Methods of treating duchenne muscular dystrophy

Abstract

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

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 364,260, filed on May 5, 2022, and U.S. Provisional Patent Application No. 63 / 387,733, filed on December 16, 2022. The disclosure of each of the above-mentioned applications is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted herewith as an XML file named "035105WO.xml" with a size of 11,646 bytes created on April 26, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0005] Provided herein are methods of treating or delaying the onset of Duchenne muscular dystrophy (DMD). In one embodiment, the method comprises administering a modified oligonucleotide to a subject having DMD. Background Art

[0006] Antisense oligonucleotides (AONs) are in the clinical (pre) development stage for many diseases and disorders, including cancer, inflammatory diseases, cardiovascular diseases, and neurodegenerative and neuromuscular disorders. Their mechanism of action is directed to the degradation of target RNA in the nucleus or cytoplasm mediated by various targets such as RNase H, splicing regulation in the nucleus (exon inclusion or skipping), or translation inhibition achieved by steric hindrance of ribosomal subunit binding in the cytoplasm. Splicing regulation or splicing switching AONs were first described for correction of abnormal splicing in human β-globin precursor mRNA (Dominski and Kole PNAS, 1993, 90 (18): 8673-8677), and are currently being studied for use in a variety of genetic disorders.

[0007] AONs have been extensively studied in the treatment of the neuromuscular disorders Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common forms of childhood muscular dystrophy. DMD is a severe, fatal neuromuscular disorder that leads to wheelchair dependence before the age of 12, and patients usually die of respiratory or heart failure before the age of 30. It is caused by a reading frameshift deletion (~67%) or duplication (~7%) of one or more exons or by a point mutation (~25%) in the 2.24Mb dystrophin gene, resulting in a lack of functional dystrophin. BMD is also caused by mutations in the dystrophin gene, but these mutations maintain the open reading frame, produce semi-functional dystrophin, and lead to a generally milder phenotype and longer lifespan.

[0008] 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 for the treatment of only a small fraction of DMD patients.

[0009] Therefore, there remains a need for treatments for DMD. Summary of the invention

[0010] Provided herein are methods of treating DMD or delaying its onset by administering a modified oligonucleotide to a subject suffering from DMD. In one embodiment, the modified oligonucleotide is an antisense oligonucleotide (AON). BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Shown are AON1 plasma concentrations over time.

[0012] Figure 2 AON1 concentrations in heart, quadriceps, liver, diaphragm, gastrocnemius, and kidney tissues are shown.

[0013] Figure 3 showed that after 25 weeks of treatment, AON1 achieved sustained dose-dependent exon 51 skipping in muscle tissues (quadriceps, gastrocnemius, heart, and diaphragm).

[0014] Figure 4 Dystrophin wt% in heart and quadriceps muscle are shown, and overall gait scores for mice treated with AON1 are shown.

[0015] Figure 5We showed that AON1 treatment alleviated hDMD del52(+ / +) model-associated muscle pathology and maintained the pharmacological effect for 12 weeks after 25 weeks of dosing.

[0016] Figure 6 AON1 treatment alleviated hDMD del52(+ / +) model-related cardiac pathology and maintained the pharmacological effects for 12 weeks.

[0017] Figure 7 Shown is dystrophin expression in the heart and quadriceps of hDMDΔ52 / mdx mice treated with AON1 with TEG linked to the oligonucleotide via a phosphorothioate group (AON1 PS, 9.4, 18.7, or 37.5 mg / kg) 14 days (filled circles) or 28 days (open circles) after QW, Q2W, or Q4W dosing.

[0018] Figure 8 Shown are % exon 51 skipping in hearts of hDMDΔ52 / mdx mice treated with AON1 PS (9.4, 18.7, or 37.5 mg / kg) 14 days (filled circles) or 28 days (open circles) after QW, Q2W, or Q4W dosing.

[0019] Fig. 9 Shown are % exon 51 skipping in quadriceps, gastrocnemius, heart and diaphragm of hDMDΔ52 / mdx mice treated with 18.7 mg / kg AON1 (13 weeks QW) at 4 and 8 weeks post-dosing.

[0020] Fig.10 Shown is dystrophin expression in the heart and quadriceps of hDMDΔ52 / mdx mice treated with AON1 (18.7 mg / kg) 4 or 8 weeks after QWx13W dosing.

[0021] Fig.11 Shown are the overall gait scores of mice treated with AON1.

[0022] Fig.12 Shown are the effects of AON1 PS and AON2 (AON1 without the 5'-TEG group) on the complement parameter Bb in human and monkey sera.

[0023] Fig.13 Shown are the effects of AON1 PS and AON2 on the complement parameter C3a in human and monkey sera.

[0024] Fig.14 Exon skipping in biceps, gastrocnemius and heart tissues of NHPs treated with AON1 for 39 weeks is shown (Example 8). DETAILED DESCRIPTION

[0025] I. Definitions

[0026] To facilitate understanding of the disclosure set forth herein, a number of terms are defined below.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. If multiple definitions exist for a term in this article, the definition in this section shall prevail unless otherwise stated.

[0028] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0029] As used herein, a "subject" is an animal, such as a mammal, including a human, such as a patient.

[0030] As used herein, biological activity refers to the in vivo activity of a compound or the physiological response produced when a compound, composition or other mixture is administered in vivo. Thus, biological activity encompasses the therapeutic effects and pharmacokinetic behavior of such compounds, compositions and mixtures. Biological activity can be observed in an in vitro system designed to test such activity.

[0031] As used herein, treatment means any manner in which one or more symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use for the treatment of DMD.

[0032] As used herein, amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any relief, whether permanent or temporary, lasting or transient, attributable to or associated with administration of the compound or pharmaceutical composition.

[0033] As used herein, unless otherwise indicated, the terms "management, managing, and management" encompass preventing a particular disease or condition from recurring in a subject who has suffered from the disease or condition, and / or prolonging the time a subject with the disease or condition remains in a state of remission. These terms encompass modulating the threshold, development, and / or duration of a disease or condition, or changing the way a subject responds to a disease or condition.

[0034] II. AONs for use in the methods provided herein

[0035] In one embodiment, the AON used in the methods provided herein is reverse complementary to a portion of exon 51 of the human dystrophin pre-mRNA. In another embodiment, the AON used in the methods provided herein has the sequence 5'-gguaaguucugu ccaagc-3' (SEQ ID NO: 1) and contains modifications. In another embodiment, the AON used in the methods provided herein has the sequence 5'-gguaaguuc*uguc*c*aagc*-3' (SEQ ID NO: 2), wherein c* is 5-methylcytosine and g and c* are LNA nucleotides. As used herein, LNA nucleotides have the following structure:

[0036]

[0037] wherein B is a nucleobase and L is a phosphate or phosphorothioate bond to another nucleotide.

[0038] In another embodiment, the AON used in the methods provided herein has the sequence TEG-5'-gguaaguuc*uguc*c*aagc*-3', wherein c* is 5-methylcytosine, g and c* are LNA nucleotides, and TEG is a triethylene glycol group. In another embodiment, the AON used in the methods provided herein has the sequence TEG-5'-gguaaguuc*uguc*c*aagc*-3', wherein c* is 5-methylcytosine, g and c* are LNA nucleotides, TEG is a triethylene glycol group attached to the 5' end by a phosphate group, the internucleoside bond is a thiophosphate bond, and the non-LNA nucleotide is a 2'-OMe nucleotide (referred to herein as "AON1"). See WO 2022 / 069511 A1 and US2022 / 0098586A1.

[0039] III. Methods for treating DMD

[0040] Provided herein is a method for treating DMD or delaying the onset of DMD by administering a modified oligonucleotide to a subject suffering from 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, a method for treating DMD by administering AON1 to a subject suffering from DMD is provided. In another embodiment, a method for delaying the onset of DMD by administering AON1 to a subject suffering from DMD is provided.

[0041] In certain embodiments, the AON is administered at a dosage 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.

[0042] In certain embodiments, AON1 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, 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.

[0043] In another embodiment, AON1 is administered at 6 mg / kg QWx25W. In another embodiment, AON1 is administered at 18 mg / kg QWx25W.

[0044] In another embodiment, a method of treating DMD by administering AON1 at 6 mg / kg QWx24W is provided. In another embodiment, a method of treating DMD by administering AON1 at 18 mg / kg QWx24W is provided. In another embodiment, a method of delaying the onset of DMD by administering AON1 at 6 mg / kg QWx24W is provided. In another embodiment, a method of delaying the onset of DMD by administering AON1 at 18 mg / kg QWx24W is provided.

[0045] In certain embodiments, compared with the existing methods for treating DMD or delaying its onset, the methods provided herein produce more excellent results. In one embodiment, the method provided herein induces relevant disease modification biomarkers (disease modifying biomarker) to change in the direction of normalization. In another embodiment, the method provided herein induces the clinical and anatomical muscle pathology associated with the disease to change in the direction of normalization. In another embodiment, the method provided herein partially or almost completely rescues the DMD phenotype, as measured in MotoRater / gait scoring. In another embodiment, the method provided herein increases the production of dystrophin. In another embodiment, the method provided herein increases the skipping of exon 51 of human dystrophin precursor mRNA. In another embodiment, the method alleviates one or more symptoms of DMD.

[0046] Using AON provided herein to alleviate one or more symptoms of DMD in an individual can be assessed by any of the following assays: extension of time to loss of walking ability, improvement in muscle strength, improvement in weight-lifting ability, improvement in time to get up, improvement in nine-meter walking time, improvement in time to climb four steps, improvement in leg function grade, improvement in lung function, improvement in cardiac function, improvement in quality of life. Each of these assays is known to a skilled technician. For each of these assays, once a detectable improvement or extension of the parameters measured in the assay is found, this will generally mean that one or more symptoms of DMD have been alleviated in an individual using AON provided herein. As described in Hodgetts et al. Neuromuscular Disorders 2006; 16: 591-602, detectable improvement or extension is a statistically significant improvement or extension in one embodiment. Alternatively, the alleviation of one or more symptoms of DMD can 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 features of one or more muscle cells from a DMD patient are improved. Such symptoms or characteristics may be assessed at the cellular, tissue level or in the patient himself.

