H2O to effectively remove the chiral auxiliary. This process converts the stereodefined phosphorothioate triesters into stereodefined phosphorothioate diesters, as shown above.

[0128] Step 3: Cleavage and global nucleobase deprotection

[0129] The crude oligonucleotide is then cleaved from the solid support by treatment with ammonium hydroxide in a suitably sized pressure-rated vessel. This reaction can provide overall deprotection (e.g., removal of protecting groups such as acetyl, benzoyl, isobutyryl, etc.) for the cyanoethyl phosphate group (which may have been deprotected in an earlier step) and the exocyclic amino group. In Formula P, each M + In some embodiments, each M + Yes H + (Such compounds are "acids of Formula P"). In some embodiments, each M + 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.

[0130] Typically, after deprotection, one or more WVE-N531 salts are provided. In some embodiments, the salt is an ammonium salt. In some embodiments, the deprotected product composition comprises one or more WVE-N531 salts. In some embodiments, the deprotected product composition comprises an ammonium salt of WVE-N531. Stage 3: Purification by anion exchange chromatography

[0131] Purification of crude oligonucleotide solution is completed by AEX-HPLC. Crude oligonucleotide solution is loaded onto a purification column filled with TSK-GEL Super Q-5PW culture medium. Purification run is performed using sodium hydroxide buffered eluent. Sodium chloride gradient is used to elute oligonucleotide from the column. Purification is performed at ambient temperature. The elution curve is monitored by ultraviolet (UV) spectrophotometry. Fractions are collected and simulated pools are evaluated by IP-RP-UPLC. Pools containing oligonucleotides at or above the target purity level are passed through the next step in the process. Stage 4: Concentration and desalination (final UF / DF)

[0132] The selected fraction pool is then concentrated and filtered with purified water to remove the purification buffer by tangential flow filtration (TFF) using a regenerated cellulose membrane box. The pool is concentrated and the pH is adjusted to 6.5-7.5 with hydrochloric acid or sodium hydroxide. The concentrated oligonucleotides are filtered with purified water and concentrated to a target value of 1200-1400 OD / mL, and the desalted oligonucleotides are then collected. The system is rinsed with purified water to maximize the yield, and the concentrated desalted oligonucleotides are mixed with the rinse solution to achieve a target final desalting concentration of 878±68OD / mL (32.5±2.5mg / mL). Stage 5: Filtration and Packaging

[0133] The oligonucleotide solution was filtered through a 0.2 micron filter and then filled into drug substance storage containers. The drug substance was packaged in sterile high density polyethylene (HDPE) bottles, each of which was labeled and sealed in a Mylar foil bag and stored at -20°C. Starting Materials

[0134] Some possible starting materials are listed below: Abbreviations: CoA = Certificate of Analysis; NF = National Formulary; CIP = Cleaning in Place; USP = United States Pharmacopeia; ACS = American Chemical Society

[0135] The starting materials include 9 phosphoramidites, a controlled pore glass (CPG) solid support, and 2-azido-1,3-dimethylimidazolium hexafluorophosphate. The reactive exocyclic groups on the nucleobases are appropriately protected during oligonucleotide synthesis to render them non-reactive, and the 5'-hydroxyl function is protected as 4,4'-dimethoxytrityl ether (DMTr). The purity level of the phosphoramidites is about 90%, about 95%, or in some cases about 98% or more (RP-HPLC (area %) at about 260 nm and / or 31 P NMR integration), about 97% or higher P(III) purity (by 31 P NMR integration), and a water content of less than 0.2% (w / w). The particle size of the controlled pore glass-5'-ODMTr-2'-F-dC(N4-Ac) succinate solid support was about 120-200 mesh (analytical screening) and the pore size was about (mercury intrusion method), density is about 0.20-0.24 g / cc (tapped density), pore volume ≥ about 0.8 cc / g (mercury intrusion method), surface area Hg intrusion ≥ about 75 m 2 / g (mercury porosimetry), and 70-80 μmol / g estimated by DMT ligand assay (spectrophotometry at 498 nm). The purity of 2-azido-1,3-dimethylimidazolium hexafluorophosphate is about 98.0% or more (HPLC), and the nitrogen content is about 23.70%-24.80% (elemental analysis).

[0136] Stereochemistry can be established by controlling the starting materials of the synthesis as well as the synthetic process. Stereodefined phosphoramidites are prepared from appropriately protected nucleosides and chiral auxiliaries such as (L)-DPSE or (L)-PSM. The chemical purity of stereodefined phosphoramidites can be determined by 31 P NMR determination. The configuration can be 31 P NMR, 1 H NMR and 13 Since the stereochemical configurations of the protected nucleoside and the chiral auxiliary are fixed, the coupling reaction may produce two diastereomers (trans and cis), of which the trans form is dominant and the cis form exists as a trace impurity.

[0137] During the manufacturing process, various in-process controls are applied. After completing the synthesis and cutting and deprotection stages, the presence of WVE-N531 drug substance in the obtained crude oligonucleotide is identified by LC / MS, and its purity is quantified by IP-RP-UPLC. During purification, the purity and impurities of a single fraction and a selected simulation pool are evaluated by IP-RP-UPLC. Once the selected fractions are collected and concentrated, the desalination of the resulting solution will be controlled by conductivity, pH and concentration measurements during the process. In some embodiments, the manufacture of drug substances or drug products meets one or more or all of the following standards. In some embodiments, drug substances (e.g., WVE-N531 or its salt form) have a certain level of purity, as described herein. In some embodiments, WVE-N531 hexasodium salt has a certain level of purity, as described herein. In some embodiments, WVE-N531 or its salt has a certain level of purity in a composition (e.g., a pharmaceutical composition). In some embodiments, WVE-N531 or its salt has a certain level of purity in a drug product. In some embodiments, WVE-N531 or its salt is WVE-N531 hexasodium salt. In some embodiments, the purity level of WVE-N531 is about 70%-90%, about 70%-85%, about 70%-80%, about 75%-85%, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% or more as determined by IP-RP-UPLC described herein. Characterization

[0138] Among other things, the present disclosure provides techniques for evaluating WVE-N531 or compositions thereof.

[0139] The product can be characterized by various techniques. For example, the WVE-N531 preparation can be characterized by mass spectrometry. For one preparation, electrospray ionization mass spectrometry (ESI-MS) analysis was performed, and the experimentally determined 6722Da value was consistent with the theoretical average mass (free acid form) (6723Da) of the WVE-N531 drug substance. In some embodiments, the sequence of WVE-N531 or its salt is confirmed by ESI-MS / MS sequencing. For example, in an assessment, within the 5ppm error of each calculated (expected) mass, at least one sequence-related fragment ion of 19 nucleobases in the sequence was observed. Since the monoisotopic mass of the full-length oligonucleotide is also verified in the ESI-MS / MS analysis, the identity and position of all 20 nucleobases have been demonstrated and the sequence has been confirmed.

[0140] The stereochemical properties include the combination of the absolute stereochemical configuration (Rp or Sp) of each chiral bonded phosphorus in the oligonucleotide (for each bonded phosphorus in the phosphorothioate or phosphoramide group of WVE-N531 or its salt). This combination can produce a single diastereomer, in 2 n The invention relates to a novel chiral phosphodiester. The chiral phosphodiester is unique in each of the diastereomers (n = number of chiral phosphorus bonds). In the case of WVE-N531 or its salts, this combination can be represented by 5'-SSRSSRSSOSSSOSSSRSS-3', where S, P and O represent Sp phosphorothioate bonds, Rp N-(1,3-dimethylimidazolidine-2-vinylidene)phosphoramide bonds and phosphate bonds, respectively. WVE-N531 or its salts also contain 2'-fluoro (F) and 2'-O-methyl (OMe) modifications.

[0141] The establishment of stereochemical properties includes controlling the starting materials of the synthesis. Synthetic coupling of phosphoramidites prepared from (L)-DPSE or (L)-PSM produces phosphorothioate linkages of Sp chirality and phosphoramide linkages of Rp chirality, respectively, and synthetic coupling of phosphoramidites prepared from (D)-DPSE or (D)-PSM produces phosphorothioate linkages of Rp chirality and phosphoramide linkages of Sp chirality. Consistent use of stereo-defined phosphoramidites for each coupling reaction allows control of the stereochemical properties of each phosphorothioate linkage or phosphoramide linkage.

[0142] Stereochemical properties can be confirmed using a variety of techniques in accordance with the present disclosure. For example, they can be confirmed by measuring using several different techniques (these techniques are used in combination) and by comparing with appropriate standards (which can provide accurate information and confirmation about this property). Available analytical techniques include NMR ( 1 H. 19 F. 31 P, multidimensional, etc.) and enzyme digestion. In some cases, NMR is performed in phosphate buffer (e.g., pH 7.0). 1 H. 19 F and 31 The results of P NMR and enzyme digestion of various preparations are consistent with the product structure. In some embodiments, NMR involves water based on DSS standards. The following describes the observed data of certain experiments. Preparation of WVE-N531 Drug Substance (Hexadecasodium Salt) 31 P NMR data were collected and recorded at 161.98 MHz. Preparation of WVE-N531 Drug Substance (Hexadecasodium Salt) 19F NMR data were collected and recorded at 348K.

[0143] In some embodiments, the stereochemistry established by UPLC is determined as part of a batch release using reference standards (e.g., standards characterized by NMR, enzyme digestion, etc.). In some embodiments, provided technology provides reference standards.

[0144] The FTIR spectrum of solid WVE-N531 hexadecasodium salt as drug substance was recorded. 1643 cm -1 and 1682cm -1 The main absorption bands at 3200-3300cm are consistent with the stretching vibrations of amide C=O, C=N and C=C. -1 The broad peaks in the region are also consistent with oligonucleotide functional groups.

[0145] Counterions can be analyzed according to the present disclosure.For example, the sodium content value of a WVE-N531 drug substance batch formulation has been determined by ICP-OES to be 5.1%, which is consistent with the theoretical sodium content value of 5.1% (w / w) (hexasodium salt).

[0146] The prepared WVE-N531 drug substance solution was a clear (no visible matter, essentially free of particulate matter) colorless solution. The pH of the WVE-N531 drug substance in purified water was found to be about 7.3.

[0147] The molar extinction coefficient of the WVE-N531 drug substance was determined to be 186486 M in water using multiple experiments. -1 cm -1 A molecular weight of 6918.72 g / mol (free acid form) was used, which corresponds to an absorption coefficient of 27.0 OD / mg.

[0148] 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.

[0149] The WVE-N531 drug substance formulation (including relevant impurities) was subjected to GLP evaluation in in vitro genotoxicity studies and in vivo micronucleus studies. No genotoxicity was observed.

[0150] Various formulations of the WVE-N531 drug substance already meet regulatory limits for residual solvents including acetonitrile, toluene, and pyridine (ICH Class 2) and elemental impurities including various elements of ICH Class 1, 2A, 3, etc.

[0151] During oligonucleotide synthesis, the fidelity of transferring chiral information (diastereoselectivity) from each stereodefined phosphoramidite to its stereodefined Sp phosphorothioate or Rp N-(1,3-dimethylimidazolidine-2-vinylidene)phosphoramide (PN) linkage in WVE-N531 or its salts is very high. The overall stereochemical purity of the oligonucleotide can be represented by the product of the diastereoselectivity introduced in each of the 17 stereodefined internucleotide linkages.

[0152] In some embodiments, stereo purity is assessed using dimer modeling. 14 dimer units (fGn001RfU, fG*SfG and fU*SfC dimers each present twice) representing stereo-defined phosphorothioate linkages or PN internucleotide linkages in WVE-N531 drug substances are independently synthesized and assessed for stereochemical purity. The stereochemical purity of WVE-N531 or its salt can be estimated as the product of the diastereoselectivity of the 17 individual dimers measured. The synthesis conditions for producing each dimer are the same as those for the WVE-N531 drug substance. Compared with the full-length WVE-N531 drug substance, these model dimer units are easier to analyze by standard analytical methods (e.g., IP-RP-UPLC), and both Rp and Sp diastereomers can be easily separated and quantified by the technology. The IP-RP-UPLC data of the model dimer are presented below.

[0153] Modeling studies showed that the stereochemical fidelity at each position was excellent, with stereochemical purity values ​​of ≥98% at each position in the sequence. The overall stereochemical purity (based on the product of the stereochemical purity of each individual component dimer) was 85% (84.9% = the product of the above 17 diastereoselective fidelity values).

[0154] In some embodiments, the present disclosure provides WVE-N531 with high stereo purity, for example, as assessed using dimer modeling as described herein. In some embodiments, the stereo purity is about 80% or more. In some embodiments, the stereo purity is about 81% or more. In some embodiments, the stereo purity is about 82% or more. In some embodiments, the stereo purity is about 83% or more. In some embodiments, the stereo purity is about 84% or more. In some embodiments, the stereo purity is about 85% or more. In some embodiments, the stereo purity is about 86% or more. In some embodiments, the stereo purity is about 87% or more. In some embodiments, the stereo purity is about 88% or more. In some embodiments, the stereo purity is about 89% or more. In some embodiments, the stereo purity is about 90% or more. In some embodiments, the stereo purity is about 80%-90%. In some embodiments, the stereo purity is about 83%-87%.

[0155] The published specifications for the WVE-N531 drug substance may include one or more specifications described herein, such as appearance (e.g., visual), sequence identity (e.g., determined by MS / MS), molecular weight, purity (e.g., area % determined by IP-RP-UPLC), impurities (e.g., area % determined by IP-RP-UPLC), sodium content (e.g., determined by ICP-OES, etc.), concentration (free acid form) (e.g., determined by UV spectrophotometry), pH (e.g., pH of a solution in purified water), residual solvents (e.g., determined by gas chromatography), elemental impurities (e.g., determined by ICP-MS), stereochemical properties (e.g., determined by UPLC), bacterial endotoxins (e.g., determined by USP <85> , European Pharmacopoeia 2.6.14), bioburden (total microbial aerobic count, total yeast and colony count, etc. (determined by USP<61 and / or European Pharmacopoeia 2.6.12)), etc.

[0156] The following describes the parameters available for determining identity (molecular weight) by LC MS: In some embodiments, for WVE-N531, system suitability is confirmed when the molecular weight (deconvoluted mass) of the full-length product (FLP) after 3 initial injections of the analytical reference material should be 6923±3 Da; in addition, the blank chromatogram has no other interfering peaks except the solvent front and gradient offset.

[0157] The identity of WVE-N531 or its salt can be determined by liquid chromatography mass spectrometry (LC-MS). Samples were prepared in water and injected onto a Waters Acquity BEH C18 column. The analysis used 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.

[0158] In some embodiments, the present disclosure provides techniques for evaluating the purity and / or impurities of WVE-N531. In some embodiments, the purity and impurities of WVE-N531 can be determined by ion pair reversed phase UPLC (IP-RP-UPLC) (e.g., using a Waters BEH C18 column). A usable procedure (Scheme A) is described below by way of example. 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 set of usable parameters (Group A) is provided below, which have been used to determine the purity / impurity levels of various preparations described herein. Various impurities can be separated using this method, including diastereomeric impurities (of the 17 WVE-N531 diastereomers, each diastereomer is different from WVE-N531 on a single bonded phosphorus (DS1-DS17), 6 diastereomers are completely separated, and 9 are partially separated). In some embodiments, the present disclosure provides a method for characterizing WVE-N531 or a salt thereof or a composition comprising WVE-N531 or a salt thereof, the method comprising HPLC or UPLC analysis comprising one or more or all parameters (e.g., column, one or more solvents in mobile phase A, one or more solvents in mobile phase B, a mobile phase comprising one or more or all solvents in mobile phase A, a mobile phase comprising one or more or all solvents in mobile phase B, mobile phase A, mobile phase B, flow rate, run time, gradient, etc.).

