How to Treat Duchenne Muscular Dystrophy

Abstract

Provided herein are methods of treating or delaying the onset of Duchenne muscular dystrophy using modified antisense oligonucleotides.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 364,260, filed May 5, 2022, and U.S. Provisional Patent Application No. 63 / 387,733, filed December 16, 2022. The disclosures of each of the above referenced applications are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a sequence listing, which is submitted herewith as an XML file, entitled "035105WO.xml", created on April 26, 2023, is 11,646 bytes in size, and is incorporated by reference in its entirety.

[0003] Provided herein is a method for treating or delaying the onset of Duchenne muscular dystrophy (DMD). In one embodiment, the method comprises administering a modified oligonucleotide to a subject with DMD. [Background technology]

[0004] Antisense oligonucleotides (AONs) are in (pre)clinical development for many diseases and conditions, including cancer, inflammatory conditions, cardiovascular diseases, and neurodegenerative and neuromuscular disorders. Their mechanism of action is directed at various targets, e.g., degradation of target RNA via RNaseH in the nucleus or cytoplasm, splice regulation (exon inclusion or skipping) in the nucleus, or translation inhibition by steric hindrance of ribosomal subunit binding in the cytoplasm. Splice regulation or splice switching AONs were first described for the correction of aberrant splicing in human β-globin pre-mRNA (Dominski and Kole PNAS, 1993, 90(18):8673-8677) and are currently being investigated for various genetic diseases.

[0005] AONs have been widely studied in the treatment of neuromuscular disease disorders Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD), the most common pediatric forms of muscular dystrophy. DMD is a severe and fatal neuromuscular disorder that renders patients wheelchair-assisted by age 12, and patients often die before age 30 from respiratory or cardiac failure. DMD is caused by frameshift deletions (~67%) or duplications (~7%) or point mutations (~25%) of one or more exons in the 2.24 Mb dystrophin gene, resulting in the absence of functional dystrophin. BMD is also caused by mutations in the dystrophin gene, but the open reading frame is maintained and a semi-functional dystrophin protein is produced, typically resulting in a much milder phenotype and longer survival times.

[0006] To date, four AONs have been approved for the treatment of DMD: eteplirsen, golodirsen, casimersen, and viltolarsen. However, these drugs have limited efficacy and are each approved to treat only a small proportion of DMD patients.

[0007] Thus, there is a continuing need for methods to treat DMD. Summary of the Invention

[0008] Provided herein is a method for treating or delaying the onset of DMD by administering a modified oligonucleotide to a subject with DMD. In one embodiment, the modified oligonucleotide is an antisense oligonucleotide (AON). [Brief description of the drawings]

[0009] [Figure 1] 1 shows plasma concentrations of AON1 over time. [Diagram 2]AON1 concentrations in heart, quadriceps, liver, diaphragm, gastrocnemius, and kidney tissues are shown. [Diagram 3] We show that AON1 achieved sustained dose-dependent exon 51 skipping in muscle tissues (quadriceps, gastrocnemius, heart and diaphragm) after 25 weeks of treatment. [Figure 4] The weight percent of dystrophin in the heart and quadriceps is shown, as well as the total ambulatory score of AON1-treated mice. [Diagram 5] We show that AON1 treatment attenuates muscle pathology associated with the hDMD del52(+ / +) model and maintains pharmacological effects for 12 weeks after 25 weeks of treatment. [Figure 6] We show that AON1 treatment attenuates cardiac pathology associated with the hDMD del52(+ / +) model, with pharmacological effects sustained for 12 weeks. [Figure 7] Dystrophin expression in heart and quadriceps muscle of hDMDΔ52 / mdx mice treated with AON1 with TEG linked to the oligonucleotide by a phosphorothioate group (AON1 PS, 9.4, 18.7, or 37.5 mg / kg) at 14 days (filled circles) or 28 days (open circles) after QW, Q2W, or Q4W administration. [Figure 8] Percentage of exon 51 skipping in hearts of hDMDΔ52 / mdx mice treated with AON1 PS (9.4, 18.7, or 37.5 mg / kg) at 14 days (filled circles), or 28 days (open circles) after QW, Q2W, or Q4W dosing. [Figure 9] Shows % exon 51 skipping in quadriceps, gastrocnemius, heart and diaphragm of hDMDΔ52 / mdx mice treated with 18.7 mg / kg AON1 (QW 13 weeks) at 4 and 8 weeks post-treatment. [Figure 10] 1 shows dystrophin expression in the heart and quadriceps of hDMDΔ52 / mdx mice treated with AON1 (18.7 mg / kg) at 4 or 8 weeks after QWx13W administration. [Figure 11]The total ambulatory scores of AON1-treated mice are shown. [Figure 12] The effect of AON1 PS and AON2 (AON1 without a 5'-TEG group) on the complement parameter Bb in human and monkey serum is shown. [Figure 13] The effect of AON1 PS and AON2 on the complement parameter C3a in human and monkey serum is shown. [Figure 14] 1 shows exon skipping in biceps, gastrocnemius, and heart tissues in NHPs treated with AON1 for 39 weeks (Example 8). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] I. Definition To facilitate understanding of the disclosure set forth herein, several terms are defined below.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, the definition in this section shall prevail unless otherwise stated.

[0012] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0013] As used herein, a "subject" is an animal, such as a mammal, including a human patient.

[0014] As used herein, biological activity refers to the in vivo activity of a compound or the physiological response that occurs upon in vitro administration of a compound, composition, or other mixture. Thus, biological activity encompasses the therapeutic effects and pharmacokinetic behavior of such compounds, compositions, and mixtures. Biological activity may also be observed in in vitro systems designed to test such activity.

[0015] As used herein, treatment refers to any manner in which one or more of the symptoms of a disease or disorder are alleviated or otherwise beneficially altered.Treatment also includes any pharmaceutical use of the compositions herein, such as use to treat DMD.

[0016] As used herein, alleviation of symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition means any relief, whether lasting or temporary, permanent or transient, that is due to or may involve administration of the compound or pharmaceutical composition.

[0017] As used herein, unless otherwise indicated, the terms "manage," "managing," and "management" include preventing the recurrence of a particular disease or disorder in a subject already suffering from the disease or disorder and / or extending the time that a subject suffering from the disease or disorder is in remission. The term also includes adjusting the threshold, onset, and / or duration of the disease or disorder, or altering a patient's response to a disease or disorder.

[0018] II. AONs for use in the methods provided herein In one embodiment, the AON for use in the methods provided herein is reverse complementary to a portion of exon 51 of human dystrophin pre-mRNA. In another embodiment, the AON for use in the methods provided herein has the sequence 5'-gguaaguucuguccaagc-3' (SEQ ID NO: 1) and contains a modification. In another embodiment, the AON for use in the methods provided herein has the sequence [ka] wherein c * 5-methylcytosine and [ka] is an LNA nucleotide. As used herein, an LNA nucleotide has the following structure: [ka] where B is a nucleobase and L is a phosphate or phosphorothioate linkage to another nucleotide.

[0019] In another embodiment, the AON for use in the methods provided herein comprises the sequence [ka] wherein c * is 5-methylcytosine, [ka] is an LNA nucleotide, and TEG is a triethylene glycol group. In another embodiment, the AON for use in the methods provided herein has the sequence [ka] wherein c * is 5-methylcytosine, [ka] is an LNA nucleotide, TEG is a triethylene glycol group attached to the 5'-terminus via a phosphate group, the internucleoside linkage is a phosphorothioate linkage, and the non-LNA nucleotide is a 2'-OMe nucleotide (referred to herein as "AON1"). See WO2022 / 069511A1 and US2022 / 0098586A1.

[0020] III. Methods of Treating DMD Provided herein is a method for treating DMD or delaying the onset of DMD by administering a modified oligonucleotide to a subject with DMD. In one embodiment, the modified oligonucleotide is an antisense oligonucleotide (AON). In another embodiment, the AON is an AON disclosed herein. In another embodiment, the AON is AON1. In another embodiment, provided is a method for treating DMD by administering AON1 to a subject with DMD. In another embodiment, provided is a method for delaying the onset of DMD by administering AON1 to a subject with DMD.

[0021] In certain embodiments, the AON is administered at a dose of 0.4 mg / kg, 0.6 mg / kg, 0.8 mg / kg, 1.5 mg / kg, 3 mg / kg, 6 mg / kg, 9 mg / kg, 12 mg / kg or 18 mg / kg. In another embodiment, the AON is administered QW. In another embodiment, the AON is administered QWx15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35W. In another embodiment, the AON is administered QWx24W or QWx25W. In another embodiment, the AON is administered QWx25W. In another embodiment, the AON is administered QWx24W.

[0022] In certain embodiments, AON1 is administered at a dose of 0.4mg / kg, 0.6mg / kg, 0.8mg / kg, 1.5mg / kg, 3mg / kg, 6mg / kg, 9mg / kg, 12mg / kg or 18mg / kg. In another embodiment, AON1 is administered QW. In another embodiment, AON1 is administered QWx15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35W. In another embodiment, AON1 is administered QWx24W or QWx25W. In another embodiment, AON1 is administered QWx25W. In another embodiment, AON1 is administered QWx24W.

[0023] In another embodiment, AON1 is administered at 6 mg / kg QWX25W. In another embodiment, AON1 is administered at 18 mg / kg QWx25W.

[0024] In another embodiment, a method of treating DMD is provided by administering AON1 at 6 mg / kg QW x 24W. In another embodiment, a method of treating DMD is provided by administering AON1 at 18 mg / kg QW x 24W. In another embodiment, a method of delaying the onset of DMD is provided by administering AON1 at 6 mg / kg QW x 24W. In another embodiment, a method of delaying the onset of DMD is provided by administering AON1 at 18 mg / kg QW x 24W.

[0025] In certain embodiments, the methods provided herein provide superior outcomes compared to conventional methods of treating or delaying the onset of DMD. In one embodiment, the methods provided herein induce normalizing changes in associated disease modified biomarkers. In another embodiment, the methods provided herein induce normalizing changes in disease-associated clinical and anatomical muscle pathology. In another embodiment, the methods provided herein provide partial to near-complete rescue of the DMD phenotype as measured by MotoRater / Walking Score. In another embodiment, the methods provided herein increase dystrophin production. In another embodiment, the methods provided herein increase exon 51 skipping of human dystrophin pre-mRNA. In another embodiment, the methods alleviate one or more symptoms of DMD.

[0026] The alleviation of one or more symptoms of DMD in an individual using the AONs described herein may be assessed by any of the following assays: prolongation of time to loss of ambulation, improvement in muscle strength, improvement in ability to lift weights, improvement in time to stand from supine position, improvement in time to walk 9 meters, improvement in time to climb 4 flights of stairs, improvement in leg function grade, improvement in pulmonary function, improvement in cardiac function, improvement in quality of life. Each of these assays is known to those skilled in the art. For each of these assays, a detectable improvement or prolongation of the parameter measured in the assay indicates that one or more symptoms of DMD have been alleviated in an individual using the AONs provided herein. A detectable improvement or prolongation is, in one embodiment, a statistically significant improvement or prolongation as described in Hodgetts et al. (Neuromuscular Disorders 2006;16:591-602). Alternatively, the alleviation of one or more symptoms of DMD may be assessed by measuring improvements in muscle fiber function, integrity and / or survival. In another embodiment, one or more symptoms of a DMD patient are alleviated and / or one or more characteristics of one or more muscle cells of a DMD patient are improved, such symptoms or characteristics may be assessed at the cellular or tissue level or in the patient themselves.