[0047] The alleviation of one or more characteristics of the muscle cells from the patient can be assessed by performing any of the following assays on the myogenic cells or muscle cells from the patient: decreased calcium uptake by the muscle cells, decreased collagen synthesis, morphological changes, altered lipid biosynthesis, decreased 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.

[0048] Improvement in muscle fiber function, integrity and / or survival can be assessed using at least one of the following assays: a detectable decrease in creatine kinase in the blood, a detectable decrease in myofiber necrosis in a cross-section of a biopsy of suspected dystrophic muscle, and / or a detectable increase in uniformity of myofiber diameter in a cross-section of a biopsy of suspected dystrophic muscle. Each of these assays is known to the skilled artisan.

[0049] As described by Hodgetts et al. (2006), creatine kinase can be detected in the blood. A detectable decrease in creatine kinase can mean a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in the same DMD patient before treatment.

[0050] Typically, a biopsy cross section is used to assess the detectable reduction of myofiber necrosis in a muscle biopsy, such as described by Hodgetts et al. (2006). A detectable reduction in necrosis can be a reduction of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area in which a biopsy cross section has been used to identify necrosis. The reduction is measured by comparing the necrosis assessed in the same DMD patient before treatment.

[0051] A detectable increase in the uniformity of myofiber diameter is typically assessed in a cross section of a muscle biopsy, such as described by Hodgetts et al. (supra). The increase is measured by comparison with the uniformity of myofiber diameter in the same DMD patient prior to treatment.

[0052] In one embodiment, the AON provided herein provides functional or semi-functional dystrophin to the individual and is capable of at least partially reducing the production of abnormal dystrophin in the individual.

[0053] In one embodiment, providing a subject with a functional or semi-functional dystrophin protein refers to an increase in the production of a functional or semi-functional dystrophin protein. Increasing the production of functional or semi-functional dystrophin mRNA or functional or semi-functional dystrophin refers to a detectable increase or increase of 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, such as 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 2014;9(9):e107494). 2018;13(4):e0195850) assay (protein). In another embodiment, the initial amount is the amount of functional or semi-functional mRNA or functional or semi-functional dystrophin when exon skipping of dystrophin pre-mRNA is induced in cells, organs, tissues and / or individuals using a compound of the present invention.

[0054] Reducing the production of abnormal dystrophin mRNA or abnormal dystrophin 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 can still be detected 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 when the AON of the present invention is used to start inducing exon skipping in dystrophin precursor mRNA in cells, organs, tissues and / or individuals. Abnormal dystrophin mRNA or protein is also referred to herein as poorly functional (compared to wild-type functional dystrophin) or non-functional dystrophin mRNA or protein. Non-functional dystrophin is a dystrophin that cannot bind to actin and / or DGC protein complex members. A non-functional dystrophin protein or dystrophin mRNA typically does not have or encodes a dystrophin protein with an intact C-terminus of the protein. Detection of functional or semi-functional dystrophin mRNA or protein can be performed as for detection of abnormal dystrophin mRNA or protein.

[0055] Once a functional or semi-functional dystrophin is provided to a DMD patient, at least part of the cause of DMD is eliminated. Therefore, the symptoms of DMD are then expected to be at least partially alleviated, or the rate of symptom deterioration is reduced, resulting in a slower decline. The enhanced jump frequency also increases the level of functional or semi-functional dystrophin produced in the muscle cells of DMD individuals.

[0056] IV. Pharmaceutical Compositions for Use in the Methods

[0057] The pharmaceutical compositions provided herein comprise a therapeutically effective amount of one or more AONs provided herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0058] AON can be formulated into suitable pharmaceutical preparations, such as solutions, suspensions, powders, sterile solutions or suspensions for ophthalmic or parenteral administration, and transdermal patches. Typically, AON as described herein are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition 1999).

[0059] In the composition, effective concentrations of one or more compounds or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical carrier or vehicle. In certain embodiments, the concentration of the AON in the composition is effective for delivering an amount that treats, prevents, or improves one or more symptoms and / or progression of a disease or condition disclosed herein when administered.

[0060] Typically, the composition is formulated for single-dose administration. In order to prepare the composition, a certain weight fraction of the compound is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration so that the disease being treated is alleviated or improved. The pharmaceutical carrier or vehicle suitable for administering the AON provided herein includes any such carrier known to those skilled in the art to be suitable for a specific mode of administration.

[0061] In addition, AON can be formulated as the only active pharmaceutical ingredient in the composition or can be combined with other active ingredients. Liposomal suspensions, including tissue-targeted liposomes, can also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome preparations can be prepared as known in the art. In short, liposomes such as multilamellar vesicles (MLVs) can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (molar ratio of 7:3) inside a flask. Add a solution of the compound provided herein in phosphate buffered saline (PBS) lacking divalent cations and shake the flask until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compounds, precipitated by centrifugation, and then resuspended in PBS.

[0062] The active compound is contained in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect on the treated subject without producing undesirable side effects. The therapeutically effective concentration can be determined empirically by testing the AON in the in vitro and in vivo systems described herein, and then the dosage for humans is extrapolated therefrom. In some embodiments, the AON is administered in a certain manner to achieve a therapeutically effective concentration of the drug. In some embodiments, a companion diagnosis (see, e.g., Olsen D and Jorgensen JT, Front. Oncol., May 16, 2014, 4: 105, doi: 10.3389 / fonC.2014.00105) is used to determine the therapeutic concentration and safety characteristics of the active compound in a specific subject or subject population.

[0063] The concentration of AON in the pharmaceutical composition will depend on the absorption, tissue distribution, inactivation and excretion rate of the active compound, the physicochemical characteristics of the compound, the dosage schedule and the amount administered, and other factors known to those skilled in the art. For example, the amount delivered is sufficient to improve one or more symptoms of a disease or condition disclosed herein.

[0064] In certain embodiments, the therapeutically effective dose should produce an active ingredient serum concentration of about 0.1 ng / mL to about 50-100 μg / mL. In one embodiment, the pharmaceutical composition provides a dosage of about 0.001 mg to about 2000 mg of compound / kg body weight / day. The drug dosage unit form is prepared to provide a basic active ingredient or basic ingredient combination of about 10 to about 500 mg per dosage unit form and about 10 to about 500 mg in certain embodiments.

[0065] AON can be used once, or it can be divided into multiple smaller doses to be used at intervals. It should be understood that the exact dosage and treatment duration depend on the disease being treated, and can be determined empirically using known test protocols or by extrapolating from in vivo or in vitro test data. It should be noted that concentration and dosage values ​​can also change with the severity of the illness to be alleviated. It should also be understood that for any particular subject, a specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the personnel administering the composition or supervising the administration of the composition, and the concentration ranges set forth herein are only exemplary and are not intended to limit the scope or practice of the claimed composition.

[0066] Therefore, an effective concentration or effective amount of one or more AONs described herein or pharmaceutically acceptable salts thereof is mixed with a pharmaceutical carrier or vehicle suitable for systemic, local or local regional administration to form a pharmaceutical composition. The amount of AON included is effective to improve one or more symptoms, or to treat, delay progression or prevent. The concentration of the active compound in the composition will depend on the absorption, tissue distribution, inactivation, excretion rate, dosage schedule, dosage, specific formulation and other factors known to those skilled in the art of the active compound.

[0067] The compositions are intended to be administered by any suitable route including, but not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, transdermal, transdermal, or buccal.

[0068] Solutions or suspensions for parenteral, intradermal or subcutaneous application may include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol, dimethylacetamide or other synthetic solvents; antimicrobial agents such as benzyl alcohol and methyl parabens; 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 tonicity such as sodium chloride or dextrose. Parenteral formulations may be packaged in ampoules, injection pens, disposable syringes or single-dose or multi-dose vials made of glass, plastic or other suitable materials.

[0069] In the event that the AON exhibits insufficient solubility, methods of dissolving the AON 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 dimethyl sulfoxide (DMSO), the use of surfactants such as ) or dissolved in aqueous sodium bicarbonate solution.

[0070] After mixing or adding the AON, the resulting mixture can be a solution, suspension, emulsion, etc. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the AON in the selected carrier or vehicle. The effective concentration is sufficient to improve the symptoms of the disease, disorder or condition being treated and can be determined empirically.

[0071] Pharmaceutical compositions are provided in unit dosage forms for administration to humans and animals, such as powders, granules, sterile parenteral solutions or suspensions, and oil-water emulsions containing an appropriate amount of AON or a pharmaceutically acceptable salt thereof. AON and its salts with pharmaceutical therapeutic activity are prepared and administered in unit dosage forms or multiple dosage forms. Unit dosage forms as used herein refer to physically discrete units that are suitable for human and animal subjects and packaged separately as known in the art. Each unit dose contains a predetermined amount of therapeutically active compounds sufficient to produce the desired therapeutic effect, and a desired pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. Unit dosage forms can be administered in fractions or multiples thereof. Multiple dosage forms are multiple identical unit dosage forms packaged in a single container that will be administered in separate unit dosage forms. Examples of multiple dosage forms include bottles of vials, tablets, or capsules, or pints or gallons of bottles. Therefore, multiple dosage forms are multiple unit doses that are not separated in packaging.

[0072] Sustained release formulations can also be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers comprising compounds provided herein, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include iontophoresis patches, polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT TM ) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are able to release molecules for more than 100 days, some hydrogels release proteins for shorter time periods. When encapsulated compounds remain in the body for a long time, they can denature or aggregate due to exposure to a humid environment at 37°C, resulting in loss of biological activity and possible changes in their structure. Depending on the mechanism of action involved, rational stabilization strategies can be designed. For example, if the aggregation mechanism is found to be intermolecular S--S bond formation achieved through thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling the moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0073] Dosage forms or compositions containing active ingredients in the range of 0.005% to 100% with the remainder consisting of non-toxic carriers can be prepared. Such compositions include solutions, suspensions, powders and sustained release formulations, such as 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, etc. Methods for preparing these compositions are known to those skilled in the art. Contemplated compositions may contain about 0.001% to 100% of active ingredients, in certain embodiments, about 0.1 to 85% or about 75-95%.