[0159] In some embodiments, all individual impurities ≥ 0.10 area % (as a function of the relative retention time of the major peak) and all impurities total ≥ 0.10 area % are included in the analysis. In some embodiments, the identity of WVE-N531 is confirmed by comparing the relative retention time to system suitability criteria.

[0160] In some embodiments, the present disclosure provides a technique for confirming the stereochemical properties of WVE-N531 or its diastereomers with respect to one or more bonded phosphorus. In some embodiments, the stereochemical properties of WVE-N531 are determined by IP-RP-UPLC (using Waters BEH C18 columns). The following describes a usable procedure (Scheme B) by way of example. The separation is completed using a gradient of mobile phase A (hexafluoroisopropanol [HFIP] and DIPEA in water) and mobile phase B (30% acetonitrile in water). A summary of method parameters (Group B) is provided below. In some embodiments, the present disclosure provides a method for characterizing WVE-N531 or its salt or a composition comprising WVE-N531 or its salt, the method comprising HPLC or UPLC analysis, the analysis comprising one or more or all parameters (e.g., columns, one or more solvents in mobile phase A, one or more solvents in mobile phase B, mobile phases comprising one or more or all solvents in mobile phase A, mobile phases comprising one or more or all solvents in mobile phase B, mobile phase A, mobile phase B, flow rate, run time, gradient, etc.).

[0161] Among other things, the present disclosure provides WVE-N531 of high purity as described herein. In some embodiments, the present disclosure provides WVE-N531 of high stereochemical purity, for example, as determined using IP-RP-UPLC with Group A and / or Group B parameters. In some embodiments, the present disclosure provides a composition comprising WVE-N531 and one or more of DS1 to DS17 ("first group"), without WVE-N531 and one or more of DS1 to DS17 ("second group"), and optionally reduced levels of WVE-N531 and one or more of DS1 to DS17 ("third group"), wherein each reduced level is independently lower (e.g., about or no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%; in some embodiments, about or no more than about 50%) than the level of one or more or each of the members of the first group. In some embodiments, the present disclosure provides a composition comprising WVE-N531 and one of DS1 to DS17, wherein the composition does not include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) of the rest of WVE-N531 and DS1 to DS17. In some embodiments, the composition does not include one of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include two of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include three of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include four of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include five of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include six of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include seven of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include eight of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include nine of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include 10 of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include 11 of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include 12 of WVE-N531 and the rest of DS1 to DS17. In some embodiments, the composition does not include 13 of WVE-N531 and the rest of DS1 to DS17.In some embodiments, the composition does not include WVE-N531 and 14 of the rest of DS1 to DS17. In some embodiments, the composition does not include WVE-N531 and 15 of the rest of DS1 to DS17. In some embodiments, the composition does not include WVE-N531 and 16 of the rest of DS1 to DS17. In some embodiments, the composition does not include WVE-N531 and 17 of the rest of DS1 to DS17. In some embodiments, the composition includes WVE-N531. In some embodiments, the combination includes DS1. In some embodiments, the combination includes DS2. In some embodiments, the combination includes DS3. In some embodiments, the combination includes DS4. In some embodiments, the combination includes DS5. In some embodiments, the combination includes DS6. In some embodiments, the combination includes DS7. In some embodiments, the combination includes DS8. In some embodiments, the combination includes DS9. In some embodiments, the combination includes DS10. In some embodiments, the composition includes DS11. In some embodiments, the composition includes DS12. In some embodiments, the composition includes DS13. In some embodiments, the composition includes DS14. In some embodiments, the composition includes DS15. In some embodiments, the composition includes DS16. In some embodiments, the composition comprises DS17. In some embodiments, at least WVE-N531 is missing from the composition. In some embodiments, at least DS1 is missing from the composition. In some embodiments, at least DS2 is missing from the composition. In some embodiments, at least DS3 is missing from the composition. In some embodiments, at least DS4 is missing from the composition. In some embodiments, at least DS5 is missing from the composition. In some embodiments, at least DS6 is missing from the composition. In some embodiments, at least DS7 is missing from the composition. In some embodiments, at least DS8 is missing from the composition. In some embodiments, at least DS9 is missing from the composition. In some embodiments, at least DS10 is missing from the composition. In some embodiments, at least DS11 is missing from the composition. In some embodiments, at least DS12 is missing from the composition. In some embodiments, at least DS13 is missing from the composition. In some embodiments, at least DS14 is missing from the composition. In some embodiments, at least DS15 is missing from the composition. In some embodiments, at least DS16 is missing from the composition. In some embodiments, at least DS17 is missing from 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., techniques capable of completely or partially separating diastereomers).For example, in some embodiments, when a peak of one of WVE-N531 and DS1 to DS17 is completely separated from one or more peaks of one or more of WVE-N531 and DS1 to DS17, the peak represents a composition comprising one of WVE-N531 and DS1 to DS17 but not comprising one or more of WVE-N531 and DS1 to DS17. In some embodiments, presence, absence and / or level are assessed using provided techniques (e.g., IP-RP-UPLC as described herein).

[0162] In some embodiments, the present disclosure provides a composition comprising WVE-N531, wherein the composition does not include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of DS1 to DS17. In some embodiments, the composition does not include one of DS1 to DS17. In some embodiments, the composition does not include two of DS1 to DS17. In some embodiments, the composition does not include three of DS1 to DS17. In some embodiments, the composition does not include four of DS1 to DS17. In some embodiments, the composition does not include five of DS1 to DS17. In some embodiments, the composition does not include six of DS1 to DS17. In some embodiments, the composition does not include seven of DS1 to DS17. In some embodiments, the composition does not include eight of DS1 to DS17. In some embodiments, the composition does not include nine of DS1 to DS17. In some embodiments, the composition does not include 10 of DS1 to DS17. In some embodiments, the composition does not include 11 of DS1 to DS17. In some embodiments, the composition does not include 12 of DS1 to DS17. In some embodiments, the composition does not include 13 of DS1 to DS17. In some embodiments, the composition does not include 14 of DS1 to DS17. In some embodiments, the composition does not include 15 of DS1 to DS17. In some embodiments, the composition does not include 16 of DS1 to DS17. In some embodiments, the composition does not include 17 of DS1 to DS17. In some embodiments, at least DS1 is missing from the composition. In some embodiments, at least DS2 is missing from the composition. In some embodiments, at least DS3 is missing from the composition. In some embodiments, at least DS4 is missing from the composition. In some embodiments, at least DS5 is missing from the composition. In some embodiments, at least DS6 is missing from the composition. In some embodiments, at least DS7 is missing from the composition. In some embodiments, at least DS8 is missing from the composition. In some embodiments, at least DS9 is missing from the composition. In some embodiments, at least DS10 is missing from the composition. In some embodiments, at least DS11 is missing from the composition. In some embodiments, at least DS12 is missing from the composition. In some embodiments, at least DS13 is missing from the composition. In some embodiments, at least DS14 is missing from the composition. In some embodiments, at least DS15 is missing from the composition. In some embodiments, at least DS16 is absent from the composition. In some embodiments, at least DS17 is absent from the composition.

[0163] Similar to WVE-N531, DS1 to DS17 can each independently be in various forms. In some embodiments, in combination, in one form; in some embodiments, in two or more forms. In some embodiments, the form is a salt form. In some embodiments, the form is a pharmaceutically acceptable salt form. In some embodiments, each form is independently a salt form. In some embodiments, each form is independently a pharmaceutically acceptable salt form. In some embodiments, the form is a hexasodium salt. In some embodiments, in a liquid composition (e.g., optionally a buffered solution), WVE-N531 and / or D1 to DS17 are dissolved and can dissociate to produce cations and anions.

[0164] Liquid WVE-N531 drug substance is packaged in sterile high-density polyethylene (HDPE) bottles with polypropylene screw caps, labeled and sealed in Mylar foil pouches that provide a gas / humidity barrier with a high level of abrasion and puncture resistance.

[0165] Various batches of WVE-N531 hexadecasodium salt have been manufactured as drug substances, in some cases at about 20 mmol scale (e.g., in one case at 2 x 23 mmol scale). Certain formulations were used in nonclinical studies, including GLP toxicology studies. Certain formulations were manufactured for clinical studies. The manufactured product was stable: no significant changes in stability (indicative properties) were observed for multiple batches of GMP drug substances after storage for 24 months at long-term storage conditions of -20°C ± 5°C and 14 days at accelerated conditions of 5°C ± 3°C. Pharmaceutical products

[0166] In some embodiments, the present disclosure provides a pharmaceutical composition comprising or delivering WVE-N531 or a pharmaceutically acceptable salt form thereof and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides a pharmaceutical composition comprising WVE-N531 hexasodium salt and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides a WVE-N531 drug product. In some embodiments, the WVE-N531 drug product comprises a WVE-N531 drug substance, such as WVE-N531 hexasodium salt manufactured using the above method.

[0167] In some embodiments, the pharmaceutically acceptable carrier is or comprises a buffer solution. In some embodiments, the pharmaceutically acceptable carrier is or comprises a phosphate buffer solution. In some embodiments, the pharmaceutically acceptable carrier is or comprises an isotonic phosphate buffer solution. In some embodiments, the WVE-N531 drug product is or comprises a WVE-N531 drug substance in an isotonic phosphate buffer solution. In some embodiments, the pharmaceutical composition or drug product closely matches the physiological conditions in the blood in terms of pH and tonicity. In some embodiments, it is used for intravenous (IV) administration.

[0168] Certain WVE-N531 drug products and useful manufacturing techniques are described below as examples.

[0169] In some embodiments, the WVE-N531 pharmaceutical composition (e.g., drug product) is packaged in a 10 mL USP Type 1 clear glass vial with a cap having coated chlorobutyl rubber stopper and aluminum outer seal with white flip cap. In some embodiments, each drug product 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 drug product include or are WVE-N531 drug substance, potassium dihydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride in water for injection (WFI). Sodium hydroxide and / or hydrochloric acid can also be used to adjust the pH of the formulated solution to the desired range.

[0170] An example composition of a WVE-N531 drug product is presented below: Abbreviations: USP = United States Pharmacopoeia, NF = National Formulary, Ph.Eur. = European Pharmacopoeia, JP = Japanese Pharmacopoeia, BP = British Pharmacopoeia; qs = sufficient, N / A = not applicable

[0171] In some embodiments, the WVE-N531 drug product is a colorless solution with no visible matter and substantially no particles. In some embodiments, a 6 mg / mL solution in phosphate buffered saline solution has a pH of 7.0-7.8 and an osmolality in the range of 280-320 mOsm / kg. In some embodiments, the WVE-N531 drug product has a density of 1.0 g / mL.

[0172] The WVE-N531 drug product batch was manufactured at a concentration of 36 mg / vial (free acid form). The 36 mg strength was achieved by filling 6.35 mL (including an overfill of 0.35 mL over the 6 mL nominal volume) of the 6 mg / mL formulation. All material contact components were sterilized prior to use. The container closure system for the WVE-N531 drug product consisted of or consisted of: 10 mL USP Type 1 clear glass vial with The container is sealed with a coated rubber stopper and an aluminum outer seal with a white flip-top. This container closure protects the sterilized product from microbial contamination.

[0173] The WVE-N531 drug product is typically a sterile solution. In some embodiments, the WVE-N531 drug product can be diluted; in some embodiments, it is diluted with 0.9% sodium chloride or 0.45% sodium chloride as a sterile, preservative-free solution prior to intravenous (IV) administration. Drug product batches can be prepared by pooling multiple drug substance batches.

[0174] In some embodiments, the drug product has a purity as described herein, e.g., the purity described for WVE-N531 or a salt thereof (e.g., hexadecasodium salt), WVE-N531 drug substance, etc. In some embodiments, purity is assessed using the IP-RP-UPLC method for purity described herein.

[0175] Figure 1 A flow chart of the method for manufacturing the WVE-N531 drug product is provided as an example.

[0176] The WVE-N531 drug substance containers can be thawed at 2-8°C for approximately 48 hours. While the drug substance thawing occurs, prepare 1x and 2x strength phosphate buffered saline (PBS) solutions, wherein the 1x solution contains 10 mM sodium / potassium phosphate and 142 mM sodium chloride, a pH of 7.4±0.2 and an osmolality of 285-315 mOsm / kg. Pool the contents of the thawed drug substance in each container into the formulation container and mix thoroughly.

[0177] If a single batch of drug substance is used for formulation, the published purity result is used as the purity correction factor. If multiple batches of drug substance are to be pooled, a purity correction factor based on the amount of active ingredient in each drug substance batch is determined.

[0178] 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. Samples are collected in process to measure concentration (by ultraviolet (UV) spectrophotometry) and density.

[0179] Based on the results of WVE-N531 drug substance concentration and solution quality, calculate the volume of 1x PBS required to achieve a WVE-N531 drug substance concentration of 6.0 mg / mL (WVE-N531 free acid form). Then add the required amount of 1x PBS directly to the formulation container and mix the contents of the container to ensure solution homogeneity.

[0180] In some embodiments, a final in-process sample is collected from the formulation vessel for determination of concentration (by UV spectrophotometry), density, osmolality, and pH. In some embodiments, the concentration of WVE-N531 in the drug product is about 6 mg / mL. In some embodiments, it is about 5.5-6.5 mg / mL (free acid form, corrected for purity). The final formulation is clarified filtered.

[0181] In some embodiments, an in-process pre-filtered bioburden sample is collected prior to sterile filtration. In some embodiments, the final formulation is sterile filtered through two 0.2 μm filters in series prior to filling. In some embodiments, the filter integrity of the sterile filter is checked by the bubble point method prior to and after filtration.

[0182] Based on the final density measurement, the target fill weight (to achieve a target fill volume of 6.35 mL / vial) as well as alarm and action limits are determined. Filling is performed with regular fill weight checks. The vials are aseptically filled, plugged and sealed in a Grade A environment. Finished bottles are visually inspected prior to release testing, bulk packaging and / or stability studies.

[0183] In some embodiments, process controls and in-process control tests and acceptance limits applied during the manufacture of WVE-N531 drug product include WVE-N531 concentration, pH, osmolality, bioburden, filter integrity, fill weight, appearance, etc.

[0184] In some embodiments, the manufacture of the WVE-N531 drug product includes in-process concentration measurements via ultraviolet (UV) spectrophotometry. In some embodiments, in-process concentration measurements are made upon initial dilution of the WVE-N531 drug substance with an equal amount of 2x phosphate buffered saline (PBS). The results are used to determine the amount of 1x PBS needed to be added to achieve the final concentration measured prior to sterile filtration and filling.