[0027] Alleviation of one or more characteristics of muscle cells from a patient may be assessed by any of the following tests on myoblasts or muscle cells from a patient: decreased calcium uptake by muscle cells, decreased collagen synthesis, changes in morphology, changes in lipid biosynthesis, reduced oxidative stress, and / or improved muscle fiber function, integrity, and / or survival. These parameters are typically assessed using immunofluorescence and / or histochemical analysis of cross-sections of muscle biopsies.

[0028] Improved muscle fiber function, integrity and / or survival may be assessed using at least one of the following tests: a detectable decrease in creatine kinase in the blood, a detectable decrease in muscle fiber necrosis in a biopsy cross-section of a muscle suspected to be dystrophic, and / or a detectable increase in uniformity of muscle fiber diameter in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to those of skill in the art.

[0029] Creatine kinase can be detected in the blood as described in Hodgetts et al. (2006). A detectable decrease in creatine kinase can mean a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater decrease compared to the concentration of creatine kinase in the same DMD patient before treatment.

[0030] A detectable reduction in muscle fiber necrosis is typically assessed in muscle biopsies using biopsy cross sections as described by Hodgetts et al. (2006). A detectable reduction in necrosis can be a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in the area of ​​necrosis identified using biopsy cross sections. This reduction is measured by comparison to necrosis assessed in the same DMD patient prior to treatment.

[0031] A detectable increase in muscle fiber diameter uniformity is typically assessed in muscle biopsy cross sections as described by Hodgetts et al. (see above), measured by comparison with the muscle fiber diameter uniformity in the same DMD patients prior to treatment.

[0032] In one embodiment, the hydroxyalkoxylated AONs provided herein can provide a functional or semi-functional dystrophin protein to the individual and at least partially reduce the production of abnormal dystrophin protein in the individual.

[0033] In one embodiment, providing an individual with a functional or semi-functional dystrophin protein means increasing the production of a functional or semi-functional dystrophin protein. By increased production of functional or semi-functional dystrophin mRNA or functional or semi-functional dystrophin protein is meant a detectable increase or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200% or more compared to the initial amount of functional or semi-functional mRNA or functional or semi-functional dystrophin protein, as determined by RT digital droplet PCR (mRNA) (Verheul et al., PLoS ONE 2016,11(9):e0162467) or immunofluorescence (Beekman et al., PLoS ONE 2014;9(9):e107494), Western blot, or capillary Western immunoassay (Beekman et al., PLoS ONE 2018;13(4):e0195850) analysis (protein). In another embodiment, the initial amount is the amount of functional or semi-functional mRNA or functional or semi-functional dystrophin protein upon initiation of exon skipping of dystrophin pre-mRNA in a cell, organ, tissue, and / or individual using a compound described herein.

[0034] A reduction in the production of abnormal dystrophin mRNA or abnormal dystrophin protein means that 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of abnormal dystrophin mRNA or abnormal dystrophin protein is still detectable by RT digital droplet PCR (mRNA) or immunofluorescence, Western blot, or capillary Western immunoassay (Wes) analysis (protein). In one embodiment, the initial amount is the amount of abnormal dystrophin mRNA or abnormal dystrophin protein when the hydroxyalkoxylated AON described herein is used to initiate induction of exon skipping of dystrophin pre-mRNA in cells, organs, tissues, and / or individuals. An abnormal dystrophin mRNA or protein is also referred to herein as hypofunctional (compared to wild-type functional dystrophin protein as defined herein above) or nonfunctional dystrophin mRNA or protein. A nonfunctional dystrophin protein is a dystrophin protein that is unable to bind to actin and / or members of the DGC protein complex. Non-functional dystrophin proteins or dystrophin mRNAs typically do not have or do not code for dystrophin proteins that include the C-terminus of the intact protein. Detection of functional or semi-functional dystrophin mRNAs or proteins can be performed in the same manner as abnormal dystrophin mRNAs or proteins.

[0035] When functional or semi-functional dystrophin protein is provided to DMD patients, at least part of the cause of DMD is eliminated.Therefore, it is expected that the symptoms of DMD are at least partially alleviated or the rate at which symptoms worsen is reduced and the decline is slowed down.Increasing skipping frequency also increases the level of functional or semi-functional dystrophin protein produced in muscle cells of DMD individuals.

[0036] IV. Pharmaceutical Compositions for Use in the Methods Pharmaceutical compositions provided herein comprise a therapeutically effective amount of one or more AONs provided herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0037] AONs can be formulated into suitable pharmaceutical preparations, such as solutions, suspensions, powders, sterile solutions or suspensions for ophthalmic or parenteral administration, and transdermal patch preparations.Typically, AONs described herein are formulated into pharmaceutical compositions using techniques and procedures known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition 1999).

[0038] In the composition, an effective concentration of one or more compounds or pharma- ceutically acceptable salts are mixed with a suitable pharmaceutical carrier or vehicle. In certain embodiments, the concentration of the AON in the composition is effective to deliver an amount that, upon administration, treats, prevents, or alleviates one or more of the symptoms and / or progression of a disease or disorder disclosed herein.

[0039] Typically, composition is formulated for single dosage administration.To formulate composition, the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in selected vehicle at effective concentration so that the pathology to be treated is relieved or alleviated.The pharmaceutical carrier or vehicle suitable for administration of AON provided herein includes any carrier known to those skilled in the art to be suitable for specific administration mode.

[0040] In addition, AONs can be formulated as the only pharmacoactive ingredient in the composition or can be combined with other active ingredients. Liposomal suspensions, including tissue-targeted liposomes, can also be suitable as pharmacologic acceptable carriers. They can be prepared according to methods known to those skilled in the art. For example, liposomal formulations can be prepared as known in the art. Briefly, liposomes such as multilamellar vesicles (MLVs) can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7:3 molar ratio) inside a flask. A solution of a compound provided herein in phosphate buffered saline (PBS) lacking divalent cations is added, and the flask is shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compound, pelleted by centrifugation, and then resuspended in PBS.

[0041] The active compound is included in a pharma- ceutically acceptable carrier in an amount sufficient to provide a therapeutically useful effect to the subject being treated without undesirable side effects. Therapeutically effective concentrations can be empirically determined by testing the AON in the in vitro and in vivo systems described herein, and then human dosages can be extrapolated therefrom. In some embodiments, the AON is administered in such a way as to achieve a therapeutically effective concentration of the drug. In some embodiments, a companion diagnostic (e.g., Olsen D and Jorgensen JT, Front. Oncol., 2014 May 16, 4:105, doi:10.3389 / fonC.2014.00105) is used to determine the therapeutic concentration and safety profile of the active compound in a particular subject or population of subjects.

[0042] The concentration of the AON in the pharmaceutical composition will depend on the absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical properties of the compound, the administration schedule, and the dosage, as well as other factors known to those skilled in the art. For example, the amount delivered will be sufficient to alleviate one or more of the symptoms of a disease or disorder disclosed herein.

[0043] In certain embodiments, a therapeutically effective dosage should produce a serum concentration of the active ingredient of about 0.1 ng / mL to about 50-100 μg / mL. In one embodiment, the pharmaceutical composition provides a daily dosage of about 0.001 mg to about 2000 mg of compound per kg of body weight. Pharmaceutical unit dosage forms are prepared to provide about 1 mg to about 1000 mg, and in certain embodiments, about 10 to about 500 mg of the essential active ingredient or combination of essential ingredients per unit dosage form.

[0044] AON may be administered at once or divided into several smaller doses and administered at time intervals. It is understood that the exact dosage and duration of treatment varies according to the disease being treated and can be empirically determined using known test protocols or by extrapolation from in vivo or in vitro test data. It is also noted that concentration and dosage values ​​may change as the severity of the condition is alleviated. It is further understood that for any particular subject, individual dosage regimens must be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the concentration ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0045] Thus, an effective concentration or amount of one or more of the AONs described herein or their pharma- ceutically acceptable salts are mixed with a pharmaceutical carrier or vehicle suitable for systemic, local, or topical administration to form a pharmaceutical composition. The AONs are included in an amount effective to alleviate or treat, delay progression, or prevent one or more symptoms. The concentration of the active compound in the composition depends on the absorption, tissue distribution, inactivation, and excretion rates of the active compound, the administration schedule, and the amount administered, the particular formulation, and other factors known to those skilled in the art.

[0046] The compositions are intended to be administered by a suitable route, including, but not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, cutaneous, transdermal, or buccal.

[0047] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain any of the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, dimethylacetamide, or other synthetic solvents; antimicrobial agents such as benzyl alcohol and methylparaben; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for adjusting isotonicity such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, pens, disposable syringes, or single- or multi-dose vials made of glass, plastic, or other suitable materials.

[0048] In cases where the AON exhibits insufficient solubility, methods for solubilizing the compound may be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of co-solvents such as dimethylsulfoxide (DMSO), the use of surfactants such as TWEEN®, or dissolution in aqueous sodium bicarbonate.

[0049] Upon mixing or addition of the AON, the resulting mixture may be a solution, suspension, emulsion, etc. The form of the resulting mixture will depend upon several factors, including the intended mode of administration and the solubility of the compound AON in the selected carrier or vehicle. The effective concentration is an amount sufficient to alleviate the symptoms of the disease, disorder, or condition being treated and may be empirically determined.

[0050] Pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms such as powders, granules, sterile parenteral solutions or suspensions, and oil-water emulsions containing a suitable amount of AON or its pharma- ceutically acceptable salts. Pharmaceutically, therapeutically active AONs and their salts are formulated and administered in unit dosage forms or multiple dosage forms. Single dosage forms as used herein refer to physically separate units suitable for human and animal subjects and packaged individually as known in the art. Each single dosage contains a predetermined amount of therapeutically active compound sufficient to produce the desired therapeutic effect, together with the required pharmaceutical carrier, vehicle, or diluent. Examples of single dosage forms include ampoules and syringes, as well as individually packaged tablets or capsules. Single dosage forms may be administered in portions or multiples thereof. Multiple dosage forms are multiple identical single dosage forms packaged in a single container to be administered in divided single dosage forms. Examples of multiple dosage forms include vials, bottles of tablets or capsules, or bottles of pints or gallons. Thus, a multiple dose form is an undivided multiple of a unit dose when packaged.

[0051] Sustained release preparations can also be prepared.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the compounds provided herein, which matrices are in the form of shaped articles, such as films or microcapsules.Examples of sustained release matrices include iontophoretic patches, polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microparticles composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time. If encapsulated compounds remain in the body for extended periods of time, they may denature or aggregate as a result of exposure to moisture at 37°C, leading to loss of biological activity and possible changes in their structure. Depending on the mechanism of action involved, rational strategies can be taken for stabilization. For example, if the mechanism of aggregation is found to be intermolecular S--S bond formation via thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0052] Dosage forms or compositions containing the active ingredient in the range of 0.005%-100% with the balance of non-toxic carriers can be prepared. Such compositions include solutions, suspensions, powders, and sustained release formulations, including but not limited to implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like. Methods for preparing these compositions are known to those skilled in the art. Contemplated compositions may contain about 0.001%-100% active ingredient, and in certain embodiments, about 0.185% or about 75-95% active ingredient.