[0074] Active AON or pharmaceutically acceptable salts can be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings.

[0075] The composition may contain other active AONs to obtain a desired combination of properties. The AONs provided herein or pharmaceutically acceptable salts thereof as described herein may also be advantageously administered together with another agent known in the general art to be valuable for treating one or more of the above-mentioned diseases or medical conditions (such as diseases associated with oxidative stress) for therapeutic or preventive purposes. It should be understood that such combination therapies constitute another aspect of the compositions and methods of treatment provided herein.

[0076] A. Injections, solutions and emulsions

[0077] Parenteral administration is also contemplated herein, typically characterized by injection, i.e., subcutaneous injection, intramuscular injection, or intravenous injection. Injections can be prepared in conventional forms, i.e., prepared as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or 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 include a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubilizers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins. Implantation of a slow release or sustained release system is also contemplated herein to maintain a constant dosage level. Briefly, the AONs provided herein are dispersed in a solid internal matrix, for example, polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, silicone carbonate copolymer, or the like. The solid inner matrix is ​​surrounded by an outer polymer membrane that is insoluble in body fluids, such as polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber, epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol terpolymer and ethylene / vinyloxyethanol copolymer. The AON diffuses through the outer polymer membrane in the release rate controlling step. The percentage of active AON contained in such parenteral compositions is highly dependent on its specific properties as well as the activity of the compound and the needs of the subject.

[0078] The parenteral administration of the composition includes intravenous, subcutaneous and intramuscular administration. Preparations for parenteral administration include sterile solutions prepared for injection, sterile dry soluble products (such as lyophilized powders) (including subcutaneous injection tablets) prepared for mixing with solvents just before use, sterile suspensions prepared for injection, sterile dry insoluble products prepared for mixing with vehicles just before use, and sterile emulsions. The solution can be aqueous or non-aqueous.

[0079] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[0080] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, sequestering or chelating agents, and other pharmaceutically acceptable substances.

[0081] 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 plant origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents that inhibit bacteria or inhibit fungal concentrations must be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, 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. Emulsifiers include polysorbate 80 ( 80). Metal ion sequestering agents or chelators include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol and propylene glycol for water-miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0082] The concentration of the pharmaceutically active AON is adjusted so that injection provides an effective amount to produce the desired pharmacological effect. As is known in the art, the exact dose depends on the age, weight and condition of the subject or animal.

[0083] The unit dose parenteral preparation is packaged in an ampoule, a vial or a syringe with a needle. As is known and practiced in the art, all preparations for parenteral administration must be sterile.

[0084] For example, intravenous or intraarterial infusion of a sterile aqueous solution containing active AON is an effective mode of administration. Another embodiment is the injection of a sterile aqueous or oily solution or suspension containing active AON as needed to produce the desired pharmacological effect.

[0085] Injection is designed for local and systemic administration. Generally, the therapeutically effective dose is formulated to contain a concentration of at least about 0.1% w / w to a maximum of about 90% w / w or higher, such as more than 1% w / w of active AON, to the treated tissue. Active AON can be applied once, or it can be divided into multiple smaller doses to be applied at intervals. It should be understood that the exact dosage and treatment duration depend on the treated tissue, and can be determined empirically using known test protocols or by extrapolating from in vivo or in vitro test data. It is worth noting that concentration and dosage values ​​can also vary with the age of the individual treated. It should also be understood that for any particular subject, a specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the personnel administering the preparation or supervising the administration of the preparation, and the concentration ranges set forth herein are only exemplary and are not intended to limit the scope or practice of the claimed preparation.

[0086] The AON can be suspended in micronized or other suitable form, or can be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture depends on many 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 improve the symptoms of the disease and can be determined empirically.

[0087] B. Freeze-dried powder

[0088] Also provided herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels.

[0089] Sterile lyophilized powder is prepared by dissolving the AON or its pharmaceutically acceptable salt provided herein in a suitable solvent. The solvent may include excipients that improve the stability of the powder or the reconstituted solution prepared from the powder or other pharmacological components. Available excipients include but are not limited to dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose or other suitable agents. The solvent may also include a buffer, such as citrate, sodium phosphate or potassium phosphate or other such buffers known to those skilled in the art, in one embodiment, about neutral pH. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to obtain the desired formulation. In general, the resulting solution will be dispensed into vials for freeze-drying. Each vial will contain a single dose (including but not limited to 10-1000 mg or 100-500 mg) or multiple doses of the compound. The freeze-dried powder can be stored under appropriate conditions, such as about 4 ° C to room temperature.

[0090] The lyophilized powder is reconstituted with water for injection to obtain 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 an amount can be determined empirically.

[0091] C. Topical application

[0092] Topical mixtures are prepared as described for topical and systemic administration. The resulting mixtures may be solutions, suspensions, emulsions, etc., and formulated as creams, gels, ointments, lotions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, douches, sprays, suppositories, bandages, skin patches, or any other formulation suitable for topical administration.

[0093] The compounds can be formulated for topical or local application, such as in the form of gels, creams and lotions for topical application to the skin and mucous membranes, such as to the eyes, as well as for application to the eyes or for intracisternal or intraspinal applications. Topical administration is contemplated for transdermal delivery, as well as for application to the eyes or mucous membranes, or for inhalation therapy. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients may also be administered.

[0094] These solutions, particularly those intended for ophthalmic use, can be formulated as 0.01%-10% isotonic solutions, pH about 5-7, containing appropriate salts.

[0095] D. Sustained Release Compositions

[0096] AONs provided herein can be administered by controlled release means or by delivery devices well known to those skilled in the art. Examples include, but are not limited to, U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and U.S. Pat. Nos. 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, , 6,740,634, each of which is incorporated herein by reference. Such dosage forms can be used to provide slow or controlled release of one or more active ingredients by using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof in varying proportions to provide the desired release profile. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients provided herein.

[0097] All controlled release pharmaceutical products have the common goal of improving drug therapy, making it exceed the drug therapy achieved by its non-controlled counterpart. In one embodiment, the use of the optimally designed controlled release formulation in medical treatment is characterized by the minimum amount of drug substance used to cure or control the disease in the shortest time. In certain embodiments, the advantages of controlled release formulations include extended drug activity, reduced frequency of administration and increased subject compliance. In addition, controlled release formulations can be used to affect the onset time or other characteristics, such as the blood level of the drug, and thereby can affect the occurrence of side effects (e.g., adverse reactions).

[0098] Most controlled-release formulations are designed to initially release a certain amount of drug (active ingredient) to rapidly produce the desired therapeutic effect, and then gradually and continuously release additional amounts of drug to maintain this level of therapeutic or preventive effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug metabolized and excreted from the body. The controlled release of AONs can be subject to various conditions, including but not limited to stimulation by pH, temperature, enzymes, water or other physiological conditions or compounds.

[0099] In certain embodiments, AON can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In one embodiment, a pump can 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 polymer material can be used. In another embodiment, a controlled release system can be placed near the therapeutic target, i.e., so that only a portion of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, Vol. 2, pp. 115-138 (1984).

[0100] Other controlled release systems are discussed in a review by Langer (Science 249: 1527-1533 (1990). The AONs can be dispersed in a solid inner matrix, e.g., polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, organosilicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic acid and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate, which is surrounded by an outer polymer membrane that is insoluble in body fluids. Films such as polyethylene, polypropylene, ethylene / propylene copolymers, ethylene / ethyl acrylate copolymers, ethylene / vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber, epichlorohydrin, ethylene / vinyl alcohol copolymers, ethylene / vinyl acetate / vinyl alcohol terpolymers and ethylene / vinyloxyethanol copolymers. The AON then diffuses through the outer polymer film in a release rate controlling step. The percentage of active ingredient contained in such parenteral compositions is highly dependent on its specific properties and the needs of the experimenter.

[0101] E. Targeted agents

[0102] AON or its pharmaceutically acceptable salt provided herein can also be formulated into specific tissues, receptors or other body regions targeting the subject to be treated, including delivery systems based on liposomes, resealed erythrocytes and antibodies. Many such targeting methods are well known to those skilled in the art. It is contemplated herein that all such targeting methods are used in the compositions of the present invention. For non-limiting examples of targeting methods, see, e.g., U.S. Pat. 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; 6,060,082; 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.

[0103] In one embodiment, the antibody-based delivery system is an antibody-drug conjugate ("ADC"), e.g., as described in Hamilton GS, Biologicals, Sep 2015, 43(5):318-32; Kim EG and Kim KM, Biomol. Ther. (Seoul), Nov 2015, 23(6):493-509; and Peters C and Brown S, Biosci. Rep., Jun 12, 2015, 35(4) pii:e00225, each of which is incorporated herein by reference.

[0104] In one embodiment, liposome suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, liposome preparations can be prepared as described in U.S. Patent No. 4,522,811. In brief, liposomes, such as multilamellar vesicles (MLVs), can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (molar ratio of 7:3) inside a flask. A solution of AON 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, precipitated by centrifugation, and then resuspended in PBS.

[0105] F. Products

[0106] The AON or a pharmaceutically acceptable salt thereof can be packaged as a product comprising packaging material, an AON or a pharmaceutically acceptable salt thereof provided herein (for treating, preventing, or ameliorating one or more symptoms or progression of a disease or condition disclosed herein), and a label indicating that the compound or a pharmaceutically acceptable salt thereof is used to treat, prevent, or ameliorate one or more symptoms or progression of a disease or condition disclosed herein.