[0185] Release specifications for WVE-N531 drug product may include one or more specifications described herein, such as appearance (e.g., visual), identity (e.g., determined by retention time by IP-RP-UPLC), purity (e.g., area % determined by IP-RP-UPLC), impurities (e.g., area % determined by IP-RP-UPLC), % labeled value (e.g., determined by UV; e.g., + / - 10%), pH value (e.g., USP <791> and / or Ph. Eur. 2.2.3), osmolality (e.g., USP <785> and / or European Pharmacopoeia 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), dose uniformity (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 capacity (e.g., USP <697> ), container closure integrity testing (e.g., USP <1207> ) etc. In some embodiments, identity (retention time), purity and / or impurities are assessed using IP-RP-UPLC techniques utilizing one or more or all of Group A parameters. In some embodiments, the purity level is about 70%-85%, as described herein. In some embodiments, the purity level is about 70% or higher. In some embodiments, the purity level is about 71% or higher. In some embodiments, the purity level is about 72% or higher. In some embodiments, the purity level is about 73% or higher. In some embodiments, the purity level is about 74% or higher. In some embodiments, the purity level is about 75% or higher. In some embodiments, the purity level is about 76% or higher. In some embodiments, the purity level is about 77% or higher. In some embodiments, the purity level is about 78% or higher. In some embodiments, the purity level is about 79% or higher. In some embodiments, the purity level is about 80% or higher. In some embodiments, the purity level is about 81% or higher. In some embodiments, the purity level is about 82% or higher. In some embodiments, the purity level is about 83% or higher. In some embodiments, the purity level is about 84% or more. In some embodiments, the purity level is about 85% or more. In some embodiments, the purity level is measured by IP-RP-UPLC area % at 260 nm using Set A parameters.

[0186] The WVE-N531 drug product may contain impurities similar to the WVE-N531 drug substance, at levels as described herein.

[0187] In some embodiments, the container closure system of the WVE-N531 drug product comprises or consists of a USP1 glass vial (10 mL capacity, 20 mm opening) with a Coated elastomer formula 4432 / 50 grey moulded stopper with aluminium outer seal and white flip top.

[0188] In some embodiments, the storage conditions of the WVE-N531 drug product are -20±5° C. In some embodiments, conditions of 5° C.±3° C. are used, for example, for relatively short-term storage.