[0053] The active AON or pharma- ceutically acceptable salt may be prepared with carriers that protect the compound against rapid elimination from the body, such as time-release formulations or coatings.

[0054] The composition may contain other active AONs to obtain a desired combination of properties. The AONs provided herein, or pharma- ceutically acceptable salts thereof as described herein, may also be administered for therapeutic or prophylactic purposes, advantageously with another pharmacological agent generally known to be beneficial in treating one or more of the above-mentioned diseases or medical conditions, such as diseases associated with oxidative stress. It is understood that such combination therapy constitutes a further aspect of the compositions and methods of treatment provided herein.

[0055] A. Injectables, Solutions and Emulsions Parenteral administration, generally characterized by injection, either subcutaneously, intramuscularly, or intravenously, is also contemplated herein. Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, in solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. In some embodiments, the suspension is a suspension of microparticles or nanoparticles. In some embodiments, the emulsion is an emulsion of microparticles or nanoparticles. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins. Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained, is also contemplated herein. Briefly, the AONs provided herein are dispersed in a solid internal matrix, such as polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrophilic polymers (e.g., hydrogels of esters of acrylic and methacrylic acid), collagen, cross-linked polyvinyl alcohol, and partially hydrolyzed cross-linked polyvinyl acetate, which are insoluble in body fluids. The polymeric material is surrounded by an outer polymer membrane such as polyethylene, polypropylene, ethylene / propylene copolymers, ethylene / ethyl acrylate copolymers, ethylene / vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl acetate, vinylidene chloride, copolymers of ethylene and vinyl chloride with propylene, ionomeric polyethylene terephthalate, butyl rubber, epichlorohydrin rubber, ethylene / vinyl alcohol copolymers, ethylene / vinyl acetate / vinyl alcohol terpolymers, and ethylene / vinyloxyethanol copolymers.The AON diffuses through the outer polymeric membrane in a release rate controlling step. The percentage of active AON contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.

[0056] Parenteral administration of the composition includes intravenous, subcutaneous, and intramuscular administration. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products such as freeze-dried powders, including subcutaneous tablets ready for use, sterile suspensions ready for injection, sterile dry insoluble products ready for use, and sterile emulsions. Solutions can be either aqueous or non-aqueous.

[0057] If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0058] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances.

[0059] Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations should be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0060] The concentration of the pharma- ceutically active AON is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect, the exact dose depending on the age, weight, and condition of the subject or animal, as is known in the art.

[0061] Unit dose parenteral preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration must be sterile, as known and practiced in the art.

[0062] By way of illustration, intravenous or intraarterial infusion of a sterile aqueous solution containing an active AON is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing the active AON injected as needed to obtain the desired pharmacological effect.

[0063] Injectable drugs are designed for local and systemic administration. Typically, a therapeutically effective dose is prepared to contain a concentration of active AON of at least about 0.1% to about 90% or more by weight, e.g., greater than 1% by weight, relative to the tissue being treated. The active AON may be administered at once or divided into several smaller doses to be administered at time intervals. It is understood that the exact dosage and duration of the therapeutic agent will vary depending on the tissue being treated and can be empirically determined using known testing protocols or by extrapolation from in vivo or in vitro test data. It is also noted that concentration and dosage values ​​may vary with the age of the individual being treated. It is further understood that for any particular subject, individual dosing regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the formulation, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed formulations.

[0064] The AONs may be suspended in micronized or other suitable form, or may be derivatized to produce more soluble active products or prodrugs. The form of the resulting mixture depends on several factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to alleviate the symptoms of the condition and may be empirically determined.

[0065] B. Freeze-dried powder Also provided herein are lyophilized powders that can be reconstituted for administration into solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels.

[0066] Sterile lyophilized powders are prepared by dissolving the AONs provided herein, or pharma- ceutically acceptable salts thereof, in a suitable solvent. The solvent may contain excipients to improve stability, or other pharmacological components of the powder, or a solution for preparation of a solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other buffer as known to those of skill in the art, in one embodiment, a buffer with a pH of about neutral. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those of skill in the art, yields the desired formulation. Generally, the resulting solution is apportioned into vials for lyophilization. Each vial contains a single dose (including, but not limited to, 10-1000 mg or 100-500 mg) or multiple doses of the compound. The lyophilized powders may be stored under appropriate conditions, such as at about 4°C to room temperature.

[0067] Preparation of a solution of this lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, about 1-50 mg, about 5-35 mg, or about 9-30 mg of lyophilized powder is added per mL of sterile water or other suitable carrier. The exact amount depends on the compound selected. Such amounts can be empirically determined.

[0068] C. Topical Administration Topical mixtures are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and is formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, irrigate, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0069] The compound can be formulated for topical or local application, for example, topical application to the skin and mucous membranes, for example, the eye, in the form of gels, creams, and lotions, and for application to the eye, or for intracapsular or intrathecal application.Local administration is contemplated for transdermal delivery, as well as for administration to the eye or mucous membranes, or for inhalation therapy.Nasal drops of the active compound can also be administered alone or in combination with other pharma- ceutically acceptable excipients.

[0070] These solutions, particularly those intended for ophthalmic use, may be formulated as 0.01%-10% isotonic solutions, pH about 5-7, containing appropriate salts.

[0071] D. Sustained Release Compositions The AONs provided herein can be administered by controlled release means or delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, U.S. Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, 5,639,480, 5,733,566, 5,739,108, 5,891,474, 5,922,356, 5,9 Nos. 72,891, 5,980,945, 5,993,855, 6,045,830, 6,087,324, 6,113,943, 6,197,350, 6,248,363, 6,264,970, 6,267,981, 6,376,461, 6,419,961, 6,589,548, 6,613,358, 6,699,500 and 6,740,634, each of which is incorporated herein by reference in its entirety. Such dosage forms can be used to provide a sustained or controlled release of one or more active ingredients, for example, by using hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide desired release profiles at various rates.Suitable controlled release formulations known to those skilled in the art, including those described herein, can be easily selected for use with the active ingredients provided herein.

[0072] All controlled release pharmaceutical products have a common goal of improving drug therapy beyond that achieved by their non-controlled counterparts. In one embodiment, the use of optimally designed controlled release preparations in treatment is characterized by the minimum amount of drug substance used to cure or control the pathology in the minimum amount of time. In certain embodiments, the advantages of controlled release formulations include extended drug activity, reduced dosing frequency, and improved subject compliance. In addition, controlled release formulations can be used to affect other characteristics such as the time of onset of action or blood concentration of the drug, and therefore can affect the occurrence of side (e.g., adverse) effects.

[0073] Many controlled release formulations are designed to initially release an amount of drug (active ingredient) that quickly produces the desired therapeutic effect, and then slowly and continuously release another amount of drug to maintain this level of therapeutic or prophylactic effect over a long period of time.To maintain this constant drug level in the body, the drug must be released from the dosage form at a rate that will compensate for the amount of drug that is metabolized and excreted from the body.The controlled release of AONs can be stimulated by various conditions, including but not limited to pH, temperature, enzymes, water, or other physiological conditions or compounds.

[0074] In certain embodiments, the AONs may be administered using intravenous injection, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment, a polymeric material may be used. In yet another embodiment, the controlled release system is placed in close proximity to the therapeutic target, i.e., so that only a fraction of the systemic dose may be required (see, e.g., Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)).

[0075] Other controlled release systems have been reviewed by Langer (Science 249:1527-1533 (1990)). AONs may be dispersed in a solid internal matrix, e.g., hydrophilic polymers such as polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and partially hydrolyzed cross-linked polyvinyl acetate, which are insoluble in body fluids. The AON is surrounded by an outer polymeric membrane, such as polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylic acid copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride copolymer with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer terephthalic acid polyethylene, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol terpolymer, and ethylene / vinyloxyethanol copolymer. The AON then diffuses through the outer polymeric membrane in a release rate control step. The percentage of active ingredient contained in such parenteral compositions is highly dependent on their specific nature and the needs of the subject.

[0076] E. Targeted Formulations The AONs or pharma- ceutically acceptable salts thereof provided herein may also be formulated to target specific tissues, receptors, or other areas of the body of the subject being treated, including liposome-based, resealed red blood cell-based, and antibody-based delivery systems. Many such targeting methods are known to those skilled in the art. All such targeting methods are contemplated herein for use with the present compositions. Non-limiting examples of targeting methods are described, for example, in U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,071,570 ... See Nos. 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874.

[0077] In one embodiment, the antibody-based delivery system is an antibody-drug conjugate ("ADC"), e.g., as described in Hamilton GS, Biologicals, 2015 September, 43(5):318-32; Kim EG and Kim KM, Biomol. Ther. (Seoul), 2015 November, 23(6):493-509, and Peters C and Brown S, Biosci. Rep., 2015 June.12, 35(4) pii:e00225, each of which is incorporated by reference herein in its entirety.

[0078] In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharma- ceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art. For example, liposomal formulations can be prepared as described in U.S. Pat. No. 4,522,811. Briefly, liposomes, such as multilamellar vesicles (MLVs), can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7:3 molar ratio) on the inside of a flask. A solution of AONs provided herein in phosphate-buffered saline (PBS) lacking divalent cations is added, and the flask is shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compounds, pelleted by centrifugation, and then resuspended in PBS.

[0079] F.Product The AON or pharma- ceutically acceptable salt may be packaged as an article of manufacture comprising packaging materials, the AON or pharma- ceutically acceptable salt thereof provided herein for use in treating, preventing, or ameliorating one or more symptoms or progression of a disease or disorder disclosed herein, and a label indicating that the compound or pharma- ceutically acceptable salt thereof is used to treat, prevent, or ameliorate one or more symptoms or progression of a disease or disorder disclosed herein.

[0080] The product provided herein includes packaging materials. Packaging materials used in packaging pharmaceutical products are well known to those skilled in the art. For example, see U.S. Patent Nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, pens, bottles, and any packaging materials suitable for the selected formulation and intended mode of administration and treatment. A wide range of formulations of the AONs and compositions provided herein are contemplated.

[0081] In certain embodiments, kits are also provided herein that can facilitate use by a medical practitioner in administering an appropriate amount of an AON to a subject. In certain embodiments, the kits provided herein include a container and a dosage form of an AON provided herein, or a pharma- ceutically acceptable salt, solvate, or prodrug thereof.

[0082] In certain embodiments, the kit comprises a container containing a dosage form of an AON or a pharma- ceutically acceptable salt, solvate, or prodrug thereof provided herein within a container that contains one or more other therapeutic agents described herein.

[0083] The kits provided herein may further comprise a device used to administer the AON. Examples of such devices include, but are not limited to, syringes, needleless injector drip bags, patches, and inhalers.