[0107] Articles provided herein include packaging materials. Packaging materials for packaging pharmaceutical products are well known to those skilled in the art. See, for example, 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, injection pens, bottles and any packaging materials suitable for selected formulations and intended modes of administration and treatment. A wide variety of formulations of AONs and compositions provided herein are contemplated.

[0108] In certain embodiments, kits are also provided herein that, when used by a medical practitioner, can simplify the administration of an appropriate amount of AON in a subject. In certain embodiments, kits provided herein include a container and a dosage form of an AON or a pharmaceutically acceptable salt, solvate, or prodrug thereof provided herein.

[0109] In certain embodiments, a kit includes a container comprising a dosage form of an AON provided herein, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, contained within the container one or more additional therapeutic agents described herein.

[0110] The kits provided herein may also include a device for administering the AON. Examples of such devices include, but are not limited to, syringes, needle-free syringe drip bags, patches, and inhalers.

[0111] The kit provided herein may also include a pharmaceutically 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 must be reconstituted for parenteral administration, the kit may include a sealed container of a suitable vehicle in which the active ingredient can be dissolved to form a particle-free sterile solution suitable for parenteral administration. Examples of pharmaceutically acceptable vehicles include, but are not limited to: aqueous vehicles, including but not limited to water for injection USP, 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 ethanol, 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.

[0112] VII. Combination Therapy with a Second Active Agent

[0113] Also provided herein are methods of combination therapy using AON disclosed herein and other therapeutic agents that can be used to treat DMD or delay its onset. In another embodiment, provided herein are methods of combination therapy using AON1 and other therapeutic agents that can be used to treat DMD or delay its onset. In these methods, AON disclosed herein, such as AON1, ​​is administered as described elsewhere herein, such as QWx24W.

[0114] As used herein, the term "combination" includes the use of more than one therapy (e.g., one or more prophylactic and / or therapeutic agents). However, the use of the term "combination" does not limit 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 to the subject 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 before), concurrently with, or after (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) the second therapy (e.g., a prophylactic or therapeutic agent) is administered to the subject. Triple therapy is also contemplated herein.

[0115] The AON disclosed herein and one or more second active agents can be administered to a subject simultaneously or sequentially via 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 decomposing before entering the bloodstream) and the disease or condition being treated.

[0116] The route of administration of the AON disclosed herein is independent of the route of administration of the second therapy. In another embodiment, the AON disclosed herein is administered intravenously. Therefore, according to these embodiments, the AON disclosed herein is administered intravenously, and the second therapy can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectal, buccal, intranasal, liposomes, by inhalation, vaginally, intraocularly, by local delivery of a catheter or stent, subcutaneously, intrafatally, intraarticularly, intrathecally, or in a slow release dosage form. In one embodiment, the AON disclosed herein and the second therapy are administered by the same mode of administration, for example, by IV. In another embodiment, the AON disclosed herein is administered by one mode of administration (e.g., by IV), and the second dose is administered by another mode of administration (e.g., oral).

[0117] 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 and is administered once or twice a day. 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 AON or its derivatives provided herein, as well as any optional additional active agents administered to the subject at the same time.

[0118] One or more second active ingredients or agents can be used in the methods and compositions provided herein together with AON or derivatives thereof provided herein. The second active agent can be a macromolecule (e.g., protein) or a small molecule (e.g., a synthetic inorganic molecule, an organometallic molecule, or an organic molecule).

[0119] Examples of macromolecular active agents include, but are not limited to, hematopoietic growth factors, cytokines, AONs, and monoclonal and polyclonal antibodies. Typical macromolecular active agents are biomolecules, such as naturally occurring or synthetic or recombinant proteins. Specific examples of AONs used as second active agents herein include idepursen, casimersen, golodisen, vitolassen, and SRP-5051 (Sarepta Therapeutics).

[0120] Examples of small molecules include corticosteroids, such as deflazacort.

[0121] Other therapies that can be combined with the AONs disclosed herein include gene therapy (e.g., SRP-9001, GALGT2, or GNT0004 (Sarepta Therapeutics)), gene editing (e.g., CRISPR / CAS9 (Sarepta Therapeutics)), and cell therapy (e.g., CAP-1002 (Capricor Therapeutics / Nippon Shinyaku Co. Ltd.)).

[0122] VIII. Examples

[0123] The following examples are intended to illustrate certain embodiments provided herein, but are not intended to limit the scope of the present disclosure.

[0124] Example 1

[0125] AON1 administration in del52hDMD / mdx mice for 25 weeks

[0126] hDMD del52 / mdx(+ / +) and C57BL / 6J wild-type (WT) mice (~7 weeks old on day 1) were divided into four groups and dosed as follows:

[0127]

[0128] A dose of 6 mg / kg / dose is equivalent to 0.92 μmol / kg / dose (the molecular weight of AON1 is 6517 g / mol). A dose of 18 mg / kg / dose is equivalent to 2.76 μmol / kg / dose. Therefore, the total amount of AON1 administered over 25W is 150 mg / kg (23.00 μmol / kg) for the 6 mg / kg dose level and 450 mg / kg (69.00 μmol / kg) for the 18 mg / kg dose level.

[0129] After administration, plasma concentration, tissue distribution, concentration, exon skipping, overall gait score, and cytoprotection were analyzed. Figure 1-6 Shown in.

[0130] In simple terms, Figure 1 Shown are AON1 plasma concentrations over time. Figure 2 AON1 concentrations in heart, quadriceps, liver, diaphragm, gastrocnemius, and kidney tissues are shown. Figure 3 showed that after 25 weeks of treatment, AON1 achieved sustained dose-dependent exon 51 skipping in muscle tissues (quadriceps, gastrocnemius, heart, and diaphragm).

[0131] Figure 4 Dystrophin wt% in heart and quadriceps are shown, and overall gait score for AON1 is shown (mean ± SEM; N = 16-18 (baseline, week 29) or 10-12 (week 37). 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). After 25 weeks of treatment, AON1 improved and maintained dystrophin and gait scores. References for fine motor and motor gait testing: NA Datson et al. (2020), NUCLEIC ACID THERAPEUTICS. DOI: 10.1089 / nat.2019.0824.

[0132] Figure 5AON1 treatment was shown to reduce muscle pathology associated with the hDMD del52 (+ / +) model and maintain pharmacological effects for 12 weeks after 25 weeks of dosing. Gastrocnemius muscles from vehicle-treated mice showed mild atrophy, minimal necrosis, minimal inflammation (anucleated cell infiltration), and minimal fibrosis. In contrast, gastrocnemius muscles from AON1-treated mice showed minimal atrophy and no other effects.

[0133] Figure 6 AON treatment was shown to reduce cardiac pathology associated with the hDMD del52 (+ / +) model and maintain pharmacological effects for 12 weeks. Myocardium from vehicle-treated mice showed minimal atrophy, minimal necrosis, minimal inflammation (monocyte infiltration), and minimal fibrosis. In contrast, myocardium from AON1-treated mice showed minimal inflammation (monocyte infiltration) and minimal fibrosis with no other effects.

[0134] Example 2

[0135] Synthesis of AON1

[0136] Oligonucleotide AON1 is synthesized by solid phase organic synthesis. No intermediates are isolated or characterized during the synthesis. After the synthesis sequence is completed, the oligonucleotide is cleaved from the solid support, deprotected and purified by ultrafiltration and preparative anion exchange (AEX) chromatography. After the HPLC fractions are pooled, the oligonucleotide AON1 solution is concentrated and desalted by a second ultrafiltration step. Subsequent lyophilization and homogenization provide the product AON1 as a solid material, which is stored at -20±5°C before shipment.

[0137] The synthesis cycle performed at ambient temperature is schematically shown below (the first cycle is used for illustration purposes). The oligonucleotide chain of AON1 is synthesized on a solid support. After the sequence is completed, the oligonucleotide is cleaved from the resin, purified, and the resulting solution is lyophilized to obtain the final drug substance AON1.

[0138]

[0139] Stage 1: Solid Phase Synthesis

[0140] The oligonucleotide portion of AON1 is manufactured by solid phase organic synthesis using platform synthesis. The synthesis is automated and carried out using coupled cycle technology on a synthesizer equipped with a closed column reactor and does not involve the separation of intermediates. A solid support with a universal adapter is loaded into a flow-through column reactor and synthesis is started. Each synthesis cycle consists of four sequential chemical steps followed by washing until a full-length oligonucleotide is established.

[0141] Step 1: Unprotect

[0142] In the first step, called deblocking or deprotection, the solid support is treated with dichloroacetic acid in toluene to remove the UnyLinker from the solid support. TM In all subsequent cycles of step 1, deprotection involves removal of the DMT protecting group from the 5'-OH of each sequentially introduced phosphoramidite or locked amidite to generate a 5'-DMT-off oligonucleotide.

[0143] Step 2 Activation and coupling

[0144] The second step, called "activation and coupling," is the chain extension step, in which the next nucleotide is added to the sequence, growing from the 3' end to the 5' end. UnyLinker is treated with a solution of the corresponding phosphoramidite in acetonitrile or dichloromethane / acetonitrile in the presence of a suitable activator solution (e.g., benzylthiotetrazolyl in acetonitrile). TM (1st cycle) or the unprotected alcohol group of oligonucleotide chain [n] (all subsequent cycles) with 5'-DMT removal to build the extended DMT onto oligonucleotide chain [n+1]. The final chain extension step incorporates a TEG group into the 5' end of AON1 using DMT-TEG-phosphoramidite without sulfurization.

[0145] Step 3 Oxidation / sulfurization

[0146] Depending on the desired backbone chemistry, the corresponding phosphorothioate or phosphodiester is introduced at this step by sulfurization or oxidation. Oligonucleotide chain [n+1] carrying DMT (DMT-on) is 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). In the manufacture of AON1, ​​only the last cycle involves an oxidation step, all other cycles involve sulfurization reactions.