[0189] Among other things, this disclosure provides the following example embodiments: 1. A method for treating muscular dystrophy, the method comprising administering WVE-N531 to a subject having the disease, the dose administered being 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 DMD gene mutation suitable for exon 53 skipping. 2. A method for treating muscular dystrophy in a subject having muscular dystrophy, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds 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, and wherein the subject has a DMD gene mutation suitable for exon 53 skipping. 3. A method comprising administering to a subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 4. A method comprising administering to a subject one or more doses of WVE-N531, wherein each dose independently corresponds 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 providing DMD exon 53 skipping in a subject, the method comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 6. A method for providing DMD exon 53 skipping in a subject, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 7. A method for restoring the DMD RNA reading frame in a subject, the method comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 8. A method for restoring the DMD RNA reading frame in a subject, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 9. A method for providing a DMD polypeptide in a subject, the method comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to wild-type DMD protein. 10. A method for providing a DMD polypeptide in a subject, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to wild-type DMD protein. 11. The method of any one of embodiments 9-10, wherein the DMD polypeptide is encoded by exon 53 skipped DMD mRNA. 12. The method of any one of embodiments 9-11, wherein the DMD polypeptide provides one or more functions of a wild-type DMD protein. 13. A method for providing an increased level of DMD function in a subject, the method comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 14. A method for providing an increased level of DMD function in a subject, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form. 15. The method of any one of embodiments 3-14, wherein the subject suffers from muscular dystrophy. 16. The method of any one of embodiments 3-15, wherein the subject suffers from DMD. 17. The method of any one of embodiments 3-16, wherein the subject has a DMD gene mutation suitable for 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 the form of one or more pharmaceutically acceptable salts. 20. The method of any one of embodiments 18-19, wherein one form is WVE-N531 hexadecasodium 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 pharmaceutically acceptable carrier. 22. A method for treating muscular dystrophy, the method comprising administering to a subject having the disease a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, the dosage administered being 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 DMD gene mutation suitable for exon 53 skipping. 23. A method comprising administering to a subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form. 24. A method for providing DMD exon 53 skipping in a subject, the method comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, the dose administered being 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 for restoring the DMD RNA reading frame, the method comprising administering to a subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, the dose administered being 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 for providing a DMD polypeptide in a subject, the method comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, the dose administered being 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 wild-type DMD protein. 27. The method of embodiment 26, wherein the DMD polypeptide is encoded by DMD mRNA with exon 53 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 providing an increased level of DMD function in a subject, the method comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, the dose administered being 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 suffers from muscular dystrophy. 31. The method of any one of embodiments 23-30, wherein the subject suffers from DMD. 32. The method of any one of embodiments 23-31, wherein the subject has a DMD gene mutation suitable for exon 53 skipping. 33. The method of any 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 of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 1 mg / kg WVE-N531 free acid form. 35. The method of any 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 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 of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 4 mg / kg WVE-N531 free acid form. 38. The method of any 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 of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 6 mg / kg WVE-N531 free acid form. 40. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 7 mg / kg WVE-N531 free acid form. 41. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 8 mg / kg WVE-N531 free acid form. 42. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 9 mg / kg WVE-N531 free acid form. 43. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 10 mg / kg WVE-N531 free acid form. 44. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 11 mg / kg WVE-N531 free acid form. 45. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 12 mg / kg WVE-N531 free acid form. 46. ​​The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 13 mg / kg WVE-N531 free acid form. 47. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 14 mg / kg WVE-N531 free acid form. 48. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 15 mg / kg WVE-N531 free acid form. 49. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 16 mg / kg WVE-N531 free acid form. 50. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 17 mg / kg WVE-N531 free acid form. 51. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 18 mg / kg WVE-N531 free acid form. 52. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 19 mg / kg WVE-N531 free acid form. 53. The method of any of the preceding embodiments, wherein the WVE-N531 in the dose is equivalent to about 20 mg / kg WVE-N531 free acid form. 54. The method of any one of embodiments 33-53, wherein the is about ± 1%. 55. The method of any one of embodiments 33-53, wherein the % is about ± 2%. 56. The method of any one of embodiments 33-53, wherein the % is about ± 3%. 57. The method of any one of embodiments 33-53, wherein the % is about ± 4%. 58. The method of any one of embodiments 33-53, wherein the % is about ± 5%. 59. The method of any one of embodiments 33-53, wherein the % is about ± 6%. 60. The method of any one of embodiments 33-53, wherein the % is about ± 7%. 61. The method of any one of embodiments 33-53, wherein the is about ± 8%. 62. The method of any one of embodiments 33-53, wherein the is about ± 9%. 63. The method of any one of embodiments 33-53, wherein the % is about ± 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 or more doses 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 pharmaceutically acceptable carrier. 67. The method of any one of embodiments 64-66, wherein each dose has about 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 amount of 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 amount of 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 amount of 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 amount of 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 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 amount of 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 amount of 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 amount of 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 amount of 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 amount of 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 amount of 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 amount of 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 amount of 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 amount of 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 amount of 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 amount of 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 amount of 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 the is about ± 1%. 90. The method of any one of embodiments 67-88, wherein the % is about ± 2%. 91. The method of any one of embodiments 67-88, wherein the % is about ± 3%. 92. The method of any one of embodiments 67-88, wherein the is about ± 4%. 93. The method of any one of embodiments 67-88, wherein the % is about ± 5%. 94. The method of any one of embodiments 67-88, wherein the is about ± 6%. 95. The method of any one of embodiments 67-88, wherein the is about ± 7%. 96. The method of any one of embodiments 67-88, wherein the is about ± 8%. 97. The method of any one of embodiments 67-88, wherein the is about ± 9%. 98. The method of any one of embodiments 67-88, wherein the % is about ± 10%. 99. The method of any preceding embodiment, wherein WVE-N531 is present in the pharmaceutical composition in the form of one or more pharmaceutically acceptable salts. 100. The method of embodiment 99, wherein the pharmaceutically acceptable salt form is the hexadecanoic acid salt. 101. The method of any preceding embodiment, wherein the pharmaceutical composition is a liquid composition comprising dissolved WVE-N531. 102. The method of any one of the preceding embodiments, wherein the pharmaceutically acceptable carrier is or comprises a phosphate buffered solution. 103. The method of any of the preceding embodiments, wherein the components of the pharmaceutical composition are WVE-N531, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and water, and hydrochloric acid and / or sodium hydroxide for pH adjustment. 104. The method of any preceding embodiment, wherein the pharmaceutical composition is isotonic. 105. The method of any preceding embodiment, wherein the pH of the pharmaceutical composition is about 7-8. 106. The method of any preceding embodiment, wherein the pH of the pharmaceutical composition is about 7.3. 107. The method of embodiment 105, wherein the pH of the pharmaceutical composition is about 7.4. 108. The method of any one of the preceding embodiments, wherein two or more consecutive 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. 109. The method of any one of the preceding embodiments, wherein 10 or more consecutive 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 weekly, or about every 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or about every 1, 2, 3, 4, 5, 6, or more months. 111. The method of any one of the preceding embodiments, wherein two or more consecutive doses are administered approximately every two weeks. 112. The method of any one of the preceding embodiments, wherein three consecutive doses are administered approximately every two weeks. 113. The method of any one of the preceding embodiments, wherein three or more consecutive doses are administered approximately every two weeks. 114. The method of any one of the preceding embodiments, wherein 10 or more consecutive doses are administered approximately every two weeks. 115. The method of any one of the preceding embodiments, wherein all doses are administered approximately every two weeks. 116. The method of any one of embodiments 1-114, wherein two or more consecutive doses are administered approximately every four weeks. 117. The method of any one of embodiments 1-114, wherein 10 or more consecutive doses are administered approximately every four weeks. 118. The method of any one of embodiments 108-117, wherein each of the consecutive doses independently corresponds to about 10 mg / kg WVE-N531 free acid form. 119. The method of any one of embodiments 1-110, wherein all doses are administered approximately every four 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 starting from a first dose, followed by a dose administered about every 3, 4, 5, 6, 7, 8, 9, or 10 weeks. 121. The method of any one of embodiments 1-110, wherein the dose is first administered about every two weeks, and then after about 4 weeks, 8 weeks, 12 weeks, or 16 weeks from the first dose, the dose is administered about every four weeks. 122. The method of any one of embodiments 1-110, wherein the dose is first administered about every two weeks, and then after about 4 weeks, 8 weeks, 12 weeks, or 16 weeks from the first dose, the dose is administered about monthly. 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 approximately 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 approximately 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 approximately 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 approximately monthly. 125. The method of any one of embodiments 123-124, wherein the two or more doses administered about every two weeks are administered prior to the two or more doses administered about every four weeks or monthly. 126. The method of any preceding embodiment, wherein the composition has a purity of about 70%-85%. 127. The method of any preceding embodiment, wherein the composition has a purity of about 70%-90%. 128. The method of any preceding embodiment, wherein the composition has a purity of about 70%-80%. 129. The method of any preceding embodiment, wherein the composition has a purity of about 70% or greater. 130. The method of any preceding embodiment, wherein the composition has a purity of about 71% or greater. 131. The method of any preceding embodiment, wherein the composition has a purity of about 72% or greater. 132. The method of any preceding embodiment, wherein the composition has a purity of about 73% or greater. 133. The method of any preceding embodiment, wherein the composition has a purity of about 74% or greater. 134. The method of any preceding embodiment, wherein the composition has a purity of about 75% or greater. 135. The method of any preceding embodiment, wherein the composition has a purity of about 76% or greater. 136. The method of any preceding embodiment, wherein the composition has a purity of about 77% or greater. 137. The method of any preceding embodiment, wherein the composition has a purity of about 78% or greater. 138. The method of any preceding embodiment, wherein the composition has a purity of about 79% or greater. 139. The method of any preceding embodiment, wherein the composition has a purity of about 80% or greater. 140. The method of any preceding embodiment, wherein the composition has a purity of about 81% or greater. 141. The method of any preceding embodiment, wherein the composition has a purity of about 82% or greater. 142. The method of any preceding embodiment, wherein the composition has a purity of about 83% or greater. 143. The method of any preceding embodiment, wherein the composition has a purity of about 84% or greater. 144. The method of any preceding embodiment, wherein the composition has a purity of about 85% or greater. 145. The method of any preceding embodiment, wherein the composition has a purity of about 86% or greater. 146. The method of any preceding embodiment, wherein the composition has a purity of about 87% or greater. 147. The method of any preceding embodiment, wherein the composition has a purity of about 88% or greater. 148. The method of any preceding embodiment, wherein the composition has a purity of about 89% or greater. 149. The method of any preceding embodiment, wherein the composition has a purity of about 90% or greater. 150. The method of any one of embodiments 126-149, wherein the purity is measured by IP-RP-UPLC using area % at 260 nm. 151. The method of any one of embodiments 126-150, wherein the purity is measured by IP-RP-UPLC using area % at 260 nm and Group A parameters. 152. The method of any one of embodiments 126-150, wherein the purity is measured by the IP-RP-UPLC method for purity as described herein. 153. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about 10%-30%. 154. The method of any preceding embodiment, wherein the impurities in the composition do not exceed about 15%-30%. 155. The method of any preceding embodiment, wherein the impurities in the composition do not exceed about 20%-30%. 156. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 30%. 157. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 29%. 158. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 28%. 159. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 27%. 160. The method of any preceding embodiment, wherein the impurities in the composition do not exceed about or are about 26%. 161. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 25%. 162. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 24%. 163. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 23%. 164. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 22%. 165. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 21%. 166. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 20%. 167. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 19%. 168. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 18%. 169. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 17%. 170. The method of any preceding embodiment, wherein the impurities in the composition do not exceed about or are about 16%. 171. The method of any preceding embodiment, wherein the impurities in the composition do not exceed about or are about 15%. 172. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 14%. 173. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 13%. 174. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 12%. 175. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 11%. 176. The method of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are 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 Group A parameters. 179. The method of any one of embodiments 153-178, wherein the impurities are measured by the IP-RP-UPLC method for purity as described herein. 180. The method of any preceding embodiment, wherein the stereochemical purity of WVE-N531 is about 80% or greater. 181. The method of any preceding embodiment, wherein the stereochemical purity of WVE-N531 is about 85% or greater. 182. The method of any preceding embodiment, wherein the stereochemical purity of WVE-N531 is about 80%-90%. 183. The method of any 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 preceding embodiment, wherein the WVE-N531 drug substance is manufactured by the method described herein. 186. The method of any preceding embodiment, wherein the WVE-N531 drug substance is characterized by one or more of the methods described herein. 187. The method of any preceding embodiment, wherein the stereochemistry of WVE-N531 is confirmed by IP-RP-UPLC. 188. The method of any of the preceding embodiments, wherein the stereochemistry of WVE-N531 is confirmed by IP-RP-UPLC according to Group B parameters. 189. The method of any preceding embodiment, wherein the stereochemical identity of WVE-N531 is confirmed by the IP-RP-UPLC method for stereochemical characterization as described herein. 190. The method of any preceding embodiment, wherein the WVE-N531 drug substance is released by one or more of the methods described herein. 191. The method of any preceding embodiment, wherein the WVE-N531 drug substance is stored by one or more of the methods described herein. 192. The method of any one of embodiments 185-191, wherein the WVE-N531 drug substance is the hexasodium salt. 193. The method of any preceding embodiment, wherein the WVE-N531 drug product is manufactured by the method described herein. 194. The method of any preceding embodiment, wherein the WVE-N531 drug product is characterized by one or more methods described herein. 195. The method of any preceding embodiment, wherein the WVE-N531 drug product is released by one or more of the methods described herein. 196. The method of any preceding embodiment, wherein the WVE-N531 drug product is stored by one or more of the methods described herein. 197. The method of any of the preceding embodiments, wherein the pharmaceutical composition is manufactured by the method described herein. 198. The method of any of the preceding embodiments, wherein the pharmaceutical composition is characterized by one or more methods described herein. 199. The method of any of the preceding embodiments, wherein the pharmaceutical composition is delivered by one or more of the methods described herein. 200. The method of any preceding embodiment, wherein the pharmaceutical composition is stored by one or more methods described herein. 201. The method of any preceding embodiment, wherein WVE-N531 is administered intravenously. 202. The method of any one of the preceding embodiments, wherein the DMD mutation is Δ3-52, Δ4-52, Δ5-52, Δ6-52, Δ9-52, Δ10-52, Δ11-52, Δ13-52, Δ14-52, Δ15-52, Δ16-52, Δ17-52, Δ19-52, Δ21-52, Δ23-52, Δ24-52, Δ25-52, Δ26-52, Δ27-52, Δ28-52, Δ29-52, Δ30-52, Δ31-52, Δ32-52, Δ33-52, Δ34-52, Δ35-52, Δ36-52, Δ37-52, Δ38-52, Δ39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ44-52 52. Δ32-52, Δ33-52, Δ34-52, Δ35-52, Δ36-52, Δ37-52, Δ38-52, Δ39-52, Δ40-52, Δ41-52, Δ42-52, Δ43-52, Δ45- 52, Δ47-52, Δ48-52, Δ49-52, Δ50-52, Δ51-52, Δ52, Δ54-58, Δ54-61, Δ54-63, Δ54-64, Δ54-66, Δ54-76, or Δ54-77. 203. The method of any of the preceding embodiments, wherein the mutation comprises Δ52. 204. The method of any of the preceding embodiments, wherein the mutation is Δ52. 205. A method as described in Example 203, wherein the mutation is Δ45-52. 206. A method as described in Example 203, wherein the mutation is Δ47-52. 207. A method as described in Example 203, wherein the mutation is Δ48-52. 208. A method as described in Example 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 preceding embodiment, wherein the muscle dystrophin is DMD. 212. The method of any of the preceding embodiments, wherein exon 53 is skipped in DMD mRNA. 213. The method of any of the preceding embodiments, wherein the level of exon 53 skipped DMD mRNA is increased. 214. The method of any of the preceding embodiments, wherein after administration for a certain period of time or after administration of a certain number of doses, 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. 215. The method of any of the preceding embodiments, wherein a DMD polypeptide that is truncated compared to a wild-type DMD protein is produced. 216. The method of any of the preceding embodiments, wherein the level of a truncated DMD polypeptide is increased. 217. The method of any one of embodiments 215-216, wherein the truncated DMD polypeptide performs one or more functions of a wild-type DMD protein. 218. The method of any of the preceding embodiments, wherein the method increases dystrophin levels relative to baseline by about 1% or more of normal levels after administration for a certain period of time or after administration for a certain number of doses. 219. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 2% or more of normal levels. 220. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 5% or more of normal levels. 221. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 5.3% or more of normal levels. 222. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 6% or more of normal levels. 223. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 7% or more of normal levels. 224. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 8% or more of normal levels. 225. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 9% or more of normal levels. 226. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 10% or more of normal levels. 227. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 11% or more of normal levels. 228. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 12% or more of normal levels. 229. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 13% or more of normal levels. 230. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 14% or more of normal levels. 231. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 15% or more of normal levels. 232. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 16% or more of normal levels. 233. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 17% or more of normal levels. 234. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 18% or more of normal levels. 235. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 19% or more of normal levels. 236. The method of embodiment 218, wherein the increase in dystrophin levels relative to baseline is about 20% or more of normal levels. 237. The method of any one of embodiments 218-236, wherein the increase is measured within about 12 weeks from the first dose after administration. 238. The method of any one of embodiments 218-236, wherein the increase is measured within about 13 weeks from the first dose after administration. 239. The method of any one of embodiments 218-236, wherein the increase is measured within about 14 weeks from the first dose after administration. 240. The method of any one of embodiments 218-236, wherein the increase is measured within about 24 weeks from the first dose after administration. 241. The method of any one of embodiments 218-236, wherein the increase is measured within about 25 weeks from the first dose after administration. 242. The method of any one of embodiments 218-236, wherein the increase is measured within about 26 weeks from the first dose after administration. 243. The method of any one of embodiments 218-236, wherein the increase is measured within about 36 weeks from the first dose after administration. 244. The method of any one of embodiments 218-236, wherein the increase is measured within about 37 weeks from the first dose after administration. 245. The method of any one of embodiments 218-236, wherein the increase is measured within about 38 weeks from the first dose after administration. 246. The method of any one of embodiments 218-236, wherein the increase is measured within about 48 weeks from the first dose after administration. 247. The method of any one of embodiments 218-236, wherein the increase is measured within about 49 weeks from the first dose after administration. 248. The method of any one of embodiments 218-236, wherein the increase is measured within about 50 weeks from the first dose after administration. 249. The method of any one of embodiments 218-236, wherein the increase is measured within about 72 weeks from the first dose after administration. 250. The method of any one of embodiments 218-236, wherein the increase is measured within about 73 weeks from the first dose after administration. 251. The method of any one of embodiments 218-236, wherein the increase is measured within about 74 weeks from the first dose after administration. 252. The method of any one of embodiments 218-236, wherein the increase is measured within about 96 weeks from the first dose after administration. 253. The method of any one of embodiments 218-236, wherein the increase is measured within about 97 weeks from the first dose after administration. 254. The method of any one of embodiments 218-236, wherein the increase is measured within about 98 weeks from the first dose after administration. 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 of the preceding embodiments, wherein DMD function is increased. 269. The method of any of the preceding embodiments, wherein DMD function is restored. 270. The method of any of the preceding embodiments, wherein the subject's loss of walking is reduced. 271. The method of any of the preceding embodiments, wherein the subject's disease progression is delayed or slowed. 272. The method of any one of the preceding embodiments, wherein the subject's muscle weakness is delayed or slowed. 273. The method of any one of the preceding embodiments, wherein the subject's muscle mass loss is delayed or slowed. 274. The method of any of the preceding embodiments, wherein the subject's loss of lung function is delayed or slowed. 275. The method of any one of the preceding embodiments, wherein the subject improves in an assessment of muscular dystrophy. 276. The method of any of the preceding embodiments, wherein the subject improves in a 10 meter walk test. 277. The method of any of the preceding embodiments, wherein the subject improves in one or more functional assessments. 278. The method of any of the preceding embodiments, wherein the subject improves in the North Star Movement Assessment (NSAA) 2.0. 279. The method of any of the preceding embodiments, wherein the subject improves in Performance of Upper Limb (PUL) 2.0. 280. The method of any of the preceding embodiments, wherein the subject improves in one or more lower extremity motor functions as measured by a timed functional test. 281. The method of any preceding embodiment, wherein the subject improves in four-stair climbing. 282. The method of any of the preceding embodiments, wherein the subject improves in time to get up from the ground. 283. The method of any of the preceding embodiments, wherein the subject experiences improvement in proximal upper extremity strength. 284. The method of any of the preceding embodiments, wherein the subject improves in hand grip muscle measurements. 285. The method of any one of the preceding embodiments, wherein the subject improves in one or more lung 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], cough peak flow [CPF], and FVC. 287. The method of any of the preceding embodiments, wherein improvement is compared to a baseline. 288. The method of any one of the preceding embodiments, wherein the improvement is compared to not administering WVE-N531. 289. The method of any of the preceding embodiments, wherein the improvement is compared to administration of a reference composition. 290. The method of any preceding embodiment, wherein the reference composition is comparable to the administered WVE-N531 composition but does not comprise WVE-N531. 291. The method of any of the preceding embodiments, wherein the subject is a pediatric subject. 292. The method of any one of the preceding embodiments, wherein the subject is administered steroids at least about one month prior to the first dose of WVE-N531. 293. The method of any one of the preceding embodiments, wherein the subject was administered steroids for at least about six months prior to the first dose of WVE-N531. 294. The method of any one of the preceding embodiments, wherein the subject is administered a corticosteroid at least about one month prior to the first dose of WVE-N531. 295. The method of any one of the preceding embodiments, wherein the subject was administered corticosteroids for at least about six 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 pharmaceutically acceptable salt form. 299. The composition of any 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 pharmaceutically acceptable salt form. 301. The composition of any of embodiments 297-300, wherein the form of WVE-N531 in the composition is WVE-N531 hexadecanoic acid salt. 302. The composition of any preceding embodiment, wherein the composition is a pharmaceutical substance. 303. The composition of any one of embodiments 297-301, wherein the composition is a pharmaceutical product. 304. The compound of any preceding embodiment, wherein the composition is a liquid composition in which WVE-N531 is dissolved. 305. The composition of any of the preceding embodiments, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. 306. The composition of embodiment 305, wherein the pharmaceutically acceptable carrier is or comprises a phosphate buffer solution. 307. A composition as described in any of the preceding embodiments, wherein the components in the composition are WVE-N531, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water, and hydrochloric acid and / or sodium hydroxide for pH adjustment. 308. The composition of any preceding embodiment, wherein the composition is isotonic. 309. The composition of any preceding embodiment, wherein the composition has a pH of about 7-8. 310. The composition of any preceding embodiment, wherein the pH of the composition is about 7.3. 311. The composition of embodiment 309, wherein the pH of the pharmaceutical composition is about 7.4. 312. The composition of any of the preceding embodiments, wherein the concentration of WVE-N531 is equivalent to about 5-45 mg / mL 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 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 WVE-N531 free acid form. 315. The composition of embodiment 312, wherein the concentration of WVE-N531 is equivalent to about 6 mg / mL WVE-N531 free acid form. 316. The composition of embodiment 315, wherein the composition is packaged in a vial, wherein the volume of the composition in the vial is at least 6 mL. 317. The composition of any of embodiments 315-316, wherein the composition is packaged into a vial, wherein the volume of the composition in the vial is at least 6-7 mL. 318. The composition of any of embodiments 315-316, wherein the composition is packaged into a vial, wherein the volume of the composition in the vial is at least 6-6.5 mL. 319. The composition of any preceding embodiment, wherein the composition has a purity of about 70%-90%. 320. The composition of any preceding embodiment, wherein the composition has a purity of about 70%-85%. 321. The composition of any preceding embodiment, wherein the composition has a purity of about 70%-80%. 322. The composition of any of the preceding embodiments, wherein the composition has a purity of about 70% or greater. 323. The composition of any of the preceding embodiments, wherein the composition has a purity of about 71% or greater. 324. The composition of any of the preceding embodiments, wherein the composition has a purity of about 72% or greater. 325. The composition of any of the preceding embodiments, wherein the composition has a purity of about 73% or greater. 326. The composition of any of the preceding embodiments, wherein the composition has a purity of about 74% or greater. 327. The composition of any of the preceding embodiments, wherein the composition has a purity of about 75% or greater. 328. The composition of any of the preceding embodiments, wherein the composition has a purity of about 76% or greater. 329. The composition of any of the preceding embodiments, wherein the composition has a purity of about 77% or greater. 330. The composition of any preceding embodiment, wherein the composition has a purity of about 78% or greater. 331. The composition of any of the preceding embodiments, wherein the purity of the composition is about 79% or greater. 332. The composition of any of the preceding embodiments, wherein the purity of the composition is about 80% or greater. 333. The composition of any of the preceding embodiments, wherein the purity of the composition is about 81% or greater. 334. The composition of any of the preceding embodiments, wherein the purity of the composition is about 82% or greater. 335. The composition of any of the preceding embodiments, wherein the composition has a purity of about 83% or greater. 336. The composition of any of the preceding embodiments, wherein the purity of the composition is about 84% or greater. 337. The composition of any of the preceding embodiments, wherein the purity of the composition is about 85% or greater. 338. The composition of any of the preceding embodiments, wherein the purity of the composition is about 86% or greater. 339. The composition of any of the preceding embodiments, wherein the purity of the composition is about 87% or greater. 340. The composition of any preceding embodiment, wherein the composition has a purity of about 88% or greater. 341. The composition of any of the preceding embodiments, wherein the purity of the composition is about 89% or greater. 342. The composition of any of the preceding embodiments, wherein the composition has a purity of about 90% or greater. 343. The composition of any one of embodiments 319-342, wherein the purity is measured by IP-RP-UPLC using area % at 260 nm. 344. The composition of any one of embodiments 319-342, wherein the purity is measured by IP-RP-UPLC using Area % at 260 nm and Group A parameters. 345. The composition of any one of embodiments 319-342, wherein the purity is measured by the IP-RP-UPLC method for purity as described herein. 346. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed 10%-30%. 347. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed 15%-30%. 348. The composition of any of the preceding embodiments, wherein impurities in the composition do not exceed 20%-30%. 349. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 30%. 350. The composition of any preceding embodiment, wherein the impurities in the composition do not exceed about or are about 29%. 351. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 28%. 352. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 27%. 353. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 26%. 354. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 25%. 355. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 24%. 356. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 23%. 357. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 22%. 358. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 21%. 359. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 20%. 360. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 19%. 361. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 18%. 362. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 17%. 363. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 16%. 364. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 15%. 365. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 14%. 366. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 13%. 367. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 12%. 368. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are about 11%. 369. The composition of any of the preceding embodiments, wherein the impurities in the composition do not exceed about or are 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 of embodiments 346-370, wherein the impurities are measured by IP-RP-UPLC using Area % at 260 nm and Group A parameters. 372. The composition of any one of embodiments 346-371, wherein the impurities are measured by the IP-RP-UPLC method for purity as described herein. 373. The composition of any of the preceding embodiments, wherein the stereochemical purity of WVE-N531 is about 80% or greater. 374. The composition of any of the preceding embodiments, wherein the stereochemical purity of WVE-N531 is about 85% or greater. 375. The composition of any of the preceding embodiments, wherein the stereochemical purity of WVE-N531 is about 80%-90%. 376. The composition of any of the preceding embodiments, wherein stereochemical purity is assessed by dimer modeling. 377. The composition of any of the preceding embodiments, wherein the amount of WVE-N531 is measured by UV at 260 nm. 378. The composition of any 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 of the preceding embodiments, wherein the WVE-N531 drug substance in the composition is manufactured by the method described herein. 380. The composition of any preceding embodiment, wherein the WVE-N531 drug substance in the composition is characterized by one or more methods described herein. 381. The composition of any preceding embodiment, wherein the WVE-N531 drug substance in the composition is released by one or more methods described herein. 382. The composition of any preceding embodiment, 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 the hexasodium salt. 384. The composition of any of the preceding embodiments, wherein the composition is a WVE-N531 drug product. 385. The composition of any of the preceding embodiments, wherein the WVE-N531 drug product is manufactured by the method described herein. 386. The composition of any preceding embodiment, wherein the WVE-N531 drug product is characterized by one or more of the methods described herein. 387. The composition of any preceding embodiment, wherein the WVE-N531 drug product is released by one or more of the methods described herein. 388. The composition of any preceding embodiment, wherein the WVE-N531 drug product is stored by one or more of the methods described herein. 389. The composition of any of the preceding embodiments, wherein the composition is manufactured by the method described herein. 390. The composition of any of the preceding embodiments, wherein the pharmaceutical composition is characterized by one or more methods described herein. 391. The composition of any of the preceding embodiments, wherein the pharmaceutical composition is delivered by one or more of the methods described herein. 392. The composition of any of the preceding embodiments, wherein the pharmaceutical composition is stored by one or more of the methods described herein. 393. The composition of any of the preceding embodiments, wherein the composition does not comprise DS1. 394. The composition of any of the preceding embodiments, wherein the composition does not comprise DS2. 395. The composition of any of the preceding embodiments, wherein the composition does not comprise DS3. 396. The composition of any of the preceding embodiments, wherein the composition does not comprise DS4. 397. The composition of any of the preceding embodiments, wherein the composition does not comprise DS5. 398. The composition of any of the preceding embodiments, wherein the composition does not comprise DS6. 399. The composition of any of the preceding embodiments, wherein the composition does not comprise DS7. 400. The composition of any of the preceding embodiments, wherein the composition does not comprise DS8. 401. The composition of any of the preceding embodiments, wherein the composition does not comprise DS9. 402. The composition of any of the preceding embodiments, wherein the composition does not comprise DS10. 403. The composition of any preceding embodiment, wherein the composition does not comprise DS11. 404. The composition of any of the preceding embodiments, wherein the composition does not comprise DS12. 405. The composition of any of the preceding embodiments, wherein the composition does not comprise DS13. 406. The composition of any of the preceding embodiments, wherein the composition does not comprise DS14. 407. The composition of any of the preceding embodiments, wherein the composition does not comprise DS15. 408. The composition of any of the preceding embodiments, wherein the composition does not comprise DS16. 409. The composition of any of the preceding embodiments, wherein the composition does not comprise DS17. 410. A composition comprising WVE-N531 and one of DS1 to DS17, wherein the composition does not comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of the remainder of WVE-N531 and DS1 to DS17. 411. The composition of embodiment 410, wherein the composition comprises WVE-N531. 412. The composition of embodiment 410, wherein the composition comprises DS1. 413. The composition of embodiment 410, wherein the composition comprises DS2. 414. The composition of embodiment 410, wherein the composition comprises DS3. 415. The composition of embodiment 410, wherein the composition comprises DS4. 416. The composition of embodiment 410, wherein the composition comprises DS5. 417. The composition of embodiment 410, wherein the composition comprises DS6. 418. The composition of embodiment 410, wherein the composition comprises DS7. 419. The composition of embodiment 410, wherein the composition comprises DS8. 420. The composition of embodiment 410, wherein the composition comprises DS9. 421. The composition of embodiment 410, wherein the composition comprises DS10. 422. The composition of embodiment 410, wherein the composition comprises DS11. 423. The composition of embodiment 410, wherein the composition comprises DS12. 424. The composition of embodiment 410, wherein the composition comprises DS13. 425. The composition of embodiment 410, wherein the composition comprises DS14. 426. The composition of embodiment 410, wherein the composition comprises DS15. 427. The composition of embodiment 410, wherein the composition comprises DS16. 428. The composition of embodiment 410, wherein the composition comprises DS17. 429. A composition as described in any of the preceding embodiments, wherein about ± 1%. 430. A composition as described in any of the preceding embodiments, wherein about ± 2%. 431. A composition as described in any of the preceding embodiments, wherein about ± 3%. 432. A composition as described in any of the preceding embodiments, wherein about ± 4%. 433. A composition as described in any of the preceding embodiments, wherein about ± 5%. 434. A composition as described in any of the preceding embodiments, wherein about ± 6%. 435. A composition as described in any of the preceding embodiments, wherein about ± 7%. 436. A composition as described in any of the preceding embodiments, wherein about ± 8%. 437. A composition as described in any of the preceding embodiments, wherein about ± 9%. 438. A composition as described in any of the preceding embodiments, wherein about ± 10%. 439. A method of making a WVE-N531 composition according to the method described in the specification. 440. The method of embodiment 439, comprising utilizing IP-RP-UPLC to assess purity and / or impurities in the manufactured WVE-N531 composition, and releasing the formulation if the purity and / or impurities meet certain criteria. 441. The method of any one of embodiments 439-440, wherein the composition is a drug substance. 442. The method of any one of embodiments 439-440, wherein the composition is a pharmaceutical product. 443. A method for releasing a WVE-N531 formulation, the method comprising utilizing IP-RP-UPLC to assess purity and / or impurities in the WVE-N531 formulation, and releasing the formulation if the purity and / or impurities meet certain criteria. 444. A method for evaluating the purity of WVE-N531 using IP-RP-UPLC. 445. The method of any one of embodiments 440-444, wherein the IP-RP-UPLC utilizes one or more parameters described in the instructions. 446. The method of any one of embodiments 440-444, wherein the IP-RP-UPLC utilizes one or more parameters of Group A. 447. The method of any one of embodiments 440-444, wherein the IP-RP-UPLC utilizes these Group 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 for confirming the stereochemical characteristics 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 Group B parameters. 451. The method of any one of embodiments 439-449, wherein the stereochemical identity of WVE-N531 is confirmed by the IP-RP-UPLC method for stereochemical characterization 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, dosage or dosage regimen as described in the specification. Examples