[0084] The kits provided herein may further comprise a pharma- ceutically acceptable vehicle that can be used to administer one or more active ingredients.For example, if the active ingredient is provided in a solid form that needs to be prepared into a solution for parenteral administration, the kit may comprise a sealed container of a suitable vehicle in which the active ingredient can be dissolved to form a particulate-free sterile solution suitable for parenteral administration.Examples of pharma- ceutically acceptable vehicles include, but are not limited to, aqueous vehicles, including but not limited to USP Water for Injection, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles, including but not limited to ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles, including but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0085] VII. Combination Therapy with a Second Active Agent Also provided herein are methods of combination therapy using the AONs disclosed herein with other therapeutic agents useful for treating or delaying the onset of DMD. In another embodiment, provided herein are methods of combination therapy using AON1 with other therapeutic agents useful for treating or delaying the onset of DMD. In these methods, the AONs disclosed herein, such as AON1, ​​are administered, for example, QWx24W, as described elsewhere herein.

[0086] As used herein, the term "in combination" includes the use of two or more therapies (e.g., one or more prophylactic and / or therapeutic agents). However, the use of the term "in combination" does not restrict the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject with a disease or disorder. The first therapy (e.g., a prophylactic or therapeutic agent, such as an AON provided herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to), simultaneously with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the second therapy (e.g., a prophylactic or therapeutic agent) to the subject. Triple therapy is also contemplated herein.

[0087] The AONs disclosed herein and one or more second active agents can be administered to a subject simultaneously or sequentially, by the same or different routes of administration. The suitability of a particular route of administration for a particular active agent will depend on the active agent itself (e.g., whether it can be administered orally without being degraded before entering the bloodstream) and the disease or disorder being treated.

[0088] The route of administration of the AONs disclosed herein is independent of the route of administration of the second therapeutic agent. In another embodiment, the AONs disclosed herein are administered intravenously. Thus, according to these embodiments, the AONs disclosed herein are administered orally or intravenously, and the second therapeutic agent can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, bucally, intranasally, liposomally, via inhalation, intravaginally, intraocularly, via local delivery by catheter or stent, subcutaneously, intraadipose, intraarticularly, intrathecally, or in a sustained release dosage form. In one embodiment, the AONs disclosed herein and the second therapeutic agent are administered by the same mode of administration, e.g., IV. In another embodiment, the AONs disclosed herein are administered by one mode of administration, e.g., IV, while the second agent is administered by another mode of administration, e.g., orally.

[0089] In one embodiment, the second active agent is administered intravenously or subcutaneously in an amount of about 1 to about 1000 mg, about 5 to about 500 mg, about 10 to about 350 mg, or about 50 to about 200 mg once or twice daily. The specific amount of the second active agent will depend on the specific agent used, the type of disease being treated or managed, the severity and stage of the disease, and the amount of the AON or derivative thereof provided herein and any optional additional active agent(s) that are administered simultaneously to the subject.

[0090] One or more second active ingredients or agents may be used in conjunction with the AONs or derivatives thereof provided herein in the methods and compositions provided herein. The second active agent may be a large molecule (e.g., a protein) or a small molecule (e.g., a synthetic inorganic, organometallic, or organic molecule).

[0091] Examples of large molecule active agents include, but are not limited to, hematopoietic growth factors, cytokines, AONs, and monoclonal and polyclonal antibodies.Typical large molecule active agents are biomolecules such as naturally occurring, synthetic, or recombinant proteins.Specific examples of AONs as second active agents for use herein include eteplirsen, casimersen, golodirsen, viltolarsen, and SRP-5051 (Sarepta Therapeutics).

[0092] Examples of small molecules include corticosteroids such as deflazacort.

[0093] Other therapies that may be combined with the AONs disclosed herein include gene therapy agents (e.g., SRP-9001, GALGT2, or GNT0004 (Sarepta Therapeutics)), gene editing (e.g., CRISPR / CAS9 (Sarepta Therapeutics)), and cell therapy agents (e.g., CAP-1002 (Caprior Therapeutics / Nippon Shinyaku Co. Ltd.)). EXAMPLES

[0094] VIII. Examples The following examples are intended to illustrate certain embodiments provided herein, and are not intended to limit the scope of the disclosure.

[0095] Example 1 25-week administration of AON1 in del52hDMD / mdx mice

[0096] hDMD del52 / mdx(+ / +) and C57BL / 6J wild type (WT) mice (approximately 7 weeks old on day 1) were divided into four groups and dosed as follows: [Table 1]

[0097] A dose of 6 mg / kg / dose is equivalent to 0.92 μmol / kg / dose (MW of AON1 is 6517 g / mol). A dose of 18 mg / kg / dose is equivalent to 2.76 μmol / kg / dose. Thus, the total amount of AON1 administered over 25 W is 150 mg / kg (23.00 μmol / kg) at the 6 mg / kg dose level and 450 mg / kg (69.00 μmol / kg) at the 18 mg / kg dose level.

[0098] After administration, plasma concentration, tissue distribution, concentration, exon skipping, overall gait score, and cytoprotection were analyzed. The results are shown in Figures 1 to 6.

[0099] Figure 1 shows plasma concentrations of AON1 over time. Figure 2 shows AON1 concentrations in heart, quadriceps, liver, diaphragm, gastrocnemius, and kidney tissues. Figure 3 shows that AON1 achieved sustained dose-dependent exon 51 skipping in muscle tissues (quadriceps, gastrocnemius, heart, and diaphragm) over 25 weeks of treatment.

[0100] Figure 4 shows wt% dystrophin in heart and quadriceps and total gait scores for AON1 (mean ± SEM, N=16-18 (baseline, 29 weeks) or 10-12 (37 weeks). Statistical significance: #p<0.05, ####p<0.0001 compared to C57BL / 6J vehicle; *p<0.05, ***p<0.0005, ****p<0.0001 compared to Del52 / mdx vehicle). AON1 improves and maintains dystrophin and gait scores over 25 weeks of treatment. Reference for fine motor and gait testing: NA Datson et al.(2020),NUCID THERAPEUTICS. DOI:10.1089 / nat.2019.0824.

[0101] Figure 5 shows that AON1 treatment attenuates muscle pathology associated with the hDMD del52(+ / +) model and maintains pharmacological effects for 12 weeks after 25 weeks of treatment. The gastrocnemius muscles of vehicle-treated mice showed mild atrophy, minimal necrosis, minimal inflammation (non-nucleated cell infiltration), and minimal fibrosis. In contrast, the gastrocnemius muscles of AON1-treated mice showed minimal atrophy without other effects.

[0102] Figure 6 shows that AON treatment attenuates cardiac pathology associated with the hDMD del52(+ / +) model, with the pharmacological effect sustained for 12 weeks. The myocardium of vehicle-treated mice showed mild atrophy, minimal necrosis, minimal inflammation (non-nuclear cell infiltration), and minimal fibrosis. In contrast, the myocardium of AON1-treated mice showed minimal inflammation (mononuclear cell infiltration) and minimal fibrosis without other effects.

[0103] Example 2 Synthesis of AON1

[0104] The oligonucleotide, AON1, ​​was synthesized by solid-phase organic synthesis. No intermediates were isolated or characterized during the synthetic process. After completion of the synthetic sequence, the oligonucleotide was cleaved from the solid support, deprotected, and purified by ultrafiltration and preparative anion exchange (AEX) chromatography. After pooling of the HPLC fractions, the oligonucleotide AON1 solution was concentrated and desalted by a second ultrafiltration step. Subsequent lyophilization and homogenization yielded the product AON1 as a solid material, which was stored at -20±5°C prior to shipping.

[0105] The synthesis cycles performed at ambient temperature are outlined below (the first cycle is used for illustrative purposes). The oligonucleotide chains of AON1 were synthesized on a solid support. After completion of the sequence, the oligonucleotides were cleaved from the resin, purified, and the resulting solution was lyophilized to obtain the final drug substance AON1. [ka]

[0106] Stage 1: Solid-phase synthesis

[0107] The oligonucleotide portion of AON1 was produced by solid-phase organic synthesis using platform synthesis. The synthesis was automated and carried out on a synthesizer equipped with a closed column reactor using coupling recycling technology without isolation of intermediates. The solid support equipped with a universal linker was loaded into a flow-through column reactor and the synthesis was started. Each synthesis cycle consisted of four chemical steps, which were carried out sequentially, followed by washings until the full-length oligonucleotide was established.

[0108] Step 1: Deprotection

[0109] In the first step, designated as deblocking or deprotection, the solid support was treated with dichloroacetic acid in toluene to remove the protecting group, dimethoxytrityl (DMT), from the UnyLinker™ on the solid support. In all subsequent cycles of step 1, deblocking involved the removal of the DMT protecting group from the 5'-OH of each successively introduced phosphoramidite or bridged amidite to generate 5'-DMT-off oligonucleotides.

[0110] Step 2 Activation and coupling

[0111] The second step, designated "activation and coupling", was a chain extension step that added the next nucleotide to the sequence, extending from the 3' to the 5' end. Unprotected alcohol groups of either the UnyLinker™ (first cycle) or the 5'-DMT-off oligonucleotide strand [n] (all subsequent cycles) were treated with a solution of the corresponding phosphoramidite in acetonitrile or dichloromethane / acetonitrile in the presence of a suitable activator solution, e.g., benzylthiotetrazole in acetonitrile, to build an elongated DMT on the oligonucleotide strand [n+1]. In the final chain extension step, a TEG group was incorporated at the 5' end of AON1 using DMT-TEG-phosphoramidite without sulfurization.

[0112] Step 3 Oxidation / sulfurization

[0113] Depending on the desired backbone chemistry, the corresponding phosphorothioate or phosphodiester was introduced in this step by sulfurization or oxidation. The DMT-on oligonucleotide chain [n+1] was exposed to a sulfurizing agent, e.g., 5-N-(dimethylamino)methylene]amino-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine / acetonitrile, or an oxidizing agent (e.g., iodine in pyridine / water). During the preparation of AON1, ​​only the last cycle involves an oxidation step, while all other cycles involve a sulfurization reaction.

[0114] Step 4: Capping

[0115] The fourth step, designated "capping", terminated the synthesis cycle by adding a mixture of Cap A (acetonitrile / 2,6-lutidine / N-methylimidazole) and Cap B (tert-butylphenoxyacetyl acetic anhydride in acetonitrile) to the oligonucleotide on the solid support to prevent any unreacted 5'-OH oligonucleotide strand [n] from further reacting with phosphoramidites in subsequent coupling cycles. This led to the formation of truncated sequences. After the synthesis cycles for all 18 amidites were completed, the base- and backbone-protected oligonucleotides continued to be attached to the solid support.

[0116] Stage 2: Backbone deprotection

[0117] The oligonucleotides on the solid support were treated with diethylamine in acetonitrile, leading to the removal of the β-cyanoethyl protecting groups on the phosphorothioate / phosphate backbone. The reaction was followed by a washing step using acetonitrile.

[0118] Stage 3: Deprotection of the base and cleavage from the resin

[0119] The column was incubated with aqueous ammonia / ethanol at ambient temperature, then washed with aqueous ammonia / ethanol. The combined solution was incubated at elevated temperature. In this two-step procedure, crude oligonucleotide was cleaved from solid support and nucleobase protecting group was removed. The resulting solution was filtered and quenched with the filtrate containing crude oligonucleotide dissolved in deprotection solution.

[0120] Stage 4: Desalting of oligonucleotides by ultrafiltration

[0121] The solution of crude AON1 was concentrated by ultrafiltration using an ultrafiltration membrane. The resulting concentrate was diafiltered using water for injection. The resulting aqueous solution of AON1 was filtered.