[0147] Step 4: Capping

[0148] The fourth step, called "capping", ends the synthesis cycle by adding a mixture of Cap A (acetonitrile / 2,6-lutidine / N-methylimidazole) and Cap B (tert-butylphenoxyacetoacetic anhydride in acetonitrile) to the oligonucleotide on the solid support to prevent any unreacted 5'-OH oligonucleotide chain [n] from further reaction with the phosphoramidites in subsequent coupling cycles. This results in the formation of truncated sequences. After all 18 amidite synthesis cycles are completed, the base and backbone protected oligonucleotides remain bound to the solid support.

[0149] Phase 2: Main chain deprotection

[0150] Treatment of the oligonucleotides on the solid support with diethylamine in acetonitrile results in the removal of the β-cyanoethyl protecting group on the phosphorothioate / phosphate backbone. The reaction is followed by a wash step using acetonitrile.

[0151] Stage 3: Base deprotection and cleavage from resin

[0152] The tower is incubated at room temperature with ammonia / ethanol, and then washed with ammonia / ethanol. The solution merged is incubated at an elevated temperature. In this two-step process, the crude oligonucleotide is cracked from the solid support, and the core base protecting groups are removed. The resulting solution is filtered and quenched, and the filtrate containing the crude oligonucleotide is dissolved in the deprotection solution.

[0153] Stage 4: Desalting of oligonucleotides by ultrafiltration

[0154] The crude AON1 solution was concentrated by ultrafiltration using an ultrafiltration membrane. The resulting concentrate was diafiltered using water for injection. The resulting AON1 aqueous solution was filtered.

[0155] Stage 5: Purification of crude oligonucleotides by anion exchange chromatography

[0156] The reaction mixture was diluted in aqueous sodium phosphate / acetonitrile buffer and used Source TM 30Q was purified by anion exchange (AEX) chromatography.

[0157] Generate a simulated pool and analyze it by HPLC-UV / MS. Based on the analysis results, select fractions to pool and perform further downstream processing. Selected fractions are combined to provide a product pool of appropriate purity (>85%) and yield. The final product pool is analyzed again by HPLC-UV / MS.

[0158] Stage 6: Desalting of oligonucleotides by ultrafiltration

[0159] The pooled AON1 solution was concentrated by ultrafiltration using an ultrafiltration membrane. The resulting concentrate was diafiltered using water for injection. The resulting AON1 aqueous solution was filtered.

[0160] Stage 7: Oligonucleotides in lyophilized solid form

[0161] The concentrated solution from stage 6 was filtered through a 0.2 μm membrane and finally lyophilized to obtain the final drug substance. After lyophilization, the material was equilibrated in a clean room environment for at least 24 hours to obtain a stable water content in the final product. The equilibrated drug substance was obtained as a white to off-white to yellow powder (melting point (mp): ~265°C (DSC decomposition); purity was 92-93% as determined by ion-pair reversed phase HPLC-UV / MS).

[0162] Example 3

[0163] Manufacturing process of 50mg / mL AON1

[0164] Compounding of buffer solutions and optional bioburden reduction filtration

[0165] The required amounts of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and sodium chloride were weighed into approximately 80% of the total amount of water for injection (WFI) and stirred in a Class C environment under laminar air flow (LAF). After the excipients were dissolved, WFI was added to the final amount.

[0166] If the preparation of the buffer solution and the preparation of the drug product solution are not performed on the same day, the buffer solution will be filtered through a 0.45 / 0.2 μm membrane filtration unit in Class C under the LAF to reduce bioburden.

[0167] Solution balancing, formulation and bioburden reduction filtration

[0168] The lyophilized AON1 (Example 2) was equilibrated in a Grade C environment under LAF at room temperature for 12–24 hours. After equilibration, the lyophilized AON1 was weighed and dissolved in approximately 50% buffer solution by stirring to avoid agglomeration. A final AON1 concentration of 50 mg / mL solution was achieved by adding the calculated amount of buffer solution. The solution was mixed at 200 to 1000 rpm for 20-30 min, avoiding foaming. The formulated solution was filtered through a sterile 0.45 / 0.2 μm membrane filtration unit to reduce bioburden. Formulation and bioburden reduction filtration were performed in a Grade C environment under LAF.

[0169] In-line sterile filtration and aseptic filling

[0170] The AON1 solution was sterile filtered using a 0.45 / 0.2 μm filter through two consecutive in-line filtration units and aseptically filled into ready-to-use sterile vials. The vials were capped and sealed. The filled units were stored in a temperature-controlled cold room.

[0171] The AON1 composition is provided in a single-dose vial as 3 mL of isotonic sterile, preservative-free solution for intravenous infusion at a concentration of 50 mg / mL and a pH of 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-top. The composition of 5.25 L of AON1 solution is provided as follows:

[0172] Composition of 5.25L AON1 solution

[0173] Components quantity Concentration (g / L) AON1 262.5g 50 Disodium hydrogen phosphate dodecahydrate 27.0g 5.135 Sodium dihydrogen phosphate dihydrate 4.6g 0.883 Sodium chloride 24.5g 4.676 Water for injection qs qs to target volume

[0174] qs = complement

[0175] Example 4

[0176] AON1 PS1 was administered to male and female del52hDMD / mdx mice for 3 weeks and assessed 2 or 4 weeks after administration.

[0177] A total of 120 homozygous hDMD del52 / mdx+ / + male and female mice were used for the study, which were bred in Charles River, UK and genotyped by BioLytix, Switzerland. All hDMD del52 / mdx mice in this study were derived from a re-derived population. In addition, age-matched C57BL / 6J (n=20) mice (Charles River, Germany) were used as wild-type (WT) controls. When setting up different groups for this study, mice were randomly divided by sex in a way that the entire litter of mice did not fall into a single test group, and baseline weight was taken into account so that there was no group difference at the beginning of the first treatment.

[0178] The mice were weighed on each treatment day and the dose was adjusted accordingly. The entire IV injection procedure took 5-8 minutes per mouse, with actual injection time ranging from 10-60 seconds.

[0179] All compounds were formulated to ensure equimolar dosing and administered at an injection volume of 8 mL / kg. Mice were weighed on each treatment day and doses were adjusted accordingly. For example, mice weighing 30 g received 240 μL of IV injection.

[0180] Mice were administered AON1 PS for 13 weeks. At the endpoint, 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 underwent cardiac puncture and heart tissue was harvested. The tissue was immediately flash frozen by immersion in isopentane in liquid nitrogen, placed in cryovials pre-cooled on dry ice and stored at -80°C. Frozen samples were analyzed for % exon skipping and dystrophin levels by qualified methods. Results are presented in Figure 7 and 8 Shown in.

[0181] Example 5

[0182] AON1 was administered to male and female del52hDMD / mdx mice for 13 weeks and assessed 4 or 8 weeks after administration

[0183] All mice received IV injections of vehicle or AON1 once a week (QW) in the tail vein starting at 7 weeks of age for a maximum study duration of 13 weeks. The entire IV injection process took 5-8 minutes per mouse, with an actual injection time of 60 seconds. The compounds were formulated at specific concentrations and administered in an injection volume of 8 mL / kg. The mice were weighed on each treatment day and the dose was adjusted accordingly. For example, a mouse weighing 30 g received an IV injection of 240 μL.

[0184] At the end of life, 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 was collected by cardiac puncture. The following tissues were collected and snap-frozen:

[0185] Skeletal muscle: gastrocnemius (R), quadriceps (L+R); collect both sides into one vial so that there is one sample of quadriceps tissue.

[0186] Half of the heart (dissected so each half has an atrium and ventricle), entire diaphragm, liver (2x 1 / 3 of the entire liver), kidney (R).

[0187] All tissues were immediately snap frozen by immersion in isopentane in liquid nitrogen, placed in cryovials pre-chilled on dry ice and stored at -80°C until sectioning.

[0188] Exon skipping analysis was performed in 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) assay. The percentage of hDMD exon 51 skipping was calculated using the following formula:

[0189] (Human skipped exon 51 copies / 20 μL reaction) / (Human skipped exon 51 copies / 20 μL reaction + Human non-skipped exon 51 copies / 20 μL reaction)*100

[0190] The lower limit of quantification (LLOQ) of skipped and non-skipped transcripts was based on a threshold approach. Fig. 9 Dystrophin levels in the heart and quadriceps Fig.10 The overall gait score is shown in Fig.11 Shown in.

[0191] Example 6

[0192] The effects of AON1 PS and AON2 on complement activation in human and cynomolgus monkey sera were investigated.

[0193] The dosing vehicle for the test article administration is an aqueous phosphate buffer (pH 6.9-7.1) with 0.7% NaCl, which is prepared and used as a baseline control. An appropriate amount of test article (AON1 PS or AON2) is weighed and dissolved into a clear-looking solution at 10X dosing concentration using stirring / vortex mixing. No correction factor is applied to the measured weight. Before and after use, the preparation is refrigerated (4°C). Before use, all test article preparations are warmed to room temperature on a desktop for at least 30 minutes. The test article is considered stable after preparation at 4°C for one week. The actual weight and dilution are recorded in the research data. No dose analysis was performed in this study; residual preparations are discarded.

[0194] Test system

[0195] Test system 1: Individual sera from n=3 normal male human volunteers (age >18 years).

[0196] Test System 2. Individual sera from n=3 normal male cynomolgus monkeys (age > 3 years).

[0197] Route of administration

[0198] This is an in vitro study and the test article is exposed to the test system in vitro. The test article is added to the test system at a ratio of one to nine parts (1:9, v / v). This ratio maintains the appropriate concentration of the test system so that sufficient concentrations of complement control proteins are present.

[0199] Dosage regimen

[0200] The mixture was incubated at 37°C ± 2°C for 30 ± 2 minutes and stored at -80°C until analyte testing.

[0201] Objectives and research plan

[0202] The aim of this study was to evaluate the complement split product activation profiles of two antisense oligonucleotides (AON1 PS and AON2) following incubation in normal male human and male cynomolgus monkey sera.