[0190] The foregoing is a description of certain non-limiting embodiments of the present disclosure. Therefore, it should be understood that the disclosed embodiments described herein are merely illustrative of the application of the principles of the present disclosure. Reference herein to the details of the illustrated embodiments is not intended to limit the scope of any claim.

[0191] The functions and advantages of certain embodiments of the present disclosure may be more fully understood from the examples described below. The following examples are intended to illustrate certain benefits of such embodiments. Example 1. Based on in vitro and animal studies, WVE-N531 is sufficiently safe to be administered to humans

[0192] Among other things, the present disclosure provides data, including those in primates, demonstrating that WVE-N531 can be administered to human subjects according to various doses and dose regimens as described herein.

[0193] WVE-N531 is an oligonucleotide that targets human Duchenne muscular dystrophy (DMD) messenger RNA (mRNA) to induce exon 53 skipping and restore functional dystrophin in DMD patients.

[0194] Among other things, WVE-N531 induces exon skipping and protein restoration in patient-derived cell lines. In vivo exon skipping, dystrophin restoration, and improved survival were achieved using replacement molecules in mdx23 and / or double knockout (dKO) mouse models. These models have point mutations in exon 23, so murine replacement oligonucleotides with a phosphoramidite (PN) backbone that mimics the chemistry of WVE-N531 were used in these experiments, with sequences designed to specifically target mouse exon 23. In vivo target engagement was also confirmed in healthy cynomolgus monkeys (a pharmacologically relevant species) using WVE-N531. In dystrophin-deficient muscle, biodistribution and exon skipping may be greater than in healthy normal muscle, as has been observed with other oligonucleotides.

[0195] WVE-N531 was evaluated in nonclinical toxicity studies and no risk was found that would prevent the administration of WVE-N531 in humans. After repeated dosing of monkeys at 45 mg / kg for 14 weeks, these effects were considered non-adverse due to the mild severity of the results, minimal morbidity, and / or no impact on the health and well-being of the animals. The no observed adverse effect level (NOAEL) is considered to be at least 45 mg / kg / dose, which is the highest dose evaluated.

[0196] Other findings observed in mice and monkeys are consistent with those generally observed with oligonucleotides, including nonadverse reversible to partially reversible renal and liver outcomes, proinflammatory effects, and transient effects on coagulation factors [prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen]. These risks can be easily monitored in the clinic.

[0197] WVE-N531 was not associated with in vitro or in vivo genotoxicity or off-target effects resulting from human genomic hybridization. In vitro, WVE-N531 did not activate Toll-like receptor 9 (TLR9) in human reporter cells or increase proinflammatory cytokines in ex vivo human peripheral blood mononuclear cells (PBMCs).

[0198] WVE-N531 did not affect 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).

[0199] The various results observed in various nonclinical studies are consistent with the classification effects observed with other oligonucleotides.

[0200] WVE-N531 has been shown to provide DMD exon skipping and restore dystrophin in DMD patient-derived myoblast cell lines (e.g., DMDΔ45-52 and DMDΔ52). In addition, in the mdx23 mouse model of DMD, a mouse exon 23-specific oligonucleotide WV-21218 with chemistry comparable to WVE-N531 has been demonstrated to have a dose-response effect on exon 23 skipping and dystrophin recovery. This oligonucleotide also showed exon skipping, dystrophin recovery, and other biological effects, including extended lifespan in the dKO mouse model of DMD, which lacks dystrophin-related proteins and dystrophin, and develops a severe muscular dystrophy phenotype comparable to that observed in DMD patients. See, for example, WO 2021 / 237223.

[0201] Bioinformatics tools were used to identify potential off-target effects based on sequence homology to WVE-N531. Analysis showed that sequence-dependent off-target effects were minimal.

[0202] In a cell-based reporter assay, WVE-N531 was analyzed for its ability to activate the innate immune protein TLR9. Compared to water-treated cells (negative control), WVE-N531 exposure to test cells (human embryonic kidney cells overexpressing human TLR9) at concentrations up to 30 μM did not increase the activity of the TLR9 reporter. Based on these test data, the potential of WVE-N531 to activate human TLR9 is negligible.

[0203] The immune activation potential of WVE-N531 was also evaluated by incubating WVE-N531 with PBMCs from healthy human donors and then assessing cytokine release. There was no statistically significant difference between the cytokine response to WVE-N531 and the cytokine response to water for any of the following 7 analytes: IFNα2, IL12p40, IL-1β, IL-6, MIP-1α, MIP-1β, TNFα. A statistically significant decrease in MCP-1 levels was observed compared to the negative control (H2O) and the positive control (TLR7 / TLR8 agonist R848). These data suggest that the immune stimulatory potential of WVE-N531 is negligible as measured in this ex vivo PBMC assay.

[0204] The effects of WVE-N531 on cardiovascular and respiratory function in cynomolgus monkeys, and on CNS function in CD-1 mice following IV administration were evaluated in a GLP study. The following table presents an overview of the study design (IV = intravenous):

[0205] In a mouse study, the effects of WVE-N531 on CNS function were evaluated in male and female CD-1 mice following weekly IV (bolus) injections of vehicle (dPBS without calcium or magnesium) or 15, 60, or 180 mg / kg / dose of WVE-N531 on days 1, 15, 29, 43, 57, 71, 85, and 99. Based on functional observation battery (FOB) data, no adverse effects on the CNS were observed at doses up to 180 mg / kg / dose.

[0206] In a monkey study, the effects of WVE-N531 on cardiovascular and respiratory function were evaluated after a single dose of WVE-N531 in conscious male cynomolgus monkeys equipped with radiotelemetry instruments. Animals received WVE-N531 (3, 15 or 45 mg / kg) or dPBS (without calcium or magnesium) via a 60-minute IV infusion. No relevant effects of WVE-N531 on cardiovascular or respiratory function were observed after administration of up to 45 mg / kg of WVE-N531. At 15 mg / kg and 45 mg / kg WVE-N531, a dose-dependent increase in plasma concentrations of complement factors Bb and C3a was observed 2 minutes after EOI. Compared with controls, factor Bb increased 3.7-fold and 34.6-fold at 15 mg / kg and 45 mg / kg dose levels, respectively, 2 minutes after EOI; C3a increased 2.0-fold and 32.4-fold at 15 mg / kg and 45 mg / kg dose levels, respectively, and returned to baseline within 24 hours.

[0207] In another monkey study, the effects of WVE-N531 on cardiovascular function were evaluated after repeated administration of WVE-N531 in anesthetized male and female cynomolgus monkeys. Animals received vehicle (dPBS without calcium or magnesium) or 3, 15 or 45 mg / kg doses of WVE-N531 via a 1-hour IV infusion every other week. Electrocardiograms (ECGs) were collected once during the pre-dose phase, as well as 0.5 hours (± 0.25 hours) after administration, on the 15th day of the dosing phase, and on the 71st day. No effects were observed after 1 hour of IV infusion of WVE-N531 up to 45 mg / kg / dose to male or female cynomolgus monkeys.

[0208] In pharmacokinetic studies conducted in animals including mice and monkeys, WVE-N531 demonstrated pharmacokinetic characteristics suitable for administration to human subjects, including plasma protein binding, tissue distribution, metabolism, etc.

[0209] Non-GLP and GLP repeated-dose toxicity studies have been conducted, including in CD-1 mice (up to 14 weeks) and cynomolgus monkeys (up to 14 weeks) using the IV route of administration. The genotoxic potential of WVE-N531 was also evaluated in 2 in vitro assays and one in vivo study in mice. All GLP studies met the Organization for Economic Co-operation and Development (OECD) data mutual recognition criteria [OECD Decision Reference C(89)87 (final)].

[0210] A nonclinical program evaluating WVE-N531 included non-GLP repeated-dose toxicity studies in mice and monkeys, and a 14-week repeat-dose GLP toxicity study (13-week recovery) in both species for in vitro and in vivo genetic toxicology and in vivo safety pharmacology.

[0211] Mice: In male mice, some inflammatory effects were observed after 6 weeks of administration of WVE-N531 (0, 30, 90, or 180 mg / kg / dose) via weekly IV bolus injections. Overall, WVE-N531 was considered well tolerated in mice at doses up to 180 mg / kg. Repeated administration of WVE-N531 (0, 15, 60, or 180 mg / kg / dose) was evaluated in male and female CD-1 mice via IV bolus injections every other week for 14 weeks (8 doses total). The NOAEL for this study is considered to be 60 mg / kg / dose. On Day 99 of the dosing phase, the mean sex-combined C-1 phenotype was 95.3% (95% CI, 2.1-3.0) (P < 0.05). The NOAEL for this study was 60 mg / kg / dose. The ... max and AUC last The values ​​were 1002±135μg / mL and 1330±109μg·h / mL, respectively.

[0212] Monkeys: In male monkeys, WVE-N531 was considered well tolerated at dose levels up to 25 mg / kg administered weekly and up to 45 mg / kg administered every other week. Given that the 45 mg / kg / dose administered resulted in mild severity of findings, low morbidity, and / or no effects on the health and well-being of the animals, the effects at this dose were considered non-adverse. The NOAEL for this study was considered to be 45 mg / kg / dose. The dose level of 45 mg / kg / dose (administered every other week) corresponded to a C max and AUC last The values ​​were 529 μg / mL and 4380 μg·hr / mL respectively.

[0213] Results from various nonclinical studies indicate that the primary outcomes associated with WVE-N531 in mice and monkeys are consistent with class effects of oligonucleotides, including some potential systemic inflammatory and coagulative effects, decreased thymic cellularity (mice only), and effects on the liver and kidneys. No effects on cardiovascular or respiratory function were observed following 60-minute IV infusions of WVE-N531 up to 45 mg / kg / dose in male cynomolgus monkeys. WVE-N531 was not associated with genotoxicity, as assessed in standard in vitro and in vivo assays.

[0214] Among other things, these results demonstrate that WVE-N531 is sufficiently safe to be administered to human subjects according to various methods, including the various doses and dosing regimens described herein. Example 2. WVE-N531 provides safe and effective exon skipping in primates

[0215] Among other things, the present disclosure shows that WVE-N531 can provide effective exon skipping at various doses in non-human primates. Monkeys were autopsied 2 days after the last dose of 6 weekly doses. In order to detect exon 53 skipping in DMD transcripts, cDNA was prepared from RNA samples. Exon 53 skipping was assessed in monkeys receiving WVE-N531 using non-quantitative RT-PCR assays. As shown in Figure 2, "non-skipped" transcripts were detected in all samples (sub-figure A, arrows show bands), while "skipped" transcripts were only detected in animal samples treated with WVE-N531 (animal numbers 3-8; sub-figure B), demonstrating that exon skipping was achieved at all doses (3 mg / kg and above).

[0216] Compared with suwardisan (25 mg / kg twice a week), WVE-N531 (45 mg / kg every other week) provided higher plasma C maxIn nonhuman primates, WVE-N531 concentrations in muscles including the heart and diaphragm were significantly higher than those of suwardisan, in some cases several-fold higher. Example 3. Clinical study of WVE-N531.