[0122] Stage 5: Purification of crude oligonucleotides by anion exchange chromatography

[0123] The reaction mixture was diluted in aqueous sodium phosphate / acetonitrile buffer and purified by anion exchange (AEX) chromatography using SOURCE™ 30Q.

[0124] A mock pool was generated and analyzed by HPLC-UV / MS. According to the analytical results, fractions were selected for pooling and further downstream processing. The selected fractions were combined to obtain a product pool of appropriate purity (>85%) and yield. This final product pool was analyzed again by HPLC-UV / MS.

[0125] Stage 6: Desalting of oligonucleotides by ultrafiltration

[0126] The pooled solution of AON1 was concentrated by ultrafiltration using an ultrafiltration membrane. The resulting concentrate was diafiltered using water for injection. The resulting aqueous solution of AON1 was filtered.

[0127] Stage 7: Oligonucleotides as lyophilized solids

[0128] The concentrated solution obtained in stage 6 was filtered through a 0.2 μm membrane and finally lyophilized to yield the final drug substance. After lyophilization, the material was equilibrated for a minimum of 24 hours in a clean room environment to obtain a stable water content of the final product. The equilibrated drug substance was obtained as a white to off-white to yellow powder (mp: approx. 265°C (DSC decomposition); purity 92-93% by ion-pair reversed-phase HPLC-UV / MS). Example 3

[0129] Process for manufacturing 50 mg / mL AON1

[0130] Buffer formulation and optional bioburden reduction filtration

[0131] Weigh out the required amounts of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, and sodium chloride in approximately 80% of the total volume of Water for Injection (WFI) and stir under laminar air flow (LAF) in a Grade C environment. After the excipients are dissolved, add WFI to the final volume.

[0132] If the buffer blend and formulation solution blend are not performed on the same day, the buffer is filtered through a Grade C 0.45 / 0.2 μm membrane filter unit under LAF to reduce bioburden.

[0133] Solution Equilibration, Blending, and Bioburden Reduction Filtration

[0134] Lyophilized AON1 (Example 2) is equilibrated at room temperature for 12-24 hours in a Grade C environment under LAF. After equilibration, the lyophilized AON1 is weighed and dissolved in approximately 50% buffer by stirring to avoid clumping. A final AON1 concentration of 50 mg / mL of solution is achieved by adding a calculated amount of buffer. The solution is mixed at 200-1000 rpm for 20-30 minutes by avoiding foaming. The compounded solution is filtered through a sterile 0.45 / 0.2 μm membrane filter unit to reduce bioburden. Compounding and bioburden reduction filtration are performed in a Grade C environment under LAF.

[0135] In-line sterile filtration and aseptic filling

[0136] The AON1 solution is sterile filtered through two consecutive in-line filter units using 0.45 / 0.2 μm filters and aseptically filled into ready-to-use sterile vials. The vials are capped and sealed. The filled units are stored in a temperature-controlled refrigerated warehouse.

[0137] The AON1 composition is supplied in a single-dose vial as a 3 mL isotonic, sterile, preservative-free solution for intravenous infusion at a concentration of 50 mg / mL at pH 7.0. The container closure system is a Type 1 glass vial with a fluoropolymer-coated bromobutyl rubber stopper and an aluminum seal with a flip-off cap. The composition of the 5.25 L AON1 solution is shown below:

[0138] [Table 2]

[0139] Example 4 AON1 PS was administered to male and female del52hDMD / mdx mice for 13 weeks and evaluated 2 or 4 weeks after administration.

[0140] A total of 120 homozygous hDMD del52 / mdx+ / + male and female mice bred at Charles River, UK and genotyped at BioLytix, Switzerland were used for the study. All hDMD del52 / mdx mice in this study were from a re-derived colony. In addition, age-matched C57BL / 6J (n = 20) mice (Charles River, Germany) were used as wild-type (WT) controls. When setting up the different groups for this study, mice were randomized into groups by sex to ensure that complete litters of mice were not included in a single test group and to take into account baseline body weights, so that there were no group differences at the start of the first treatment.

[0141] Mice were weighed on each treatment day and doses were adjusted accordingly. The entire IV injection process took 5-8 min per mouse, with actual injection times ranging from 10-60 s.

[0142] All compounds were formulated at concentrations that ensured equimolar dosing and were administered in an injection volume of 8 mL / kg. Mice were weighed on each treatment day and doses were adjusted accordingly. For example, a mouse weighing 30 g received an IV injection of 240 μL.

[0143] Mice were administered AON1 PS for 13 weeks. At the end point 14 or 28 days after the last IV injection, mice were euthanized by deep anesthesia with sodium pentobarbital (60 mg / kg Mebunat, Orion Pharma, Finland). Mice were subjected to cardiac puncture and cardiac tissue was harvested. Tissue was immediately snap frozen by immersion in isopentane au bain marie in liquid nitrogen, placed in pre-cooled cryovials on dry ice, and stored at -80°C. Frozen samples were analyzed for exon skipping % and dystrophin levels by qualified methods. Results are shown in Figures 7 and 8.

[0144] Example 5 AON1 PS was administered to male and female del52hDMD / mdx mice for 13 weeks and evaluated 2 or 4 weeks after administration.

[0145] All mice were given IV tail vein injections once a week (QW) for a maximum study period of 13 weeks with vehicle or AON1, ​​starting at 7 weeks of age. The entire IV injection process took 5-8 minutes per mouse, with actual injection time of 60 seconds. Compounds were formulated at specific concentrations and administered in an injection volume of 8 mL / kg. Mice were weighed on each treatment day and doses were adjusted accordingly. For example, a mouse weighing 30 g received an IV injection of 240 μL.

[0146] At the end of the survival period, 4 or 8 weeks (days 113 and 141, respectively) after the last IV injection on day 85, mice were transcardially perfused with PBS to remove blood from tissues after blood collection by cardiac puncture. The following tissues were collected and flash frozen:

[0147] Skeletal muscle: gastrocnemius (R), quadriceps (L+R), collect both sides into one vial so that quadriceps tissue has one sample.

[0148] Half the heart (dissected so that both atria and ventricles are present in each half), the entire diaphragm, the liver (2 × 1 / 3 of the entire liver), and the kidney (R).

[0149] All tissues were snap frozen by immersion in isopentane au bain marie in liquid nitrogen, placed in cryovials pre-cooled on dry ice, and stored at -80°C until sectioning.

[0150] Exon skipping analysis was performed on gastrocnemius, quadriceps, heart, and diaphragm tissues from all groups. The analysis was performed using a qualified reverse transcriptase digital droplet polymerase chain reaction (RT-ddPCR) analysis method. The percentage of skipping of hDMD exon 51 was calculated by the following formula:

[0151] (51 human skipped exon copies / 20 μL reaction) / (51 human skipped exon copies / 20 μL reaction + 51 human non-skipped exon copies / 20 μL reaction)*100

[0152] The lower limit of quantification (LLOQ) of skipped and non-skipped transcripts was based on a threshold technique. The results are shown in Figure 9. Dystrophin levels in heart and quadriceps are shown in Figure 10. Total ambulation scores are shown in Figure 11.

[0153] Example 6 The effects of AON1 PS and AON2 on complement activation in human and cynomolgus monkey serum were examined.

[0154] The dose vehicle for test article administration was aqueous phosphate buffer (pH 6.9-7.1) containing 0.7% NaCl, which was prepared and used as a baseline control. The appropriate amount of test article (AON1 PS or AON2) was weighed and dissolved in a visually clear solution at 10x dose concentration using stirring / vortex mixing. No correction factor was applied to the measured weights. Formulations were stored refrigerated (4°C) before and after use. All test article formulations were warmed to room temperature for at least 30 minutes on the laboratory bench prior to use. Test articles were considered stable at 4°C storage for 1 week after preparation. Actual weights and dilutions were recorded in the study data. Dose analysis was not performed in this study. Residual formulations were discarded.

[0155] Testing Systems

[0156] Test system 1: n=3 individual sera from healthy male human volunteers (>18 years old).

[0157] Test system 2. n=3 individual sera from normal male cynomolgus monkeys (>3 years old).

[0158] Route of Administration

[0159] This is an in vitro study, where the test article was exposed to the test system in vitro. The test article was added to the test system in a ratio of 1 part to 9 parts (1:9, v / v). This ratio maintained the proper concentration of the test system and therefore the concentration of the complement control protein was sufficient.

[0160] Dosing regimen

[0161] The mixture was incubated at 37°C ± 2°C for 30 ± 2 minutes and stored at -80°C until analyte testing.

[0162] Objectives and Study Protocol

[0163] The objective of this study was to evaluate the complement split product activation profile of two antisense oligonucleotides (AON1 PS and AON2) after incubation in normal male human and cynomolgus monkey serum.

[0164] Experimental design

[0165] Testing was performed by mixing 270 μL of the test system with 30 μL of the prepared 10x test article in a 1.5 mL polypropylene microcentrifuge snap-cap tube. Once all mixtures were prepared, they were transferred to a 37°C ± 2°C water bath and incubated for 30 ± 2 minutes. After the mixtures were incubated, all samples were aliquoted and frozen at -80°C or colder.

[0166] Biological analysis of complement activation

[0167] Bb cleavage products by ELISA: Bb is generated upon cleavage of factor B upon activation of the alternative pathway of complement. The level of Bb produced during in vitro exposure to test substances can serve as a measure of the level of alternative pathway activation. Bb ELISAs are performed in duplicate wells for reporting average values. Levels of Bb were initially assessed at two dilutions to ensure that the assay was not saturated by highly activated samples.

[0168] C3a split products by ELISA: C3a is generated when C3 is cleaved at the complement midpoint. Thus, C3a cleavage can occur after activation of any of the three complement pathways. The level of C3a is also important because it is an anaphylatoxin. The level of C3a produced during in vitro exposure to the test substance can serve as a measure of the level of complement activation strong enough to reach this midpoint, as well as an indication of possible direct proinflammatory consequences of this activation. C3a ELISA is performed in duplicate wells to report average values. C3a levels were initially assessed at two dilutions to ensure that the assay was not saturated by highly activated samples.

[0169] The results are shown in Figures 12 and 13.

[0170] Example 7 26-week toxicity and bioanalysis study in young mice

[0171] In a 26-week juvenile toxicity and toxicokinetic (TK) study with 13 weeks of recovery, male CD-1 mice (20-22 / group (main toxicity study), 6 (vehicle; vehicle control buffer diluted in sterile saline), 25-31 / group (TK); 21-27 / group (recovery)) were administered 0 (vehicle control buffer), 6, 12, or 18 mg / kg AON1 by bolus IV injection once every 4 days from PND 21 to PND 203. For bioanalysis, blood was collected from AON1-treated groups on PND 21 and 203 pre-dose (PND 203 only), and 0.5, 1, 3, 8, and 24 hours post-dose. The following parameters were assessed: body weight, food intake, developmental landmarks (balanoplasty), neurobehavioral assessments (locomotor activity at ≈PND 187 and 281, acoustic startle at ≈PND 190 and 284, Morris water maze at ≈PND 193 and 287), including ophthalmological examination, clinical pathology (clinical chemistry, hematology, and coagulation), gross dissection, organ weights, and histopathology.