[0203] Experimental design

[0204] The test is 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-top tube. Once all mixtures are prepared, they are transferred to a 37°C ± 2°C water bath and incubated for 30 ± 2 minutes. After the mixtures have been incubated, all samples are aliquoted and frozen at -80°C or below.

[0205] Bioanalytical Assays for Complement Activation

[0206] Detection of Bb cleavage products by ELISA: Bb is produced when factor B is cleaved by activation of the alternative pathway of complement. The level of Bb produced during in vitro exposure to the test article can be used as a measure of the level of activation of the alternative pathway. The Bb ELISA is run with duplicate wells and the average is reported. Initially assess the level of Bb at two dilutions to verify that the assay is not saturated by samples with a higher degree of activation.

[0207] Detection of C3a cleavage products by ELISA: C3a is produced when C3 is cleaved at the complement centerpoint. Therefore, activation of any of the three complement pathways can result in C3a cleavage. The level of C3a is also important because it is an anaphylatoxin. The level of C3a produced during in vitro exposure to the test article can be used as a measure of the level of complement activation that is strong enough to reach this centerpoint, and can also be used as an indicator of the possibility of direct proinflammatory consequences of this activation. The C3a ELISA is run with duplicate wells and the average is reported. Initially evaluate the level of C3a at two dilutions to verify that the assay is not saturated by samples with a higher degree of activation.

[0208] The results are Fig.12 and 13 Shown in.

[0209] Example 7

[0210] 26-week toxicity and bioanalysis study in juvenile mice

[0211] In a 26-week juvenile toxicity and toxicokinetics (TK) study with a 13-week recovery period, male CD-1 mice (20-22 / group (primary toxicity study); 6 (vehicle; vehicle control buffer diluted with sterile saline), 25-31 / group (TK); 21-27 / group (recovery)) were dosed with 0 (vehicle control buffer), 6, 12, or 18 mg / kg AON1 by IV bolus every 4 days from PND 21 to PND203. Blood was collected from AON1-treated groups on PND 21 and 203 for bioanalysis pre-dose (PND203 only), 0.5, 1, 3, 8, and 24 hours post-dose. The following parameters were assessed: body weight, food consumption, developmental landmarks (glans preputial separation), neurobehavioral assessments (locomotor activity at ≈PND 187 and 281; auditory startle at ≈PND 190 and 284; Morris water maze at ≈PND 193 and 287), ophthalmological examination, clinical pathology (clinical chemistry, hematology, and coagulation), gross necropsy, organ weights, and histopathology.

[0212] Based on the absence of adverse findings, the no observed adverse effect level (NOAEL) for toxicity was 18 mg / kg / dose. At PND 21, the maximum observed concentration (C max ) and the area under the concentration-time curve (AUC 0-24 ) values ​​were 5280nmol / L and 11,700h*nmol / L, respectively. At PND 203, the C max and AUC 0-24 The values ​​were 5860 nmol / L and 14,600h*nmol / L respectively.

[0213] Example 8

[0214] NHP 39-week toxicity and bioanalysis study

[0215] Male cynomolgus monkeys (3 / group (primary toxicity study); 2 / group, 0, 6, 12, and 18 mg / kg QW (13-week recovery)) were dosed with 0 (vehicle: 0.9% NaCl injection, USP), 6, 12, or 18 mg / kg QW AON1 by IV infusion (60 minutes) for 39 weeks. Blood was collected for bioanalysis / TK and complement activation analysis at different study days / time points. The following parameters were evaluated: mortality, clinical signs, body weight, food consumption, 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 indicators (Bb, C3a, and sC5b-9), ophthalmology, ECG, organ weights, and histopathology.

[0216] Mean concentrations of AON1 in tissues were quantifiable for all AON1 treatment groups at the time of planned terminal euthanasia and unplanned euthanasia on day 274. Mean concentrations generally increased with increasing doses from 6 mg / kg / dose to 12 mg / kg / dose. On day 274 of the 6 to 12 mg / kg / dose dosing period, the order of mean concentrations of AON1 was liver, kidney, heart, gastrocnemius, and biceps femoris.

[0217] Exon skipping in tissues was measured by RT-ddPCR. This method quantifies the number of non-skipped DMD (dystrophin) transcripts, as well as the number of exon 51 skipped DMD transcripts; and the percentage of exon skipping (% skipping) 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 Fig.14 .

[0218] No AON1-related changes in body weight; ophthalmologic findings; veterinary observations; abnormal ECG waveforms or arrhythmias; or effects on PR interval, QRS duration, QT or corrected QT (QTc) interval, or heart rate were observed. No AON1-related clinical pathology (hematologic and coagulation) effects were observed for animals dosed up to 18 mg / kg / dose. No AON1-related macroscopic observations or organ weight effects were observed at early terminal sacrifice or scheduled terminal sacrifice for males dosed up to 18 mg / kg / dose.

[0219] On the 194th day or the 211th day of the dosing period, one male administered with 12 or 18 mg / kg / dose was sacrificed due to AON1-related moribund state. Clinical and veterinary observations in both animals were similar, including decreased activity, emaciated appearance, and depression or subcutaneous edema of the lower trunk and / or limbs. Clinical pathology findings indicated inflammation, azotemia, hypoalbuminemia, and elevated urine protein (in one measured animal). The cause of moribundity was attributed to glomerular renal damage and multisystem vascular inflammation. The AON1-related clinical pathological effects in the unplanned animals supported inflammatory reactions, fluid deficiency, possible gastrointestinal losses, excitement / physiological reactions, and / or moderate reductions in platelet counts, but lacked a clear mechanism. Microscopic findings in the unplanned animals included tubular degeneration / regeneration, vascular degeneration, hypertrophy, and inflammation of the vascular wall and / or perivascular tissue, which may lead to interstitial edema, thrombosis (lungs), and / or local degenerative changes in the affected tissues (heart). Edema may also be associated with a significant decrease in serum protein, which is caused by glomerular changes. These findings and their sequelae are consistent with complement-mediated etiology, which monkeys are prone to in the context of AON therapy (Frazier Toxicol Pathol. 2015 January; 43 (1): 78-89). The reason for the increased sensitivity to AON in NHPs is related to the genetics of the factor H component in cynomolgus monkeys, which is understood to increase the binding of factor H to AON. The reduction in the amount of free factor H required to regulate complement activation eliminates key regulators of the alternative pathway.

[0220] Low-level and transient complement activation involving the alternative and terminal pathways of complement was observed in all dose groups, including the control group. In most animals, the increase in complement activation generally decreased to near pre-dose levels by 72 hours after dosing, and activation levels trended upward throughout the study. Two animals that were terminally sacrificed early had Bb levels increased by >200% on days 113 and 204, respectively, compared to pre-dose levels. Although the complement activation profiles of these two animals were consistent with complement involved in the observed renal damage, other animals also showed increases in the measured complement fragments. Due to the presence of multiple complement regulators, the differences between animals may be due to individual differences in the ability of animals to control complement activation after initiation. The mechanism of complement activation through nonspecific interactions between AONs and factor H proteins is thought to be of very limited clinical relevance because complement activation in humans 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).

[0221] AON1-related clinical pathological effects were observed in animals that were scheduled to be sacrificed and administered ≥6 mg / kg / dose. In animals administered 18 mg / kg / dose, slight increases in alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities were observed on days 176 (except ALT), 204, 211, 216, and 218 of the dosing period, indicating hepatic disturbances. Effects supporting inflammatory responses included mild decreases in albumin concentrations and increases in globin concentrations (resulting in a decrease in the albumin:globin ratio) on days 176, 204, 211, 216, and 218 of the dosing period in animals administered 18 mg / kg / dose, and mild increases in haptoglobin concentrations on days 211, 216, and 218 of the dosing period in animals administered 18 mg / kg / dose. In addition, albumin is lost via the kidneys, as supported by a mild increase in the urine protein:urine creatinine ratio on days 211, 216, and 218 of the dosing period and an increase in the incidence of protein in the urine in animals given 18 mg / kg / dose. In animals given ≥6 mg / kg / dose, a mild to moderate increase in the microalbumin:creatinine ratio on days 211, 216, 218, 225, 239, 253, 260, 267, and 274 of the dosing period also supports albumin loss via the kidneys. In animals given 18 mg / kg / dose, a mild increase in the aggregin:creatinine ratio on days 211, 216, and 218 of the dosing period supports renal injury. Another effect was a mild, dose-independent decrease in platelet counts in animals given ≥6 mg / kg / dose, lacking a clear mechanism, on days 176, 204, 211, 216, 218, 225, 239, 253, 260, 267, and 274 of the dosing period. In the 18 mg / kg QW group, a decrease in platelet counts over time was observed in 2 animals, resulting in moderate / marked thrombocytopenia, but this was not associated with any anatomic pathology findings. No AON1-related coagulation effects were observed in animals that were sacrificed as scheduled.

[0222] Microscopic findings in two early terminally sacrificed animals and an animal scheduled for terminal sacrifice administered 18 mg / kg / dose on day 218 of the dosing period included minimal degeneration of tubular epithelial cells in one animal and minimal mixed cellular inflammation in the liver of another animal, which was associated with elevated liver enzyme values. These findings were attributed to accumulation of AON1 in tubular epithelial cells or Kupffer cells, respectively. Other AON1-related findings commonly seen in moribund and early-sacrificed animals included mild or moderate mononuclear infiltrates in heart valves and a trend toward increased incidence of mononuclear infiltrates in several tissues. These findings may reflect the presence of systemic inflammation and / or complement activation associated with dosing and, in addition to valvular changes, are considered to be an exacerbation of common incidental findings in monkeys. In all animals, observations associated with accumulation of AON1 included basophilic granules in renal tubular epithelial cells and Kupffer cells (often in a dose-related manner) and the presence of macrophages characterized by altered, foamy, and / or slightly basophilic cytoplasm in multiple tissues, but most notably in the lymph nodes. This accumulation did not appear to be associated with damage to surrounding tissues in animals given ≥12 mg / kg / dose, and macrophage changes did not appear to be associated with damage at any dose. At recovery-phase euthanasia, microscopic findings tended to be reversible, with minimal basophilic granules in the kidneys, minimal pigment in the liver Kupffer cells, and minimal to moderate vacuolated / pigmented macrophage infiltrates in the mandibular and / or mesenteric lymph nodes in animals given ≥6 mg / kg / dose. Additionally, minimal vacuolated / pigmented macrophage infiltrates were observed in the gastrointestinal tract of one animal dosed at 12 or 18 mg / kg / dose each, and in the bladder (transitional cell degeneration / vacuolation was not observed in this monkey study) and testes of one animal dosed at 18 mg / kg / dose.