[0217] The safety and efficacy of WVE-N531 are being evaluated in clinical trials.

[0218] The clinical study of WVE-N531 is a Phase 1b / 2a open-label study designed to evaluate the safety, tolerability, PK, PD, and clinical effects of IV WVE-N531 in DMD patients with mutations amenable to exon 53 skipping intervention.

[0219] 1 mg / kg (unless otherwise stated, doses are equivalent to the free acid form of WVE-N531) was shown to be safe in human subjects.

[0220] One or more various higher doses, such as 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 by a dose escalation study, and the safety characteristics and effects (e.g., DMD exon 53 skipping, DMD protein recovery, biological function recovery, etc.) of various doses and dosage regimens are evaluated. 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 dose or each dose is independently about 10 mg / kg. In some embodiments, the dose or each dose is independently about 11 mg / kg. In some embodiments, the dose or each dose is independently about 12 mg / kg. In some embodiments, the dose or each dose is independently about 13 mg / kg. In some embodiments, the dose or each dose is independently about 14 mg / kg. In some embodiments, the dose or each dose is independently about 15 mg / kg. In some embodiments, the dose or each dose is independently about 16 mg / kg. In some embodiments, the dose or each dose is independently about 17 mg / kg. In some embodiments, the dose or each dose is independently about 18 mg / kg. In some embodiments, the dose or each dose is independently about 19 mg / kg. In some embodiments, the dose 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 certain embodiments, dosage regimen comprises or is made up of one or more dosages, each dosage is independently about 3-20mg / kg, for example, 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 20mg / kg, and each dosage is independently applied for 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 (if two or more dosages are applied) after its immediately preceding dosage. In certain embodiments, each dosage in dosage regimen is approximately the same. In certain embodiments, two or more or all intervals between two consecutive doses are the same. In certain embodiments, compared with initial or earlier dosages, the application interval of one or more later dosages may be longer.

[0221] In some embodiments, the clinical trial is or includes one or more conditions, protocols, methods, standards, external metrics, designs, etc. of NCT04906460. In some embodiments, the clinical trial is NCT04906460.

[0222] The following presents available clinical research schemes with examples. In certain embodiments, clinical trials are or include one or more conditions, schemes, methods, standards, external metrics, designs, etc. of the research described below. In certain embodiments, clinical trials are conducted according to the research described below. Those skilled in the art will appreciate that the research can be adjusted.

[0223] An Open-Label Phase 1b / 2a Study of WVE-N531 in Patients with Duchenne Muscular Dystrophy

[0224] The studies evaluated, among other things, one or more of the following: the safety and tolerability of WVE-N531 in patients with Duchenne muscular dystrophy (DMD) who are suitable for exon 53 skipping intervention, the concentration of WVE-N531 in muscle tissue, the pharmacodynamic (PD) effects of WVE-N531 (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).

[0225] To participate in this study, patients must have a documented DMD gene mutation that is amenable to exon 53 skipping intervention.

[0226] The study has 2 parts. Patients in Part A (up to 5 patients) will receive escalating doses of WVE-N531 (starting at the lowest dose level) followed by ≥28 days of safety monitoring between each dose. The sponsor plans to evaluate 4 dose levels to carefully assess the acute tolerability of the escalating doses in an effort 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 and the dose levels used for multiple-dose treatment in Part B of this study. Approximately 12 patients in Part B will receive 7 administered doses at the selected dose level every other week. Patients in Part A may receive up to 3 additional doses of WVE-N531 (administered every other week at a fixed dose level), provided that they do not experience any safety events that would prevent further participation in this study. Patients in Part A will receive a maximum of 7 total doses. All dosing recommendations will be reviewed and approved by an independent Data Safety Monitoring Board (DSMB) and proposed by site investigators.

[0227] Part A patients will receive a biopsy (from the deltoid muscle) during the follow-up period after they have received 7 doses. Part B patients will receive a biopsy at baseline and during the follow-up period after completing dosing.

[0228] The criteria for dose selection in Part A and the decision to initiate Part B are described in detail below.

[0229] Part A.

[0230] Patients will initiate screening up to 6 weeks prior to the start of Part A. Part A will include an within-patient dose escalation portion and a multiple-dose portion. A minimum of 3 patients will be enrolled, and these patients will receive up to 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 biweekly doses at fixed dose level.

[0231] The first dose level evaluated is 1mg / kg. In Part A, administration at all dose levels will be carried out in a staggered manner. During Part A, no more than 1 patient will be administered every 48 hours. All patients will stay in the clinic during the 24-hour post-administration monitoring period and return for safety follow-up on the 3rd day (approximately 48 hours after administration). The decision to continue to administer the next patient at the same dose level will be based on the review of the safety data of the 48-hour observation period of the last administration patient, and the decision to continue to administer the next patient will consider the evaluation of the researcher. After at least 3 patients complete the 2-week post-administration follow-up visit with the dose level, all available safety and PK data will be reviewed. The recommended dose level for subsequent dose administration will be reviewed and approved by DSMB and proposed by on-site researchers. Before accepting the next dose level, the patient will continue to evaluate the safety, PK and functional evaluation of each dose level evaluated within a minimum of 28 days after administration. Any additional patients included after the DSMB review will receive the subsequent selected dose level for evaluation. Detailed information about the DSMB review is provided below.

[0232] Once the maximum tolerated dose (MTD) is reached, patients in Part A will receive up to 3 additional biweekly doses of WVE-N531 at a fixed dose level (e.g., 10 mg / kg), provided that they do not experience any safety events that would hinder further participation in this study. Patients will receive up to 7 doses in Part A of the study. Patients will receive safety and PK assessments during the entire treatment phase. Patients will receive open muscle biopsies 2 weeks after completing multiple dose treatment. After the last dose, patients will continue to receive safety monitoring for 10 weeks. In some embodiments, the single dose in the incremental patient includes or is about 1 mg / kg, about 3 mg / kg, about 6 mg / kg, and about 10 mg / kg. In some embodiments, 3 additional doses of about 10 mg / kg are administered every other week.

[0233] A determination on initiation of Part B will be made based on review of the safety, PK, and muscle tissue concentrations of WVE-N531.

[0234] Part B.

[0235] In Part B, approximately 12 new patients will be enrolled. Patients will start screening up to 6 weeks before dosing. Once the patient is determined to be eligible, they will undergo a baseline open muscle biopsy. The baseline biopsy should be performed at least 2 weeks before the first subsequent dose. Part B patients will receive 7 biweekly doses of WVE-N531. Part B will evaluate the safety, PK, PD, and clinical effects of WVE-N531 in all patients. In addition, patients will receive regular functional assessments, including North Star Mobility Assessment (NSAA) 2.0, Upper Limb Performance (PUL) 2.0, four-ladder climbing, hand-held muscle measurement, and pulmonary function tests. These assessments should be performed in the order specified below. Functional assessments can be performed up to 24 hours before dosing and should be performed before any other assessments (especially blood draws) on the same day. Patients will receive a second open muscle biopsy 2 weeks after the last dose is administered. After the last dose, patients will continue to be monitored for safety for 10 weeks.

[0236] Dose selection is guided primarily by clinical safety, tolerability, and PK data. In addition, it is recommended that the predicted exposure at the highest dose to be tested does not exceed the minimum plasma concentration-time curve area (AUC) from time 0 to the last quantifiable concentration value of 1330 μg·h / mL observed at the no observed adverse effect level (NOAEL) in a 14-week good laboratory practice (GLP) toxicity study in mice (60 mg / kg / dose). last ), or until the lowest maximum observed concentration (C max ) value of 852 μg / mL with a minimum area under the plasma concentration-time curve.

[0237] The dose increase at each dose level should not exceed 3 times the previous dose level.

[0238] The same dose levels from the multiple-dose portion of Part A will be evaluated in Part B; however, a different dose level may be selected based on the results of Part A. The dose used in Part B will not exceed the MTD determined in Part A.

[0239] Number of patients: Approximately 15 patients.

[0240] Study Population: Only patients who met all of the following inclusion and exclusion criteria were included.

[0241] Inclusion criteria: 1. Patients and / or parents or legal guardians must be able and willing to provide written informed consent prior to any research-related procedures. 2. DMD is diagnosed based on the clinical phenotype of increased serum creatine kinase. 3. Documented DMD-associated mutations that are amenable to exon 53 intervention. 4. The score of item 1 or item 2 of the PUL shoulder part is ≥1. 5. Ambulatory or non-ambulatory males. 6. Age ≥5 years and ≤18 years at the time of screening. 7. Willing and able to comply with scheduled visits, medication administration schedule, laboratory testing, study restrictions, and all study procedures, including undergoing muscle biopsy procedures. 8. Stable lung and heart function as measured by: a. Reproducible percentage of predicted forced vital capacity (FVC) ≥ 50%; b. Left ventricular ejection fraction (LVEF) >55% for patients <10 years and >45% for patients ≥10 years, as measured (and recorded) by echocardiography (ECHO) or cardiac magnetic resonance imaging (MRI). 9. Adequate deltoid muscle for open muscle biopsy at screening. 10. Currently receiving a stable corticosteroid regimen, defined as initiation of systemic corticosteroid therapy ≥6 months prior to screening and no dose change for ≤3 months prior to the screening visit. 11. Sexually mature males must be willing to use contraception during the study and within 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 research-related information on social media.

[0242] Exclusion criteria: 1. Clinically significant medical findings on physical examination, other than DMD, that, in the judgment of the Investigator, would render the patient unsuitable for participation and / or completion of study procedures. 2. Other previous or ongoing medical conditions, including the following: a. Acute illness within 4 weeks of the initial screening visit; b. Abnormal physical findings, other than those related to musculoskeletal findings attributable to DMD. 3. Laboratory abnormalities that the researcher believes may adversely affect the patient's safety, make the treatment course or follow-up unlikely to be completed, or affect the evaluation of the study results. These laboratory abnormalities include, but are not limited to, the following: a. Abnormal renal function, defined as an increase in serum cystatin C level >1.5 times the upper limit of normal (ULN) at the screening visit; b. Impaired liver function, glutamate dehydrogenase (GLDH) ≥ 2.5x ULN and bilirubin ≥ 2x ULN (or international normalized ratio [INR] ≥ 1.5x ULN); c. Activated partial thromboplastin time (aPTT) value ≥ 1.5x ULN; d. Platelet count is below the lower limit of normal (LLN). 4. Documented positive test for hepatitis B surface antigen or hepatitis C antibody. 5. Known to be positive for human immunodeficiency virus (HIV). 6. Severe cognitive deficits and / or behavioral problems that the researcher believes may prevent participation in this study. 7. Heart failure: a. Severe cardiomyopathy that the investigator considers precludes participation in this study; however, cardiomyopathy treated with angiotensin-converting enzyme (ACE) inhibitors or beta-blockers is acceptable if the patient meets the LVEF inclusion criteria. b. Any other evidence of clinically significant structural or functional cardiac abnormality. 8. The researcher believes that daytime mechanical ventilation or non-invasive ventilation is required, or is expected to be required. Nighttime non-invasive ventilation is allowed. 9. Changes in nutritional or herbal supplements or concomitant medications within 1 month prior to the screening visit or planned modifications (dose or regimen) during the study. 10. Currently using anticoagulants or drugs known to significantly increase the risk of bleeding, such as chronic nonsteroidal anti-inflammatory drugs (NSAIDs) and heparin. 11. Prior treatment with investigational peptide-coupled phosphorodiamidate morpholino oligomer (PPMO) or drisapersen. 12. Previously received DMD gene therapy treatment. 13. Received treatment with ataluren, vitolasen, ipresen or grodisen within 14 weeks before screening. 14. Receipt of any investigational drug within 3 months or 5 half-lives (whichever is longer) before screening. 15. Known hypersensitivity reaction to any oligonucleotide, manifested as systemic allergic reaction, such as changes in pulse, blood pressure, respiratory function, etc. 16. Parents or legal guardians directly or indirectly participate in the implementation and management of this study as researchers, deputy researchers, research coordinators, or other research staff members; or the patient is a first-degree family member, important other person, or relative living with one of the above persons directly or indirectly participating in the study.

[0243] Investigational Product, Dose, Route, Regimen: WVE-N531 will be provided as a diluted isotonic solution for infusion. Four dose levels of WVE-N531 are planned. The route of administration will be IV. The infusion should be administered over a minimum of 60 minutes; however, if necessary, the infusion time may be extended to a maximum of 3 hours to enhance tolerability.

[0244] Study Duration: Part A contains dose escalation with a safety monitoring period of ≥28 days between each dose. After selection of multiple dose levels, patients in Part A will subsequently receive up to 3 additional biweekly doses with subsequent 10-week follow-up after the final dose. Patients will receive a total of 7 doses. Part B consists of 7 biweekly doses with 10-week follow-up after the final dose.

[0245] end: Security AEs; physical examination results, including vital signs, safety laboratory tests (hematology, chemistry, coagulation, urinalysis); and electrocardiogram (ECG) will be monitored / collected to assess the safety and tolerability of WVE-N531. Pharmacokinetics and pharmacodynamics • Following multiple doses of WVE-N531, dystrophin levels (% normal dystrophin) were assessed by Western blotting of muscle tissue. ·Concentration of WVE-N531 in muscle tissue. ·PK of WVE-N531. Clinical effects NSAA (version 2.0) PUL (version 2.0) Lower extremity motor function determined by timed functional testing (including 10-meter walk / run time, four-stair climb time, and time to rise from the ground) Proximal upper limb strength assessed with handheld myometry Pulmonary function tests (peak flow rate [PFR], peak cough flow [CPF], and FVC)

[0246] Statistical methods:

[0247] No formal sample size determination was performed for Part A. A maximum of 5 patients was considered adequate for the initial evaluation of the safety, tolerability, and PK of escalating doses of WVE-N531. Part B aims to demonstrate the effect of WVE-N531 on dystrophin compared to baseline (% normal). The sample size was determined based on the following assumptions: • Single sample t-test to test whether the change in dystrophin from baseline is greater than 0. - Mean increase in dystrophin from baseline of 5 (% of normal) or more. The one-sided significance level was 2.5%. The standard deviation (SD) of the change in dystrophin levels from baseline was 4.5%. The withdrawal rate or non-assessment rate is 10%.

[0248] Analyze groups

[0249] The safety population will include all treated patients.

[0250] The dystrophin analysis population will be a subset of the safety population and include patients with post-baseline dystrophin values.

[0251] The PK population will be a subset of the safety population and include subjects with evaluable PK data.

[0252] Security Analysis

[0253] Safety assessments for WVE-N531 will include summary of TEAEs, TEAEs leading to treatment discontinuation, TEAEs by severity, TEAEs by relationship, and serious TEAEs. Changes in laboratory assessments will also be summarized.

[0254] Analysis of changes in dystrophin levels (% of normal)

[0255] Changes in dystrophin levels from baseline (% of normal) were analyzed using a 1-sample t-test. Specific study design

[0256] This is an open-label study designed to evaluate the safety, tolerability, PK, PD, and clinical effects of intravenous (IV) WVE-N531 in patients with DMD. To participate in this study, patients must have a documented DMD gene mutation that is amenable to exon 53 skipping intervention.

[0257] filter

[0258] The Screening Period allows for the determination of the patient's eligibility for the study. The Screening Period will begin when the patient and / or parent or legal guardian (as appropriate) signs the study Informed Consent Form. In addition, the patient may be required to provide approval (if applicable). Patients will initiate screening up to 6 weeks prior to dosing. Screening assessments may be performed on multiple days, provided they are within the Screening Period. The investigator will determine if the patient meets the eligibility criteria and collect demographic and medical data to fully characterize the patient.