[0172] Based on the lack of adverse findings, the no observed adverse effect level (NOAEL) was 18 mg / kg / dose. At PND21, this dose level had (AUC 0 ~24 ) values ​​were the maximum observed concentrations (C max At PND203, this dose level corresponds to a C of 5860 nmol / L and 14,600 h*nmol / L, respectively. max and AUC 0 Equivalent to a value of ~24.

[0173] Example 8 NHP 39-week toxicity and bioanalysis study

[0174] Male cynomolgus monkeys (3 / group (main toxicity study); 2 / group of 0, 6, 12, and 18 mg / kg QW (13 week recovery)) were administered 0 (vehicle: 0.9% NaCl injection, USP), 6, 12, or 18 mg / kg QW AON1 via IV infusion (60 min) for 39 weeks. At different study days / time points, blood was collected for bioanalysis / TK, and complement activation analysis. The following parameters were evaluated: mortality, clinical signs, body weight, food intake, AON1 levels in plasma and tissues (gastrocnemius, heart, biceps femoris, liver, and kidney), percentage of exon skipping in tissues (gastrocnemius, heart, biceps femoris, liver, and kidney), clinical pathology (clinical chemistry, hematology, urinalysis, urine chemistry, and urine biomarkers), complement activation indices (Bb, C3a, and sC5b-9), ophthalmology, ECG, organ weights, and histopathology.

[0175] Mean tissue concentrations of AON1 were quantifiable in all AON1 treatment groups at scheduled and unscheduled terminal euthanasia on Day 274. Mean concentrations generally increased with increasing dose up to 6-12 mg / kg / dose. The ranked mean concentrations of AON1 on Day 274 in the 6-12 mg / kg / dose dosing phase were liver, kidney, heart, gastrocnemius, and biceps femoris.

[0176] Exon skipping in tissues was measured by RT-ddPCR. This method quantified the number of non-skipped DMD (dystrophin) transcripts and the number of exon 51 skipped DMD transcripts, and the percentage of exon skipping (% skip) was calculated. The percentage of exon 51 skipping ranged from 4.5% to 40.8%, and was higher in the high dose groups (12 and 18 mg / kg / QW) than in the low dose group (6 mg / kg / QW). See also Figure 14.

[0177] No AON1-related changes in body weight; ophthalmologic findings; veterinary observations; ECG waveform abnormalities or arrhythmias; or effects on PR interval, QRS duration, QT or corrected QT (QTc) interval, or heart rate were observed. No AON1-related effects on clinical pathology (hematology and coagulation) were observed in animals dosed up to 18 mg / kg / dose. No AON1-related effects on macroscopic observations or organ weights were observed at early or scheduled sacrifice in males dosed up to 18 mg / kg / dose.

[0178] One male receiving 12 mg / kg / dose or 18 mg / kg / dose was sacrificed on days 194 or 211 of the treatment phase, respectively, for AON1-related moribundity. Clinical and veterinary observations were similar in both animals and included decreased activity, thin appearance, and pitting or subcutaneous edema of the lower trunk and / or extremities. Clinicopathological findings showed inflammation, azotemia, hypoalbuminemia, and elevated urinary protein (in one animal measured). The cause of moribundity was attributed to glomerular renal injury and multisystem vascular inflammation. AON1-related clinicopathological effects in unscheduled sacrifice animals supported inflammatory responses, water deprivation, possible intestinal loss, agitation / physiologic responses, and / or a moderate decrease in platelet count, lacking a clear mechanism. Microscopic findings in unscheduled sacrificed animals included renal tubule degeneration / regeneration, vascular degeneration, hypertrophy, and inflammation of the vascular wall and / or perivascular tissues, which may have resulted in interstitial edema, thrombosis (lungs), and / or local degenerative changes in diseased tissues (heart). Edema may also have been associated with significantly lower serum protein, which was attributed to glomerular changes. These findings and their sequelae were consistent with a complement-mediated pathogenesis predisposing monkeys to AON therapy (Frazier Toxicol Pathol. 2015,Jan;43(1):78-89). The reason for increased susceptibility to AONs in NHPs has been linked to genetics of factor H components in cynomolgus monkeys, which are understood to increase binding of factor H to AONs. The reduction in the amount of free factor H required to regulate complement activation removes the key regulator of the alternative pathway.

[0179] Low levels of transient complement activation involving the alternative and terminal pathways were observed in all treatment groups, including the control group. The increase in complement activation generally decreased to near pre-dose levels by 72 hours post-dose in most animals, although activation levels tended to increase over the course of the study. Two animals that were scheduled to be sacrificed early had >200% increases in Bb levels compared to pre-dose levels on days 113 and 204, respectively. While the complement activation profile in these two animals was consistent with the complement associated with the kidney injury observed, other animals also showed increases in measured complement fragments. Differences between animals may be due to individual differences in the ability of animals to control complement activation once induced, given the large number of complement regulators present. The mechanism of complement activation via non-specific interaction of AONs with factor H protein was observed to be of very limited clinical relevance because human complement activation occurs at much higher AON plasma concentrations (Henry Int. Immunopharmacol. 2002;2:1657-66; Henry Antisense Drug Technology: Principles, Strategies, and Applications, 2nd ed. CRC Press: Carlsbad, CA;2008:327-63; Shen J Pharmacol Exp Ther. 2014 Dec;351(3):709-17).

[0180] AON1-related clinical pathology effects were observed in scheduled sacrifice animals dosed at 6 mg / kg / dose and above. Mild increases in alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities in animals dosed at 18 mg / kg / dose on days 176 (except ALT), 204, 211, 216, and 218 of the treatment phase were suggestive of liver damage. Effects supporting an inflammatory response included mildly decreased albumin concentrations and increased globulin concentrations (resulting in a decreased albumin:globulin ratio) in animals dosed at 18 mg / kg / dose on days 176, 204, 211, 216, and 218 of the treatment phase, and mildly increased haptoglobin concentrations in animals dosed at 18 mg / kg / dose on days 211, 216, and 218 of the treatment phase. Additionally, albumin was lost via the kidney. This is supported by mildly elevated urinary protein:urinary creatine ratios and increased incidence of protein in urine in animals receiving 18 mg / kg / dose on days 211, 216, and 218 of the dosing phase. Mild to moderate increases in microalbumin:creatinine ratios in animals receiving 6 mg / kg / dose and above on days 211, 216, 218, 225, 239, 253, 260, 267, and 274 of the dosing phase also supported loss of albumin via the kidney. Mild increases in clusterin:creatinine ratios in animals receiving 18 mg / kg / dose on days 211, 216, and 218 of the dosing phase also supported kidney damage. An additional effect was a mild, non-dose-dependent decrease in platelet counts on days 176, 204, 211, 216, 218, 225, 239, 253, 260, 267, and 274 of the treatment phase in animals receiving 6 mg / kg or more. The dose lacked a clear mechanism. A decrease in platelet counts over time leading to moderate / marked thrombocytopenia was observed in two animals in the 18 mg / kg QW group, but was not associated with anatomopathological findings. No AON1-related coagulation effects were observed in scheduled sacrifice animals.

[0181] Microscopic findings in two early terminally sacrificed animals and one scheduled for planned sacrifice at 18 mg / kg / dose on day 218 of the treatment phase included minimal degeneration of renal tubular epithelium in one animal and minimal mixed cell inflammation in the liver of another animal. This correlated with increased liver enzyme values. These findings were attributed to accumulation of AON1 in renal tubular epithelium or Kupffer cells, respectively. Additional AON1-related findings common to moribund and early sacrificed animals included mild or moderate mononuclear cell infiltration in cardiac valves and a trend toward increased incidence of mononuclear infiltrates in several tissues. These findings may reflect the presence of treatment-related systemic inflammation and / or complement activation and, with the exception of valvular changes, were considered an exacerbation of incident findings commonly observed in monkeys. In all animals, findings associated with AON1 accumulation included basophilic granules in renal tubular epithelium and Kupffer cells (generally in a dose-dependent manner), and the presence of macrophages characterized by foamy and / or slightly basophilic cytoplasm, with changes in multiple tissues but most prominent in lymph nodes. The accumulation did not appear to be associated with damage to surrounding tissues in animals receiving 12 mg / kg / dose or higher, and macrophage changes did not appear to be associated with damage at any dose. Microscopic findings at the time of recovery euthanasia in mandibular and / or mesenteric lymph nodes from animals receiving 6 mg / kg / dose or higher showed a trend toward reversibility, as demonstrated by minimal basophilic granules in the kidneys, minimal pigment in Kupffer cells in the liver, and minimal to moderate infiltration of vacuolated / pigmented macrophages. In addition, minimal vacuolated / pigmented macrophage infiltration was noted in the gastrointestinal tract and urinary bladder of one animal treated with 12 or 18 mg / kg / dose, respectively (no transitional cell degeneration / vacuoles were observed in this monkey study) and in the testes of one animal treated with 18 mg / kg / dose.

[0182] Based on the severity of clinical pathology and anatomopathology findings, the no observed adverse effect level (NOAEL) for the overall effects on health and well-being of AON1-treated males was considered to be 6 mg / kg / dose, with a mean maximum observed concentration (Cmax) of 17,000 nmol / L on day 260 of the treatment phase. max ) and the area under the concentration-time curve (AUC 0-25 ) was equivalent to

[0183] Example 9 First human dose selection

[0184] The proposed dose and clinical trial duration (Example 10) were selected to ensure safety and visibility of direct benefit to each pediatric participant enrolled in the study.

[0185] The first human clinical trial (Example 10) involves sequential evaluation of up to six dose levels of AON1: IV administration of 0.6 mg / kg, 1.5 mg / kg, 3 mg / kg, 6 mg / kg, 9 mg / kg, and 12 mg / kg.

[0186] The starting dose of clinical trial (Example 10) was determined using the NOAEL of AON1 obtained from the GLP 13-week repeated dose toxicity study (18 mg / kg / week) in male cynomolgus monkeys (Example 13). The human equivalent dose (HED) of the monkey NOAEL of 18 mg / kg QW is 5.8 mg / kg QW based on the body surface area scaling approach. Therefore, the selected starting dose of 0.6 mg / kg provides a 10-fold safety margin based on monkeys, which are considered to be a more relevant species for the identification of hazardous substances because they are more sensitive to complement activation and the resulting inflammatory response. This is considered to be one of the class effects of antisense oligonucleotides (the latter being the main cause of nonclinical toxicity in monkeys) (Shen J Pharmacol Exp Ther.2014 Dec;351(3):709-17). It is widely accepted that the use of monkeys maximizes the likelihood of identifying any adverse reactions that are quantitatively and qualitatively similar to those that might be expected in humans (Farmman Toxicol Pathol. 2003;31:119-122).

[0187] Given that accumulation is expected to be low due to the short plasma half-life of AON1 compared to weekly dosing intervals, the escalation of dose levels every 2 weeks in Cohort 1A (Part 1) is considered appropriate to minimize the exposure period of individual participants at sub-therapeutic dose levels. The 2- to 2.5-fold dose increments allow for safe and efficient escalation of individual participants through doses where non-clinical data indicates a high likelihood of safety. The toxicity findings observed in the 39-week study are associated with chronic dosing and would not be expected with an initial single ascending dose in a clinical trial (Example 10).