[0223] Based on the severity of clinical pathology and anatomic pathology findings, the no-observed-adverse-effect level (NOAEL) for the overall effect on health and well-being of males administered AON1 was considered to be 6 mg / kg / dose on day 260 of the dosing period, which corresponds to a mean maximum observed concentration (C 260) of 17,000 nmol / L. max ) and the area under the concentration-time curve (AUC 0-25 ).

[0224] Example 9

[0225] First-in-human dose selection

[0226] The proposed dose and duration of the clinical trial (Example 10) were selected to ensure safety and the prospect of direct benefit for each pediatric participant enrolled in the trial.

[0227] The first human clinical study (Example 10) included a sequential evaluation of up to 6 dose levels of AON1: 0.6 mg / kg, 1.5 mg / kg, 3 mg / kg, 6 mg / kg, 9 mg / kg and 12 mg / kg administered IV.

[0228] The starting dose for the clinical study (Example 10) was determined from the NOAEL (18 mg / kg / week) of AON1 in the GLP 13-week repeat dose toxicity study using male cynomolgus monkeys (Example 13). Based on the body surface area scaling method, the human equivalent dose (HED) of the monkey NOAEL of 18 mg / kg QW is 5.8 mg / kg QW. Therefore, the selected starting dose of 0.6 mg / kg provides a 10-fold safety margin based on monkeys, which are considered a more relevant species for hazard identification because monkeys are more sensitive to complement activation and the ensuing inflammatory response, which is considered to be one of the class effects of antisense oligonucleotides (the latter being the main source of nonclinical toxicity in monkeys) (Shen J Pharmacol Exp Ther. 2014 Dec;351(3):709-17). It is generally believed that the use of monkeys will maximize the possibility of identifying any adverse reactions that are similar in quantity and quality to those that may be expected in humans (Farman Toxicol Pathol. 2003;31:119-122).

[0229] Given the short plasma half-life of AON1 and the expected lower accumulation compared to weekly dosing intervals, it is considered appropriate to increase the dose level of Cohort 1A (Part 1) every 2 weeks to minimize the duration of exposure of individual participants at subtherapeutic dose levels. 2 to 2.5-fold dose increments allow individual participants to be safely and effectively escalated to doses that nonclinical data indicate may be safe. The toxicological findings observed in the 39-week study were related to chronic dosing and were not expected to be related to the initial single ascending dose in the clinical trial (Example 10).

[0230] It has been previously demonstrated that plasma exposure to phosphorothioate oligonucleotides is similar between NHPs and DMD patients for the same mg / kg dose (Bosgra Nucleic Acid Ther. 2019; 29(6): 305-322); therefore, the use of body surface area scaling provides a more conservative approach to starting dose selection for the single ascending dose portion of clinical trials compared to body weight scaling. For the repeated dose phase of clinical trials, direct weight-based (mg / kg) extrapolation from NHPs to humans was applied, and sequential evaluation of 6, 9, and 12 mg / kg QW was proposed.

[0231] The starting dose of 6 mg / kg QW for the repeated-dose phase was selected to provide each pediatric participant with the prospect of immediate benefit because this is a pharmacologically active dose at which dystrophin expression is expected to be >10% of normal at steady state. The DMC will review safety data from the dosing cohort through Week 4 and make recommendations for dose escalation.

[0232] The maximum dose of 12 mg / kg / week, selected to maximize the potential efficacy of BMN 351 treatment with a manageable safety profile, was justified by chronic toxicology studies and provided information:

[0233] Overall, the major toxicity signals in the chronic toxicology studies (39-week monkey study (Example 8), 26-week mouse study (Example 11), and juvenile mouse study (Example 7)) were consistent with the well-known class effects of phosphorothioate oligonucleotides, which can be monitored clinically.

[0234] Dose-limiting toxicity was observed in the 39-week monkey study, where the NOAEL was determined to be 6 mg / kg / week. Dosing above the NOAEL (6 mg / kg / week) determined in the 39-week monkey study was considered appropriate because dose-limiting toxicity at ≥12 mg / kg QW appears consistent with complement-mediated effects, to which NHPs are known to have exaggerated sensitivity compared with 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). Therefore, complement-mediated effects should be considered hazard identification rather than providing a margin of clinical safety. In the 18 mg / kg QW group in the 39-week monkey study, a decrease in platelet count over time was observed in 2 animals, resulting in moderate / marked thrombocytopenia, but this was not associated with any anatomic pathology findings. Thrombocytopenia is clinically monitorable.

[0235] Based on nonclinical studies, the liver, kidneys, hematological system (thrombocytopenia), coagulation system, and vascular system (inflammatory changes) were identified as target organs of toxicity in humans.

[0236] Risk monitoring and mitigation activities will be conducted through frequent clinical and laboratory monitoring, 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 whether to escalate the dose, reduce the dose, open a new cohort, or expand a cohort.

[0237] Example 10

[0238] A Phase 1 / 2 Dose-Escalation Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of AON1 in Participants with Duchenne Muscular Dystrophy

[0239] This study will investigate the safety and tolerability of 6 ascending doses of AON1 by intravenous (IV) infusion. Given the progressive nature of Duchenne muscular dystrophy (DMD), this is an open-label study and all participants will receive AON1. The proposed dose and trial duration are selected to ensure the safety of each pediatric participant enrolled in the trial and the prospect of direct benefit. The drug product to be used is described in Example 3.

[0240] The no-observed-adverse-effect level (NOAEL) in the pivotal 13-week GLP toxicity study in monkeys was determined to be 18 mg / kg QW; therefore, the human equivalent dose (HED) was 5.8 mg / kg QW based on the body surface area scaling method. Therefore, the selected starting dose of 0.6 mg / kg provides a 10-fold safety margin based on monkeys, which are considered more relevant for hazard identification because monkeys are more sensitive to complement activation and the ensuing inflammatory response, 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). Low (2 to 2.5-fold) dose titration will further increase the likelihood of early detection of any potential dose-dependent safety signals, allowing earlier intervention.

[0241] Based on previous safety experience with this class of molecules, participants will be staggered at each of 6 dose levels (0.6mg / kg, 1.5mg / kg, 3mg / kg, 6mg / kg, 12mg / kg, and 18mg / kg). The Data Monitoring Committee (DMC) will evaluate available safety data before recommending whether to escalate the dose, de-escalate the dose, open a new cohort, or expand a cohort. The design includes careful monitoring of participants' safety prior to dose escalation, including acute safety events of known class effects of antisense oligonucleotides (AONs), and implementation of individual and study stopping rules when necessary.

[0242] This study was conducted to demonstrate proof of concept by combining safety, tolerability, and pharmacokinetics (PK) with dystrophin expression to assess dose response and provide insights for dose selection for further clinical development. Studies have shown that higher amounts of dystrophin in muscle are associated with less severe disease phenotypes (de Feraudy et al. Ann Neurol. 2021;89(2):280–292); therefore, increased dystrophin expression is a meaningful outcome of this study.

[0243] Plasma PK and muscle distribution will be assessed to investigate the relationship between dose, plasma and muscle exposure, exon skipping, and dystrophin expression.

[0244] After all participants complete 12 weeks (Cohort 1A, Part 2) or 24 weeks (Cohorts 1B, 2, and 3) of treatment at the 6mg / kg, 9mg / kg, and 12mg / kg dose levels, AON1 distribution, exon skipping, and dystrophin levels in muscle will be read to determine the best tolerated dose for future clinical development.

[0245] Once this is accomplished, participants will be enrolled in the long-term extension (LTE) study, which is planned to last at least 1 year. The concept of 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 assess functional changes in participants longitudinally and potentially compared to a matched external control group. Given the natural history of the disease, a relevant extension study is an essential component of this study design.

[0246]

[0247]

[0248] 6MWT, 6-minute walk test; AE, adverse event; AESI, adverse event of special interest; AUC 0-t , the area under the concentration-time curve from time 0 to the last time point; AUC 0-inf , area under the concentration-time curve from time 0 to infinity; CBCL, Child Behavior Checklist; CL, clearance; C max , maximum concentration; C trough , plasma trough concentration; DMD, Duchenne muscular dystrophy; ECG, electrocardiogram; mRNA, messenger RNA; NSAA, NorthStar Ambulatory Assessment; PK, pharmacokinetics; RT-ddPCR, reverse transcriptase-droplet digital polymerase chain reaction; SAE, serious adverse event; t 1 / 2 , half-life; T max, the time to reach the maximum concentration; V ss , steady-state volume of distribution

[0249] This study is a dose-escalation study designed to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of 6 escalating doses of IV AON1 in participants with DMD who are susceptible to exon 51 skipping. This is a Phase 1 / 2, open-label study; no randomization is 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:

[0250] Male and aged 4 to 10 years at screening

[0251] A clinical diagnosis of Duchenne muscular dystrophy, caused by a documented dystrophin mutation in the DMD gene that predisposes to exon 51 skipping, reviewed by a central genetic counselor

[0252] Ambulatory at screening, defined as the ability to walk independently without assistive devices and complete a timed 10-meter walk / run test in 8 seconds or less

[0253] Currently not dependent on a daytime ventilator and not expected to require daytime mechanical or non-invasive ventilation within the next year

[0254] Currently receiving oral corticosteroids, maintained 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 changes in body weight

[0255] Normal renal function based on pre-specified laboratory values

[0256] Selected key exclusion criteria included the following:

[0257] For children aged 7 years or older, forced expiratory volume ( FEV1 ) < 60% of the predicted value

[0258] Current or past liver or kidney disease

[0259] Left ventricular ejection fraction (LVEF) < 55% based on echocardiogram (ECHO) performed within 3 months prior to screening or at the screening visit

[0260] · Mean QT interval corrected by the Fridericia method (QTcF) ≥ 450 milliseconds on three screening electrocardiograms (ECGs)

[0261] Platelet count <150 x 10 at screening 9 / L

[0262] · Receipt of any exon 51 skipping therapy within 12 weeks prior to baseline (Day 1) or any gene therapy for the treatment of DMD at any time

[0263] Eligible participants will be assigned to 1 of 3 cohorts: Cohort 1 (n=6), Cohort 2 (n=3), or Cohort 3 (n=6). Participants in Cohort 1 are 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 1-week dosing holiday at each dose level while safety data is evaluated before escalating to the next higher dose, or in the case of the 6 mg / kg dose, before escalating to chronic dosing. In Cohort 1A (Part 2), participants will receive weekly doses of AON1 at 6 mg / kg, a dose that is expected to result in approximately 10% dystrophin expression at steady state based on nonclinical 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. The detailed dose escalation procedure is described below.