[0259] To participate in this study, patients must have a documented DMD gene mutation that is amenable to skipping of exon 53. This evaluation must be performed using recognized laboratory methods for mutation analysis.

[0260] deal with

[0261] Section A

[0262] Part A will consist of an within-patient dose escalation portion and a multiple-dose portion. Three patients will be enrolled, and these patients will receive up to 7 doses of WVE-N531.

[0263] Dose escalation part

[0264] In the intra-patient dose escalation part, patients will receive (starting from the lowest dose level) increasing doses of WVE-N531, with a safety monitoring period of ≥28 days between each dose. The first dose level evaluated will be 1 mg / kg. In Part A, administration at all dose levels will be performed in a staggered manner. During Part A, no more than 1 patient will be administered every 48 hours. All patients will stay in the clinic during the 24-hour post-administration monitoring period and return for safety follow-up on the 3rd day / visit 4 (approximately 48 hours after administration). The decision to continue to administer the next patient at the same dose level will be based on a review of the safety data of the 48-hour observation period of the last administered patient, and the decision to continue to administer the next patient will take into account the evaluation of the researcher. The patient will return to the clinic for weekly visits until the 22nd day / visit 7. Subsequent weekly safety monitoring will be completed by the researcher via telephone / telemedicine visits until the patient returns to receive the next dose. There must be at least 28 days between doses.

[0265] After a minimum of 3 patients complete a 2-week post-dose follow-up visit at the dose level, all available safety and PK data will be reviewed. The recommended dose level for subsequent dose administration will be reviewed and approved by the DSMB and proposed by the researchers. Before receiving the next dose level, patients will continue to evaluate the safety, PK and functional assessments of each dose level evaluated within a minimum of 28 days after administration. Any additional patients included after the DSMB review will receive the subsequent selected dose level for evaluation.

[0266] Multiple dose portion

[0267] Once the maximum tolerated dose (MTD) is reached, patients in Part A will receive up to 3 additional biweekly doses of WVE-N531 at a fixed dose level, provided that they do not experience any safety events that would prevent further participation in this study. All patients will receive a maximum of 7 total doses. Patients will receive safety and PK assessments throughout the treatment phase. Patients will receive open muscle biopsies of the deltoid muscle 2 weeks after completing multiple doses of treatment. Patients will continue to receive safety monitoring for up to 10 weeks after the last dose. Based on a review of the safety, PK, and muscle tissue concentrations of WVE-N531, the start of Part B will be determined.

[0268] Section B

[0269] In Part B, about 12 new patients will be included. Part B patients will receive 7 biweekly doses of WVE-N531 at a fixed dose level, and safety, PK, PD and clinical effects will be evaluated. Patients in Part B will receive baseline open muscle biopsy. This open muscle biopsy will be collected from the deltoid muscle and will be collected at least 2 weeks before the subsequent first dose (Part B Day 1 / Visit 2) so that recovery from surgery is obtained before baseline functional assessment. Patients will receive a second open muscle biopsy 2 weeks after the last dose is completed. All patients will receive regular safety, PK and PD assessments of WVE-N531 throughout Part B. In addition, patients will receive regular functional assessments, including NSAA2.0, PUL 2.0, four-ladder climbing, hand-held muscle measurement and pulmonary function tests. Functional assessments can be performed up to 24 hours before administration, and should be performed before any other assessments (especially blood draws) on the same day.

[0270] At Week 14 / Visit 10 in Part B (minimum 2 weeks after the last dose), all patients will undergo a second muscle biopsy to assess the PD effect of WVE-N531 on muscle tissue. In addition, the concentration of WVE-N531 in muscle tissue will be assessed. For safety reasons, all patients in Part B will be followed until Week 22 / Visit 13 (10 weeks after the last dose).

[0271] If a patient withdraws from the study prematurely, the patient should complete an Early Termination (ET) visit. Study End Points

[0272] Security Adverse events (AEs) and physical examination results (including vital signs, safety laboratory tests (hematology, chemistry, coagulation, urinalysis), and electrocardiogram (ECG)) will be monitored / collected to evaluate the safety and tolerability of WVE-N531.

[0273] Pharmacokinetics and pharmacodynamics • Following multiple doses of WVE-N531, dystrophin levels (% normal dystrophin) were assessed by Western blotting of muscle tissue. Concentration of WVE-N531 in muscle tissue ·PK of WVE-N531

[0274] Clinical effects NSAA (version 2.0) PUL (version 2.0) Lower extremity motor function determined by timed functional testing (including 10-meter walk / run time, four-stair climb time, and time to rise from the ground) Proximal upper limb strength assessed with handheld myometry Pulmonary function tests (peak flow rate [PFR], peak cough flow [CPF], and FVC)

[0275] WVE-N531 will be provided as a diluted isotonic solution for IV infusion. WVE-N531 is provided as a solution in a single-use vial. The study drug should be diluted with 0.45% Sodium Chloride Injection or 0.9% Sodium Chloride Injection prior to infusion as directed. The study drug will be administered at the study site by trained personnel. The route of administration will be IV, and the total infusion volume will be 100mL to 500mL based on patient weight. The infusion should be administered over a minimum of 60 minutes. However, if necessary, the infusion time may be extended to a maximum of 3 hours to potentially enhance tolerability. The prepared infusion solution does not contain a preservative and should be administered within 4 hours. Reconstitute / dilute WVE-N531 in a saline infusion bag / container. The patient should remain still during the infusion.

[0276] Treatment Adherence: Since this is an IV-administered medication that will be administered in the clinic, treatment non-adherence is not expected to be a problem.

[0277] Assessment methods and end points

[0278] Safety assessment. The safety assessment will include the following: AE Medical history and demographics Previous and concomitant medications Physical examination (including neurological and psychiatric) Vital signs Height and weight 12-lead ECG ECHO or cardiac MRI Clinical laboratory evaluation (including clinical chemistry, hematology, and urinalysis)

[0279] Any abnormal laboratory test result (hematology, clinical chemistry, or urine) or other safety assessment (e.g., vital sign measurement) that is clinically significant in the medical and scientific judgment of the investigator should be recorded as an AE or SAE.

[0280] Pharmacokinetic evaluation

[0281] Plasma Pharmacokinetics: Samples will be collected from all patients according to the above schedule. Blood samples will be collected by a CRO approved by the sponsor for analysis. Samples will be used only by the sponsor and / or contracted suppliers for research related to DMD treatment development and will be stored for up to 15 years. All biological materials should be stored and safeguarded in a manner that ensures that unauthorized access is prohibited and that samples are not lost, deteriorated, or accidentally or illegally destroyed. Detailed instructions for sample collection, storage, handling, and transportation will be provided in the study-specific manual.

[0282] Muscle Concentration of WVE-N531: WVE-N531 concentrations in muscle tissue may be assessed by hybridization-ligation enzyme-linked immunosorbent assay (HL-ELISA; if adequate tissue samples are available). Samples will be used only by the Sponsor and / or contracted suppliers for research related to DMD therapeutic development and will be stored for up to 15 years. All biological materials should be stored and safeguarded in a manner that ensures that unauthorized access is prohibited and that samples are not lost, deteriorated, or accidentally or unlawfully destroyed. Detailed instructions for sample collection, storage, handling, and transportation will be provided in the study-specific manual.

[0283] Immunogenicity: Immunogenicity samples will be collected according to the evaluation schedule to evaluate anti-drug and anti-dystrophin antibodies in serum. Samples will be used by the Sponsor and / or contracted suppliers only for research related to DMD therapeutic development and will be stored for up to 15 years. All biological materials should be stored and secured in a manner that ensures that unauthorized access is prohibited and that samples are not lost, deteriorated, or accidentally or unlawfully destroyed. Detailed instructions for sample collection, storage, handling, and transportation will be provided in the study-specific manual.

[0284] Pharmacodynamic Assessments: Tissue samples collected from muscle biopsies will be evaluated for changes in dystrophin levels. In addition, exploratory immunohistochemistry assessments may be performed for the presence of drug or biomarkers. Serum samples for possible biomarkers will also be collected at the time points indicated in the assessment schedule. Samples will be used only by the Sponsor and / or contracted vendor for research related to DMD therapeutic development and will be stored for a maximum of 15 years. All biological materials should be stored and safeguarded in a manner that ensures that unauthorized access is prohibited and that samples are not lost, deteriorated, or accidentally or unlawfully destroyed.

[0285] Dystrophin Quantification by Western Blot: Dystrophin levels can 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 dystrophin restored in muscle tissue will be assessed using a validated Western blot method.

[0286] Clinical Effects: All functional assessments must be performed pre-dose (on the dosing day or up to 24 hours prior to the respective dosing). Functional assessments included in the NSAA and timed functional testing will be administered only once per time point as part of the NSAA and scored based on both assessments. Functional assessments should be performed in the order specified below: NSAA 2.0 PUL 2.0 Four-ladder climbing Hand-held muscle measurement Pulmonary function tests (PFR, CPF, and FVC)

[0287] Motor function measurements will be performed according to the assessment schedule. If the patient is able to perform motor function and strength tests, they should only perform these tests. If the patient is unable to complete the tests, this should be documented on the case report form.

[0288] Polaris Ambulatory 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 use in multicenter holistic studies and is widely used internationally as a relevant functional assessment for patients with DMD. The NSAA includes 17 items ranging from standing (item 1) to running (item 17), which are necessary to maintain functional ambulation. The scale includes items that assess abilities that may be present in the early stages of the disease, such as lifting the head and standing on tiptoes, as well as other activities that are typically never fully achieved in untreated children with DMD, such as hopping on one foot or running. Each item is scored on a 3-point scale using the following simple criteria: 2-normal; achieves the goal without any assistance; 1-modified approach; does not rely on the physical assistance of others to achieve the goal; and 0-cannot be achieved independently. The total score is the sum of the scores of the individual items. Scores range from 0 (if no activity can be completed independently) to 34 (if all activities can be completed without any assistance). Typically, the scale is completed in a maximum of 15 minutes. The NSAA also includes the following timed assessments of lower extremity motor function: time to stand from supine position and time to walk 10 meters. Another timed functional test that is not part of the NSAA is the time to climb 4 standard-sized stairs.

[0289] Performance of the Upper Limb (PUL 2.0) Test: The PUL test is an assessment tool for evaluating upper limb function in ambulatory and nonambulatory DMD patients that was developed through an international collaborative group that included DMD boys and their families. The PUL test was designed using a conceptual framework that reflects the natural history of the progression of weakness and functional decline in DMD.

[0290] Four-Stair Climb: The time to climb 4 standard-sized stairs will measure lower extremity motor function in ambulatory patients.

[0291] Grip Myometry: Proximal upper extremity strength will be assessed by grip myometry.

[0292] Pulmonary Function Tests: Pulmonary function tests (PFR, CPF, and FVC) will be performed using a Microlab spirometer. PFR and CPF: Muscle weakness due to neuromuscular disorders will result in lower PFR values. This test is force dependent. Using CPF minimizes the variation associated with force. Therefore, in patients with neuromuscular weakness, measuring CPF with a peak flow meter is a reliable measure of expiratory muscle strength. FVC: During an FVC assessment, the patient inhales maximally and fills the lungs to total lung capacity (TLC), then exhales to maximal capacity. The total volume of air expelled during forced exhalation after maximal inspiration is the FVC. Values ​​will be lower in patients with neuromuscular disorders. FVC measurements will be taken when the patient is in a sitting position. If the patient is wearing a thoracolumbar device, the measurement will be taken with the device on and the patient in a sitting position.

[0293] Statistical methods

[0294] Study Drug Exposure and Adherence: The extent of study drug exposure and adherence for the safety population will be summarized.

[0295] Safety data analysis

[0296] The analysis will include Part A and Part B analyses. All safety analyses for the safety population will be conducted using the following general rules: • Baseline value is usually defined as the last available value before the first dose of study drug. The adverse event observation period is defined as follows: oA treatment-prevalent AE was an AE that developed or worsened before the first dose of study drug. o Treatment-emergent AEs are defined as AEs that develop or worsen from the first dose of study drug until study completion / discontinuation. The summary of TEAEs will include all treatment-emergent AEs. Quantitative safety parameters based on descriptive statistics of central laboratory / reading measurements will be used to summarize visit outcomes and changes from baseline values.

[0297] The amount of WVE-N531 present in plasma and muscle was assessed. Pharmacokinetic parameters included C max (maximum observed concentration), AUC last (area under the plasma concentration-time curve from time 0 to the last quantifiable concentration), AUC inf (area under the plasma concentration-time curve from time 0 to infinity), t 1 / 2 (terminal half-life), CL (total body clearance), and Vd ss (Volume of distribution at steady state). In some embodiments, provided technology provides improved pharmacokinetic properties compared to existing oligonucleotide therapeutics.

[0298] Assessing the immunogenicity of WVE-N531, including antibodies against WVE-N531 and / or dystrophin. In some embodiments, provided techniques do not induce antibodies against 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.

[0299] In some embodiments, the change in dystrophin levels from baseline (% of normal) is assessed. In some embodiments, it is analyzed using a 1 sample t-test.

[0300] In some embodiments, an interim analysis is performed. In some embodiments, an interim analysis is not performed.

[0301] In some embodiments, the study will be conducted according to the study protocol and SOPs that comply with the guidelines provided by the International Conference on Harmonization (ICH) on Good Clinical Practice (GCP) in Clinical Research and any other applicable local regulatory requirements.

[0302] In a Phase 1b / 2a open-label clinical trial, patients received a single dose of WVE-N531 and suvodisan (both at 1 mg / kg). The plasma AUC of WVE-N531 was approximately 4 times that of suvodisan, and the C max C for Suwardison max The plasma half-life of WVE-N531 was demonstrated to be much longer than that of suvodisin: while the plasma half-life of suvodisin was less than 24 hours, the half-life of WVE-N531 was estimated to be more than 1 week.