[0188] It has previously been shown that plasma exposure of phosphorothioate oligonucleotides is similar between NHP and DMD patients for the same mg / kg dose (Bosgra Nucleic Acid Ther. 2019;29(6):305-322). Thus, the use of body surface area scaling provides a more conservative approach than body weight scaling for starting dose selection for the single escalating dose portion of clinical trials. For the repeat-dose phase of clinical trials, we apply direct body weight-based (mg / kg) extrapolation from NHP to humans and suggest sequential evaluation of 6, 9, and 12 mg / kg QW.

[0189] The starting dose of 6 mg / kg QW for the repeat dosing phase will be chosen to ensure that each pediatric participant derives direct benefit because it is a pharmacologically active dose that is expected to result in steady-state dystrophin expression >10% of normal. The DMC will review safety data for dosing cohorts through week 4 and make recommendations regarding dose escalation.

[0190] The maximum dose of 12 mg / kg / week has been selected to maximize the potential efficacy of treatment with BMN351 with a manageable safety profile, and is justified and informed by chronic toxicity studies: Overall, the major toxicity signals in the chronic toxicity studies (39-week monkey study (Example 8), 26-week mouse study (Example 11), and juvenile mouse study (Example 7)) are all consistent with known class effects of phosphorothioate oligonucleotides that can be clinically monitored. ●Dose-limiting toxicity was observed in a 39-week monkey study, where the NOAEL was determined to be 6 mg / kg / week. A dose above the NOAEL identified in the 39-week monkey study (6 mg / kg / week) is considered appropriate because the dose-limiting toxicity at 12 mg / kg QW and above appears to be consistent with complement-mediated effects, to which NHPs are known to have exaggerated sensitivity compared to other preclinical species and humans. (Barbour Nephrol Dial Transplant.2013 Jul;28(7):1685-93, Shen J Pharmacol Exp Ther.2014 Dec;351(3):709-17). Thus, complement-mediated effects should be considered as identifying risk rather than providing an error for clinical safety. A decrease in platelet count over time leading to moderate / marked thrombocytopenia was observed in two animals in the 18 mg / kg QW group in the 39-week monkey study, but was not associated with anatomic pathology findings. Thrombocytopenia can be monitored clinically.

[0191] The liver, kidneys, blood (thrombocytopenia), coagulation system, and vasculature (inflammatory changes) have been identified as target organs for human toxicity based on nonclinical studies.

[0192] Risk monitoring and mitigation activities will be implemented through frequent clinical and laboratory surveillance, including the use of organ-specific toxicity biomarkers, clearly outlined DLT criteria, and study stopping criteria. Safety data will be evaluated before the Data Monitoring Committee (DMC) recommends either dose escalation, dose tapering, opening of new cohorts, or cohort expansion.

[0193] Example 10 A Phase 1 / 2 Dose-Escalation Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of AON1 in Participants With Duchenne Muscular Dystrophy

[0194] This study will investigate the safety and tolerability of AON1 at six ascending doses via intravenous (IV) infusion. Given the progressive nature of Duchenne muscular dystrophy (DMD), this study is open-label and all participants will receive AON1. The proposed dose and duration of the study will be selected to ensure safety and the prospect of direct benefit to each pediatric participant enrolled in the study. The formulation to be used is described in Example 3.

[0195] The no observed adverse effect level (NOAEL) from a pivotal 13-week GLP toxicology study in monkeys was determined to be 18 mg / kg QW. The human equivalent dose (HED) is therefore 5.8 mg / kg QW, based on a body surface area scaling approach. The selected starting dose of 0.6 mg / kg therefore provides a 10-fold monkey-based safety margin, which may be more relevant for the identification of hazardous substances, as monkeys are more sensitive to complement activation and the inevitable inflammatory response, which is one of the class effects of antisense oligonucleotides (Shen L, Frazer-Abel A, Reynolds PR, et al. Mechanistic understanding for the greater sensitivity of monkeys to antisense oligonucleotide-mediated complement activation compared with humans. J Pharmacol Exp Ther. 2014 Dec;351(3):709-17). The lower (2-2.5-fold) dosing further increases the likelihood of early detection of any potential dose-dependent safety signals, thus allowing for earlier intervention.

[0196] Dosing of participants at each of the six dose levels (0.6 mg / kg, 1.5 mg / kg, 3 mg / kg, 6 mg / kg, 12 mg / kg, and 18 mg / kg) will be staggered at intervals based on previous safety experience with this class of molecule. A Data Monitoring Committee (DMC) will evaluate available safety data before recommending whether to dose escalate, dose de-escalate, open new cohorts, or expand cohorts. This design includes careful monitoring of participant safety, including acute safety events that are known class effects of antisense oligonucleotides (AONs) prior to dose escalation, and implementation of individual and study stopping rules, as appropriate.

[0197] This study is being conducted to prove proof of concept by dystrophin expression combined with safety, tolerability, and pharmacokinetics (PK) to evaluate dose response and provide insight into dose selection for further clinical development. Studies have shown that the higher the amount of dystrophin in muscle, the less severe the disease phenotype (de Feraudy et al. Ann Neurol. 2021 ;89(2):280-292). Therefore, increased dystrophin expression is a meaningful outcome for this study.

[0198] Plasma PK and muscle distribution will be assessed to explore the relationship between dose, plasma and muscle exposure, exon skipping, and dystrophin expression.

[0199] Readouts of AON1 distribution, exon skipping, and dystrophin levels in muscle were performed after all participants completed 12 weeks (cohort 1A, part 2) or 24 weeks (cohorts 1B, 2, and 3) of treatment at 6 mg / kg, 9 mg / kg, and 12 mg / kg to determine the optimally tolerated dose for future clinical development.

[0200] Once this is completed, participants can be enrolled in a long-term extension (LTE) study with a planned duration of at least 1 year. The rationale for the extension phase is to evaluate the safety, tolerability, PK, and pharmacodynamic (PD) effects of AON1 in participants with DMD over a longer period of time, as well as to evaluate participants' functional changes longitudinally and potentially compared to a matched external control group. Given the natural history of the disease, a related extension study is a necessary component of this study design. [Table 3]

[0201] This is a dose escalation study evaluating the safety, tolerability, pharmacokinetics, and pharmacodynamics of six ascending doses of IV AON1 in participants with DMD who are suitable for exon 51 skipping. This is a Phase 1 / 2 open-label study with no randomization planned. Data will be compared to individual participant baseline data. To be eligible for the study, potential participants must meet all eligibility requirements described in the protocol, including the following selected key inclusion criteria: - Male and aged 4-10 years at the time of screening A clinical diagnosis of Duchenne muscular dystrophy caused by a documented dystrophin mutation in the DMD gene amenable to exon 51 skipping, reviewed by a core genetic counselor At the time of screening, participants were ambulatory (defined as being able to walk independently without assistive devices and complete a timed 10-meter walk / run test in 8 seconds or less) - Not currently dependent on daytime ventilation and not expected to require daytime mechanical or non-invasive ventilation within the next year Currently receiving treatment with oral corticosteroids at a stable dose for at least 12 weeks prior to baseline and must maintain a consistent dose / dosing regimen throughout the study, except for modifications to accommodate weight changes Normal renal function based on pre-specified laboratory values

[0202] Inclusion and exclusion criteria included: Forced expiratory volume (FEV) for children aged 7 years and older 1 )<60% of predicted value -Current or history of liver or kidney disease Left ventricular ejection fraction (LVEF) < 55% based on echocardiogram (ECHO) performed within 3 months prior to screening or at the screening visit A mean QT interval (QTcF) corrected by the Fridericia method on three screening electrocardiograms (ECGs) of 450 milliseconds or more Platelet count at screening was 150 x 10 9 / L Treatment with any exon 51 skipping therapy within 12 weeks prior to baseline (Day 1), or treatment with any gene therapy for the treatment of DMD at any time

[0203] Eligible participants will be assigned to one of three cohorts: Cohort 1 (n=6), Cohort 2 (n=3), or Cohort 3 (n=6). Cohort 1 participants will be further divided into Cohort 1A (n=3) and Cohort 1B (n=3). In Cohort 1A (Part 1), participants will receive single ascending doses of AON1 at 0.6 mg / kg, 1.5 mg / kg, 3 mg / kg, and 6 mg / kg, followed by a one-week washout at each dose level when assessed prior to escalation to the next higher dose or, in the case of the 6 mg / kg dose, prior to escalation to chronic dosing. In Cohort 1A (Part 2), participants will receive weekly doses of AON1 at 6 mg / kg, a dose expected to result in approximately 10% dystrophin expression at steady state based on preclinical data. Participants in Cohort 1B will receive 6 mg / kg QW, participants in Cohort 2 (n=6) will receive 9 mg / kg QW, and participants in Cohort 3 (n=6) will receive 12 mg / kg QW. Specific dose escalation procedures are described below.

[0204] The DMC will monitor safety and make dose escalation recommendations. Dose-limiting toxicity (DLT) criteria will be applicable through the 1-week assessment of single doses of 0.6 mg / kg, 1.5 mg / kg, 3 mg / kg, and 6 mg / kg (Cohort 1A (Part 1) only). For Cohorts 1A (Part 2), 1B, 2, and 3, DLT criteria will be applicable through the 4-week assessment of the last participant in the cohort. Study discontinuation criteria will apply to all participants throughout the study. Enrollment will range up to 18 participants depending on whether the maximum tolerated dose (MTD) is reached.

[0205] AON1 will be administered at a starting dose of 0.6 mg / kg to participants (n=3) in Cohort 1A (Part 1) with at least 9 days between doses for participants. Cohort 1A (Part 1) participants will not be dosed at the Week 2 visit. The DMC will review each participant's safety data through Week 2 and, in the absence of DLTs, recommend that the participant be escalated to 1.5 mg / kg (or, in the presence of DLTs, expand the cohort to 6 participants). Cohort 1A (Part 1) participants will receive 1.5 mg / kg AON1 at Week 3. Thereafter, the DMC will review each participant's safety data through Week 4 and, in the absence of DLTs, recommend that the participant be escalated to 3 mg / kg (or, in the presence of DLTs, expand the cohort to 6 participants). Cohort 1A (Part 1) participants will not be dosed at the Week 4 visit. Cohort 1A (Part 1) participants will receive 3 mg / kg AON1 at week 5. The DMC will then review each participant's safety data through week 6 and, if no DLTs are present, will recommend that the participant be titrated to 6 mg / kg (or expand the cohort to 6 participants if no DLTs are present). Cohort 1A (Part 1) participants will not be dosed at the week 6 visit. Cohort 1A (Part 1) participants will receive 6 mg / kg AON1 at week 7. The DMC will then review each participant's safety data through week 8 and, if no DLTs are present, will recommend that the participant be initiated in Part 2 at 6 mg / kg weekly dosing (or expand the cohort to 6 participants in Part 1 if no DLTs are present). Cohort 1A (Part 1) participants will not be dosed at the week 8 visit.

[0206] If a DLT occurs among a participant in Cohort 1A, an additional 3 participants will be enrolled at the dose level at which the DLT occurred and then individually dose escalated according to the dose escalation rules outlined above for Cohort 1A (Part 1).