[0303] Additional data demonstrate that WVE-N531 may provide various advantages supporting its therapeutic use as described herein. In a Phase 1b / 2a open-label clinical trial, patients received a single dose of 1 mg / kg, 3 mg / kg, or 6 mg / kg of WVE-N531, or a single dose of 1 mg / kg, 5 mg / kg, 7 mg / kg, or 10 mg / kg of suwardisan. Figure 4 and Figure 6 As shown in Table 1 below, the plasma AUC of 6 mg / kg WVE-N531 was last The AUC of 5 mg / kg suwardison last The plasma C max C of 5mg / kg suwardison max On day 7, the plasma concentration of 6 mg / kg WVE-N531 was about 43 times that of 5 mg / kg suwardisan. Figure 5 As shown, a single dose of 6 mg / kg WVE-N531 can provide quantifiable plasma concentrations (e.g., >0.002 μg / mL) for at least 105 days after infusion. Figure 6 As shown in Table 1 below, the plasma AUC of 10 mg / kg WVE-N531 was last The AUC of 5 mg / kg suwardison last The plasma concentration of 10 mg / kg WVE-N531 was about 11 times that of 5 mg / kg suvodisen on day 7. In some embodiments, the plasma concentration and other pharmacokinetic parameters of a single dose of 10 mg / kg WVE-N531 showed a half-life of about 25 days, which can support monthly dosing. Even at lower doses, WVE-N531 can provide higher plasma concentrations than suvodisen (see, for example, data for 3 mg / kg and 1 mg / kg WVE-N531, compared to data for 5 mg / kg or even 10 mg / kg suvodisen). Table 1. Clinical pharmacokinetic (PK) plasma data. BLQ = Below Limit of Quantitation

[0304] In a Phase 1b / 2a open-label clinical trial, patients received a single ascending dose of 1, 3, 6 and 10 mg / kg WVE-N531, and then received three doses of 10 mg / kg WVE-N531 every other week (week 0, week 2, week 4) after about 1 to 2 months. Two weeks after the final dose (week 6), a muscle biopsy was performed. The WVE-N531 concentration, WVE-N531 position, exon 53 skipping % and dystrophin expression of muscle biopsies were analyzed. Certain data for individual patients are shown in Table 2 below. The average WVE-N531 muscle concentration was approximately 42 μg / g (approximately 6.1 μM). A high level of exon 53 skipping was observed: the average exon 53 skipping determined by RT-PCR was approximately 53%. As far as the applicant is aware, as of December 19, 2022, this is the earliest time point for exon skipping reported in clinical trials conducted on DMD boys. The average dystrophin expression determined by Western blotting was approximately 0.27% of normal (below the quantification limit of 1%). Without wishing to be limited by theory, applicants suggest that protein production may lag behind the splicing of RNA transcripts. In some embodiments, WVE-N531 was visualized in the nuclei of muscle fibers using RNA in situ hybridization, confirming that WVE-N531 can reach the nucleus of muscle cells. Among other things, these data confirm that WVE-N531 can provide very high levels of exon 53 skipping, even at 10 mg / kg every two weeks (which is significantly lower than grodisin and / or vitrolaxan). Table 2. Clinical muscle biopsy data. * Biopsy was obtained from the biceps rather than the deltoid muscle because of scarring of surrounding tissue from a previous biopsy.

[0305] WVE-N531 appeared safe and well tolerated. Treatment-emergent adverse events (TEAEs) were mild, with the exception of a single COVID-19 infection of moderate intensity. All WVE-N531-related adverse events (AEs) (headache, prurigo) were mild, transient, and resolved without sequelae. There were no serious adverse events (SAEs), no events that met stopping criteria, no trend toward increasing TEAEs, and no evidence of oligonucleotide class-related safety events (e.g., thrombocytopenia, coagulation, complement activation, cytokine activation).

[0306] Additional clinical trials can also be designed and implemented by those skilled in the art based on the present disclosure to evaluate the safety and efficacy of WVE-N531. For example, longer time periods can be used to evaluate and confirm the production of dystrophin.

Claims

1. A method for treating muscular dystrophy, the method comprising administering WVE-N531 to a subject having the disease, the dose administered being 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 DMD gene mutation suitable for exon 53 skipping.

2. A method comprising administering to a subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or A method for providing DMD exon 53 skipping in a subject, the method comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or A method for providing DMD exon 53 skipping in a subject, the method comprising administering to the subject one or more doses of WVE-N531, wherein each dose independently corresponds to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or A method for restoring the DMD RNA reading frame in a subject, the method comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or A method for providing a DMD polypeptide in a subject, the method comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form, wherein the DMD polypeptide is truncated compared to a wild-type DMD protein; or A method for providing an increased level of DMD function in a subject, the method comprising administering to the subject WVE-N531 at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form.

3. The method of claim 2, wherein the DMD polypeptide is encoded by a DMD mRNA with exon 53 skipped, and / or wherein the DMD polypeptide provides one or more functions of a wild-type DMD protein.

4. The method of claim 2, wherein the subject has a DMD gene mutation suitable for exon 53 skipping and / or the subject suffers from muscular dystrophy, optionally wherein the subject suffers from DMD.

5. The method of any one of claims 1-4, wherein WVE-N531 is administered in one or more forms, optionally one or more pharmaceutically acceptable salt forms, optionally one of which is WVE-N531 hexadecasodium salt.

6. A method for treating muscular dystrophy, the method comprising administering a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier to a subject having the disease, the dosage of which is equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form, wherein the subject has a DMD gene mutation suitable for exon 53 skipping.

7. A method comprising administering to a subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier at a dose equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg of WVE-N531 free acid form; or A method for providing DMD exon 53 skipping in a subject, the method comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, the dosage administered being equivalent to about 1-20 (e.g., about 1-5, about 5-10, about 10-15, about 15-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) mg / kg WVE-N531 free acid form; or A method for restoring the DMD RNA reading frame, the method comprising administering to a subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, the dosage administered being 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; or A method for providing a DMD polypeptide in a subject, the method comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, the dosage administered being 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; or A method for providing an increased level of DMD function in a subject, the method comprising administering to the subject a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier, the dose administered being 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. The method of claim 7, wherein the DMD polypeptide is encoded by a DMD mRNA with exon 53 skipped, and / or wherein the DMD polypeptide provides one or more functions of a wild-type DMD protein.

9. The method of any one of claims 7-8, wherein the subject has a DMD gene mutation suitable for exon 53 skipping, and / or the subject suffers from muscular dystrophy, optionally wherein the subject suffers from DMD.

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

11. The method of any one of the preceding claims, wherein two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more) doses are administered.

12. The method of any one of claims 11, wherein each dose is administered independently in a pharmaceutical composition comprising WVE-N531 and a pharmaceutically acceptable carrier.

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

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

15. The method of any one of claims 6-14, wherein WVE-N531 is present in the pharmaceutical composition in one or more pharmaceutically acceptable salt forms, optionally wherein the pharmaceutically acceptable salt form is the hexadecasodium salt.

16. The method of any one of claims 6-15, wherein the pharmaceutical composition is a liquid composition comprising dissolved WVE-N531.

17. The method of any one of claims 6-16, wherein the pharmaceutically acceptable carrier is or comprises a phosphate buffered solution.

18. The method of any one of claims 6-17, wherein the components in the pharmaceutical composition are WVE-N531, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and water, and hydrochloric acid and / or sodium hydroxide for pH adjustment.

19. The method of any one of claims 6-18, wherein the pharmaceutical composition is isotonic.

20. The method of any one of claims 6-19, wherein the pH of the pharmaceutical composition is about 7-8, optionally wherein the pH of the pharmaceutical composition is about 7.3 or 7.

4.

21. The method of any of the preceding claims, wherein two, three, four, five, six, seven, eight, nine, ten or more consecutive 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.

22. The method of any of the preceding claims, wherein all doses are administered about weekly, or about every 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or about every 1, 2, 3, 4, 5, 6, or more months.

23. The method of claim 21 or 22, wherein each of the consecutive doses independently corresponds to about 10 mg / kg WVE-N531 free acid form.

24. The method of any of the preceding claims, wherein the composition is about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or more pure.

25. The method of claim 24, wherein the purity and / or impurities are measured by IP-RP-UPLC using area % at 260 nm, optionally using area % at 260 nm and Group A parameters.

26. The method of any of the preceding claims, wherein stereochemical purity is assessed by dimer modeling and / or wherein the stereochemical purity of WVE-N531 is about 80%, 85%, 90% or more.

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

28. The method of any of the preceding claims, wherein the stereochemistry of WVE-N531 is confirmed by IP-RP-UPLC, optionally according to Group B parameters.

29. The method of any of the preceding claims, wherein the WVE-N531 drug substance is manufactured by the methods described herein, characterized by one or more methods described herein, released by one or more methods described herein, and / or stored by one or more methods described herein.

30. The method of claim 29, wherein the WVE-N531 drug substance is the hexasodium salt.

31. The method of any of the preceding claims, wherein the WVE-N531 drug product is manufactured by the methods described herein, characterized by one or more methods described herein, released by one or more methods described herein, and / or stored by one or more methods described herein.

32. The method of any of the preceding claims, wherein the pharmaceutical composition is manufactured by the methods described herein, characterized by one or more methods described herein, released by one or more methods described herein, and / or stored by one or more methods described herein.

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

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

35. The method of any of the preceding claims, wherein the level of exon 53 skipped DMD mRNA is elevated, or wherein after administration for a certain period of time or after administration for a certain number of doses, 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.

36. The method of any of the preceding claims, wherein a truncated DMD polypeptide is produced compared to a wild-type DMD protein, optionally wherein the truncated DMD polypeptide performs one or more functions of a wild-type DMD protein.

37. The method of any of the preceding claims, wherein the level of a truncated DMD polypeptide is elevated, or wherein the method increases dystrophin levels relative to baseline by about 1%, 2%, 3%, 4%, 5%, 5.3%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more of normal levels after administration for a certain period of time or after administration for a certain number of doses.

38. The method of claim 37, wherein the increase is measured within about 12 weeks, about 13 weeks, about 14 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 72 weeks, about 73 weeks, about 74 weeks, about 96 weeks, about 97 weeks, or about 98 weeks from the first dose after administration, and / or wherein the increase is measured after 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 48, 49, or 50 doses.

39. A method as claimed in any preceding claim, wherein DMD function is increased and / or restored.

40. The method of any of the preceding claims, wherein the subject has a reduced loss of ambulation, a delayed or slowed disease progression in the subject, a delayed or slowed muscle weakness in the subject, a delayed or slowed muscle mass loss in the subject, and / or a delayed or slowed loss of lung function in the subject, and / or wherein the subject improves in a muscular dystrophy assessment and / or one or more functional assessments, and / or wherein the subject improves in a 10 meter walk test, North Star Ambulatory Assessment (NSAA) 2.0, Upper Limb Performance (PUL) 2.0, Four Stair Climb, Proximal Upper Limb Strength, Handgrip Myometry, Time to Get Up from the Ground, and / or one or more lower limb motor functions by a timed functional test, and / or wherein the subject improves in one or more lung function tests, optionally wherein the one or more lung function tests are peak flow rate (PFR), cough peak flow (CPF), and / or FVC.

41. The method of claim 39 or 40, wherein the improvement is compared to baseline, no administration of WVE-N531, or administration of a reference composition, optionally wherein the reference composition is comparable to the administered WVE-N531 composition but does not comprise WVE-N531.

42. The method of any of the preceding claims, wherein the subject is a pediatric subject.

43. The method of any of the preceding claims, wherein the subject is administered a steroid, optionally wherein the steroid is a corticosteroid, optionally wherein the corticosteroid is deflazacort, at least one month, two months, three months, four months, five months, or six months prior to the first dose of WVE-N531.

44. A composition comprising WVE-N531.

45. The composition of claim 44, wherein the form of WVE-N531 in the composition is a pharmaceutically acceptable salt form and / or WVE-N531 hexadecasodium salt.

46. ​​The composition of any one of claims 44-45, wherein each form of WVE-N531 in the composition is independently a salt form, optionally a pharmaceutically acceptable salt form and / or WVE-N531 hexadecasodium salt.

47. The composition of any one of claims 44-46, wherein the composition is a pharmaceutical substance and / or a pharmaceutical product.

48. The compound of any one of claims 44-47, wherein the composition is a liquid composition in which WVE-N531 is dissolved.

49. The composition of any one of claims 44-48, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, optionally wherein the pharmaceutically acceptable carrier is or comprises a phosphate buffered solution.

50. A composition as described in any of claims 44-49, wherein the components in the composition are WVE-N531, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and water, and hydrochloric acid and / or sodium hydroxide for pH adjustment, and / or wherein the composition is isotonic, and / or wherein the pH of the composition is about 7-8, optionally wherein the pH of the composition is about 7.3 or 7.

4.

51. The composition of any of claims 44-50, wherein the concentration of WVE-N531 is equivalent to about 5-45 mg / mL WVE-N531 free acid form, optionally wherein the concentration of WVE-N531 is equivalent to about 25-40 mg / mL WVE-N531 free acid form.

52. The composition of claim 51, wherein the concentration of WVE-N531 is equivalent to about 5-7 mg / mL WVE-N531 free acid form, optionally wherein the concentration of WVE-N531 is equivalent to about 6 mg / mL WVE-N531 free acid form.

53. The composition of claim 52, wherein the composition is packaged into a vial, wherein the volume of the composition in the vial is at least 6-7 mL, at least 6-6.5 mL, or at least 6 mL.

54. The composition of any of claims 44-53, wherein the composition is about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or more pure.

55. The composition of claim 54, wherein the purity and / or the impurities are measured by the IP-RP-UPLC method for purity as described herein and / or by IP-RP-UPLC using Area % at 260 nm, optionally using Area % at 260 nm and Group A parameters.

56. The composition of any one of claims 44-55, wherein stereochemical purity is assessed by dimer modeling and / or wherein the stereochemical purity of WVE-N531 is about 80%, 85%, 90% or more.

57. The composition of any one of claims 44-56, wherein the amount of WVE-N531 is measured by UV at 260 nm, optionally at 260 nm and 27 OD / mg.

58. The composition of any of claims 44-57, wherein the WVE-N531 drug substance in the composition is manufactured by the methods described herein, characterized by one or more methods described herein, released by one or more methods described herein, and / or stored by one or more methods described herein.

59. The composition of claim 58, wherein the WVE-N531 drug substance is the hexasodium salt.

60. The composition of any one of claims 44-59, wherein the composition is a WVE-N531 drug product.

61. The composition of any of claims 44-60, wherein the WVE-N531 drug product is manufactured by the methods described herein, characterized by one or more methods described herein, released by one or more methods described herein, and / or stored by one or more methods described herein.

62. The composition of any one of claims 49-61, wherein the pharmaceutical composition is manufactured by the methods described herein, characterized by one or more methods described herein, released by one or more methods described herein, and / or stored by one or more methods described herein.

63. The composition of any one of claims 44-62, wherein the composition does not comprise DS1, DS2, DS3, DS4, DS5, DS6, DS7, DS8, DS9, DS10, DS11, DS12, DS13, DS14, DS15, DS16 and / or DS17.

64. A composition comprising WVE-N531 and one of DS1 to DS17, wherein the composition does not comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of the remainder of WVE-N531 and DS1 to DS17.

65. The composition of claim 64, wherein the composition comprises WVE-N531.

66. The composition of claim 64, wherein the composition comprises DS1, DS2, DS3, DS4, DS5, DS6, DS7, DS8, DS9, DS10, DS11, DS12, DS13, DS14, DS15, DS16 or DS17.

67. A method for making a WVE-N531 composition according to the method described in the specification.

68. The method of claim 67, comprising utilizing IP-RP-UPLC to assess purity and / or impurities in the manufactured WVE-N531 composition, and releasing the formulation if the purity and / or impurities meet certain criteria.

69. The method of any one of claims 67-67, wherein the composition is a pharmaceutical substance or a pharmaceutical product.

70. A method for releasing a WVE-N531 formulation, the method comprising using IP-RP-UPLC to assess purity and / or impurities in the WVE-N531 formulation, and releasing the formulation if the purity and / or impurities meet certain criteria; or A method for evaluating the purity of WVE-N531 using IP-RP-UPLC.

71. The method of claim 70, wherein the IP-RP-UPLC utilizes one or more parameters described in the specification and / or one or more parameters of Group A.

72. The method of claims 67-71, wherein the stereochemistry of WVE-N531 is confirmed by IP-RP-UPLC.

73. A method for confirming the stereochemical characteristics of WVE-N531 using IP-RP-UPLC.

74. The method of any one of claims 67-73, wherein the stereochemical identity of WVE-N531 is confirmed by IP-RP-UPLC according to Group B parameters or by the IP-RP-UPLC method for stereochemical characterization as described herein.

75. The method of any one of claims 1-43 and 67-74, wherein the composition is any one of claims 44-66.

76. A compound, oligonucleotide, composition, method, process, use, dosage or dosage regimen as described in the specification or any of Example Examples 1-453.

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