[0207] The Week 8 visit or later will mark the start of Part 2 for Cohort 1A participants, with a baseline (Week 1) visit designated for the 6 mg / kg QW dose level. Separate dosing of Cohort 1A (Part 2) participants at 6 mg / kg QW by at least 9 days. After all three Cohort 1A participants have received 3 doses of 6 mg / kg QW in Part 2 and the third participant has completed the Week 4 assessment, the DMC will review the available safety data and, in the absence of DLTs, make a recommendation to open Cohort 1B for dosing at 6 mg / kg QW.

[0208] Dosing of Cohort 1B participants at 6 mg / kg QW will be separated by at least 9 days. After all 3 Cohort 1B participants have received 3 doses of 6 mg / kg QW in Part 2 and the last participant has completed the Week 4 evaluation, the DMC will review the available safety data for all 6 Cohort 1 participants and, if there is ≤1 DLT, will make a recommendation to open Cohort 2 for dosing at 9 mg / kg QW.

[0209] Cohort 2 participants (n=6) will receive AON1 at 9 mg / kg QW with at least 9 days between participants. After all 6 participants in Cohort 2 have received 3 doses and the last participant has completed the Week 4 evaluation, the DMC will review the available safety data and, if there is ≤1 DLT, make a recommendation to open Cohort 3 for dosing at 12 mg / kg QW.

[0210] AON1 will be administered at 12 mg / kg QW to all Cohort 3 participants (n=6), with participants receiving doses at least 9 days apart.

[0211] If two or more participants in a given cohort experience a DLT, the MTD has been exceeded and further dose escalation will not be pursued. The DMC, after review of the data, may recommend expansion of previous cohorts, selection of a lower intermediate dose, or stopping the study.

[0212] The primary safety endpoints are listed in the Objectives and Endpoints table above. Secondary objectives of the study include evaluation of plasma and urinary PK and intramuscular distribution of AON1. All participants will undergo muscle biopsy at screening and at week 13 (cohort 1A) or week 25 (cohorts 1B, 2, and 3). After all 6 participants in cohort 3 (12 mg / kg QW) have completed the week 25 evaluation, all data will be evaluated to determine the dose for the planned long-term extension (LTE) study, which will be open to all participants who complete the study and have a planned duration of at least 1 year. Details of the LTE study will be specified in a separate protocol. Once the dose is determined and enrollment for the LTE study begins, participants in the study will either transition into the LTE study or have a study completion visit 4 weeks after the last dose of AON1. They will continue to be dosed and evaluated in the study until the LTE study begins.

[0213] After the last participant receives their dose at week 25, the DMC will review all participant data. Based on the results and the DMC's recommendation, the sponsor will determine the dose for the LTE study. Participants are considered to have completed the study if they have completed all scheduled visits through the sponsor's LTE dose determination. Following medical authority clearance or approval, participants may begin dosing on the LTE after the participant's site receives IRB / EC approval for the LTE. The study will be completed on the date of the last visit before the participant enrolls in the LTE. During the LTE study, participants will receive AON1 at 12 mg / kg or the highest tolerated dose determined during the study. Example 11

[0214] Evaluation of AON1 26-week toxicity study in male CD-1 mice

[0215] Male CD-1 mice (10 or 15 / group (main toxicity study) and up to 5 / group of main toxicity animals in the 0 and 18 mg / kg dose groups were euthanized after a 13-week recovery period. 4 (vehicle control) and 19 / group (toxicokinetics (TK))) were administered AON1 via slow bolus IV injection twice weekly for a total of 26 weeks at 0 (vehicle, 0.9% NaCL injection, USP), 6, 12, or 18 mg / kg. For bioanalysis, blood was collected at 0 (within 2 minutes after dosing), 0.33, 1, 3, 8, and 24 hours after dosing on days 1 and 176. The following parameters were evaluated: mortality, clinical signs, body weight, food intake, ophthalmological examination, clinical pathology (i.e., clinical chemistry, hematology, and coagulation), gross dissection, organ weights, and histopathology.

[0216] Bioanalysis and tissue distribution

[0217] Plasma exposure (C max and AUC 0 ~24 The concentrations of AON1 in mice were generally dose-proportional (based on the 100% confidence interval [CI]) and increased with increasing dose. No accumulation was observed after multiple doses of AON1 in male mice.

[0218] Mean concentrations of AON1 in male mouse tissues were measurable in all treatment groups at day 178. Mean concentrations generally increased with increasing dose from 6 to 18 mg / kg / dose. The ranking of mean AON1 concentrations across dose levels was liver, kidney, heart, gastrocnemius, and biceps femoris.

[0219] The no observed adverse effect level (NOAEL) was 18 mg / kg / dose twice weekly due to the mild severity of pathological findings and no effect on the health or well-being of animals dosed up to 18 mg / kg / dose twice weekly. This was due to mean maximum observed concentrations (C max ) and area under the concentration-time curve (AUC 0~24 ) was equivalent to Example 12

[0220] Inhibition and induction of cytochrome P450

[0221] The potential of AON1 to inhibit CYP enzymes was evaluated in human liver microsomes incubated with 0.1–100 μM AON1. Assay validation was confirmed using positive controls (i.e., reference compounds) for each isoenzyme. IC for direct inhibition of CYP1A2 was 0.01–0.02. 50 The IC value for direct inhibition of CYP2C8 and CYP2C19 was 7.85 μM, while 50 After accounting for microsomal protein binding, the unbound IC values ​​for direct inhibition of CYP1A2, CYP2C8, and CYP2C19 were >100 μM. 50 The values ​​were 0.09, >1.27, and >1.08 μM, respectively, which are within the predicted human plasma C values ​​at the maximum dose of 18 mg / kg. max The inhibition of AON1 was lower than the 18 μM observed in human liver microsomes (Kazmi Drug Metab Dispos.2018 Aug;46(8):1066-1074. Available at: https: / / dmd.aspetjournals.org / content / dmd / 46 / 8 / 1066.fully.pdf). Plasma protein binding of AON1 was attempted but could not be assessed due to high levels of nonspecific binding to the filtration device. There was no direct inhibition of CYP2B6, CYP2C9, CYP2D6, or CYP3A4 / 5 (substrates = testosterone or midazolam). Based on the literature, phosphorothioate-based oligonucleotide inhibition of CYP enzymes was test system dependent. Inhibition of CYP enzymes by phosphorothioate-based oligonucleotides was observed in human liver microsomes and not in cryopreserved human hepatocytes (Kazmi Drug Metab Dispos.2018 Aug;46(8):1066-1074. Available at: https: / / dmd.aspetjournals.org / content / dmd / 46 / 8 / 1066.fully.pdf). Cryopreserved human hepatocytes provide a more clinically relevant inhibition profile for use in in vitro-in vivo estimation of drug-drug interactions for phosphorothioate-based oligonucleotide inhibition of CYP enzymes. Considering that AON1 is also a phosphorothioate-based oligonucleotide, microsomes may not be representative of in vivo conditions.

[0222] To that end, further studies will be performed using cryopreserved human hepatocytes to determine the IC 50 values ​​>100 μM demonstrated that AON1 is not a direct or time-dependent inhibitor of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 / 5.

[0223] The potential to induce CYP enzymes was evaluated in hepatocytes incubated with 100 μM AON1. The validity of the assay was confirmed using positive controls (i.e., reference compounds) for each isoenzyme. In vitro assays for CYP induction showed that CYP1A2, CYP2B6, and CYP3A4 were not induced at the transcriptional level by AON1. Example 13

[0224] Evaluation of AON1 in male cynomolgus monkeys treated for 13 weeks

[0225] Male cynomolgus monkeys (3 / group (main toxicity study); 2 / group at 0, 12, and 18 mg / kg QW (4 week recovery)) were administered AON1 via IV infusion (60 min) at 0 (vehicle: 0.9% NaCl injection, USP), 6, 12, or 18 mg / kg QW for 13 weeks. Blood was collected pre-dose, on day 1, day 85, and 0.5, 1, 2, 3, 6, 8, and 24 hours post-dose for bioanalysis, and 1, 3, 6, and 24 hours post-dose for complement activation analysis. A pre-dose sample was also collected on day 78. The following parameters were evaluated: mortality, clinical signs, body weight, food intake, AON1 levels in plasma and tissues (gastrocnemius, heart, biceps femoris, liver, and kidney), percentage of exon skipping in tissues (gastrocnemius, heart, biceps femoris, liver, and kidney), clinical pathology (clinical chemistry, hematology, urinalysis, urine chemistry, and urine biomarkers), complement activation indices (Bb, C3a, and sC5b-9), ophthalmology, ECG, organ weights, and histopathology.

[0226] Exon skipping in tissues was measured by RT-ddPCR, which quantified the number of non-skipped DMD (dystrophin) transcripts and the number of exon 51-skipped DMD transcripts, and calculated the percentage of exon skipping (% skip). The percentage of exon 51 skipping ranged from 1.7% to 14.4%, and generally increased with dose.

[0227] The present disclosure is not limited in scope by the embodiments disclosed in the examples, which are intended as merely illustrative of individual aspects, and any equivalents are within the scope of the present disclosure. Various modifications in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0228] Various references, such as patents, patent applications, and publications, are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

1. A method of treating a subject having DMD or delaying the onset of DMD in a subject, comprising administering to the subject AON1 at a dose of 0.4 mg / kg, 0.6 mg / kg, 0.8 mg / kg, 1.5 mg / kg, 3 mg / kg, 6 mg / kg, 9 mg / kg, 12 mg / kg, or 18 mg / kg, wherein the AON1 is administered QW.

2. 2. The method of claim 1, wherein the AON1 is administered QWx15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35W.

3. The method of claim 1 or claim 2, wherein the AON1 is administered QWx24W or QWx25W.

4. The method of any one of claims 1 to 3, wherein the AON1 is administered QW x 25W.

5. The method of any one of claims 1 to 3, wherein the AON1 is administered QWx24W.

6. The method of any one of claims 1 to 5, wherein the AON1 is administered at a dose of 3 mg / kg.

7. The method of any one of claims 1 to 5, wherein the AON1 is administered at a dose of 6 mg / kg.

8. The method of any one of claims 1 to 5, wherein the AON1 is administered at a dose of 9 mg / kg.

9. The method of any one of claims 1 to 5, wherein the AON1 is administered at a dose of 12 mg / kg.

10. The method of any one of claims 1 to 5, wherein the AON1 is administered at a dose of 18 mg / kg.

11. The method of any one of claims 1 to 10, which is a method for treating DMD in a subject.

12. The method of any one of claims 1 to 10, which is a method for delaying the onset of DMD in a subject.

13. The method of any one of claims 1 to 12, further comprising administering to the subject a second active agent.

14. 14. The method of claim 13, wherein the second active agent is a corticosteroid such as eteplirsen, casimersen, gologirsen, viltolarsen, SRP-5051 (Sarepta Therapeutics), deflazacort, a gene therapy agent (e.g., SRP-9001, GALGT2, or GNT0004 (Sarepta Therapeutics)), gene editing (e.g., CRISPR / CAS9 (Sarepta Therapeutics)), or a cell therapy agent (e.g., CAP-1002 (Capricor Therapeutics / Nippon Shinyaku Co. Ltd.)).

Images