[0264] The DMC will monitor safety and make dose escalation recommendations. Dose-limiting toxicity (DLT) criteria apply up to the time period evaluated 1 week after a single dose 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 apply until the Week 4 evaluation of the last participant in the cohort. Study stopping criteria apply to all participants throughout the study. A maximum of 18 participants will be enrolled, depending on whether the maximum tolerated dose (MTD) is reached.

[0265] For participants in Cohort 1A (Part 1) (n=3), AON1 will be administered at a starting dose of 0.6 mg / kg, with an interval of at least 9 days between dosing for participants. Participants in Cohort 1A (Part 1) will not receive dosing at their Week 2 visit. The DMC will review the safety data for each participant to Week 2 and recommend that the participant's dose be escalated to 1.5 mg / kg in the absence of a DLT (or if a DLT occurs, the cohort will be expanded to 6 participants). Participants in Cohort 1A (Part 1) will receive 1.5 mg / kg AON1 at Week 3, after which the DMC will review the safety data for each participant to Week 4 and recommend that the participant's dose be escalated to 3 mg / kg in the absence of a DLT (or if a DLT occurs, the cohort will be expanded to 6 participants). Participants in Cohort 1A (Part 1) will not receive dosing at their Week 4 visit. Participants in Cohort 1A (Part 1) will receive 3 mg / kg AON1 (single dose) at Week 5, after which the DMC will review safety data for each participant through Week 6 and recommend dose escalation to 6 mg / kg for the participant in the absence of a DLT (or expand the cohort to 6 participants if a DLT exists). Participants in Cohort 1A (Part 1) will not be dosed at their Week 6 visit. Participants in Cohort 1A (Part 1) will receive 6 mg / kg AON1 (single dose) at Week 7, after which the DMC will review safety data for each participant through Week 8 and recommend initiating Part 2 with weekly dosing at 6 mg / kg in the absence of a DLT (or expand the cohort to 6 participants in Part 1 if a DLT exists). Participants in Cohort 1A (Part 1) will not be dosed at their Week 8 visit.

[0266] If a DLT occurs in a participant in Cohort 1A, 3 additional participants will be enrolled at the dose level where the DLT occurred and then individually escalated according to the dose escalation rules outlined above for Cohort 1A (Part 1).

[0267] For participants in Cohort 1A, the visit after Week 8 will be the start of Part 2, designated as the baseline (Week 1) visit for the 6 mg / kg QW dose level. Dosing of 6 mg / kg QW for participants in Cohort 1A (Part 2) will be spaced at least 9 days apart. After all 3 Cohort 1A participants have received 3 doses of 6 mg / kg QW in Part 2 and the third participant completes the Week 4 assessment, the DMC will review the available safety data and, if there are no DLTs, recommend initiation of Cohort 1B at 6 mg / kg QW.

[0268] Dosing of 6 mg / kg QW for participants in Cohort 1B will be spaced at least 9 days apart. After all 3 Cohort 1B participants have received 3 doses of 6 mg / kg QW in Part 2 and the last participant completes the Week 4 assessment, the DMC will review the available safety data for all 6 Cohort 1 participants and recommend initiation of Cohort 2 dosing at 9 mg / kg QW if there is no more than 1 DLT.

[0269] For participants in Cohort 2 (n=6), AON1 will be administered at 9 mg / kg QW with an interval of at least 9 days between participants. After all 6 Cohort 2 participants have received 3 doses and the last participant completes the Week 4 assessment, the DMC will review the available safety data and recommend the opening of Cohort 3 at 12 mg / kg QW if there is no more than 1 DLT.

[0270] For all participants in Cohort 3 (n=6), AON1 will be administered at 12 mg / kg QW with an interval of at least 9 days between participants.

[0271] If 2 or more participants in a given cohort experience a DLT, the MTD will be exceeded and no further dose escalation will be performed. The DMC may recommend expanding the previous cohort, selecting a lower intermediate dose, or stopping the study after data review.

[0272] The primary safety assessment endpoints are listed in the “Objectives and Endpoints” table above. Secondary objectives of the study include evaluating the plasma and urine PK and muscle distribution of AON1. All participants will undergo muscle biopsies at screening and at Week 13 (Cohort 1A) or Week 25 (Cohorts 1B, 2, and 3). Once all 6 participants in Cohort 3 (12 mg / kg QW) complete the Week 25 assessment, 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 this study and have a planned duration of at least 1 year. The details of the LTE study will be specified in a separate protocol. Once the dose is determined and the LTE study is open for recruitment, participants in this study will transition to the LTE study or have a study completion visit 4 weeks after their last dose of AON1. Dosing and assessments will continue in this study until the LTE study is opened.

[0273] The DMC will review all participant data after the last participant is dosed at Week 25. Based on the results and DMC recommendations, the Sponsor will determine the dose for the LTE study. A participant is considered to have completed the study if they complete all scheduled visits during the Sponsor-determined LTE dose. Participants may begin dosing in the LTE following health authority clearance or approval and after the participant's site receives IRB / EC approval for the LTE. Study completion occurs on the day of the participant's last visit prior to enrollment in the LTE. During the LTE study, participants will receive 12 mg / kg of AON1 or the highest tolerated dose determined during this study.

[0274] Embodiment 11

[0275] Evaluation of a 26-week toxicity study of AON1 in male CD-1 mice

[0276] Male CD-1 mice (10 or 15 / group (primary toxicity study), and up to 5 / group primary toxicity animals in the 0 and 18 mg / kg dose groups, euthanized after a 13-week recovery period; 4 (vehicle control) and 19 / group (toxicokinetics (TK))) were dosed by slow IV bolus with 0 (vehicle: 0.9% Sodium Chloride Injection, USP), 6, 12, or 18 mg / kg AON1 twice weekly for a total duration of 26 weeks. Blood was collected for bioanalysis at 0 (within 2 minutes of dosing), 0.33, 1, 3, 8, and 24 hours post-dose on days 1 and 176. The following parameters were evaluated: mortality, clinical signs, body weight, food consumption, ophthalmic examination, clinical pathology (i.e., clinical chemistry, hematology, and coagulation), gross necropsy, organ weights, and histopathology.

[0277] Bioanalysis and tissue distribution

[0278] Plasma exposure (based on C max and AUC 0-24 ) generally increased with increasing doses in a dose-proportional manner. No accumulation was observed in male mice following multiple doses of AON1.

[0279] The mean concentrations of AON1 in male mouse tissues were measurable in all treatment groups on day 178. The mean concentrations generally increased with increasing doses from 6 mg / kg / dose to 18 mg / kg / dose. The order of mean concentrations of AON1 at different dose levels was: liver, kidney, heart, gastrocnemius, and biceps femoris.

[0280] Due to the mild severity of the pathological findings and the fact that administration up to 18 mg / kg / dose twice weekly had no effect on the health or well-being of the animals, the no observed adverse effect level (NOAEL) was 18 mg / kg / dose twice weekly on day 176 of the dosing period, which corresponded to mean maximum observed concentrations (Cmax) of 37,700 nmol / L and 14,700 h*nmol / L, respectively. max ) and the area under the concentration-time curve (AUC 0-24 )value.

[0281] Example 12

[0282] Cytochrome P450 Inhibition and Induction

[0283] The potential of AON1 to inhibit CYP enzymes was evaluated in human liver microsomes incubated with 0.1 to 100 μM AON1. The validity of the assay was confirmed by positive controls (i.e., reference compounds) for each isozyme. IC for direct inhibition of CYP1A2 50 The value was 7.85 μM, while the IC 50 Values ​​are >100 μM. After taking into account microsomal protein binding, the unbound IC values ​​for direct inhibition of CYP1A2, CYP2C8, and CYP2C19 50 The values ​​were 0.09, >1.27, and >1.08 μM, respectively, which were lower than the predicted human plasma C at the maximum dose of 18 mg / kg. max 18 μM. Plasma protein binding of AON1 was attempted but could not be assessed due to high levels of nonspecific binding to the filter device. No direct inhibition of CYP2B6, CYP2C9, CYP2D6, CYP3A4 / 5 (substrate = testosterone or midazolam). According to the literature, inhibition of CYP enzymes by phosphorothioate-based oligonucleotides is test system dependent; inhibition of CYP enzymes by phosphorothioate-based oligonucleotides was observed in human liver microsomes but 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.full.pdf). Cryopreserved human hepatocytes provide a more clinically relevant inhibition profile for in vitro to in vivo extrapolation of drug-drug interactions for inhibition of CYP enzymes by phosphorothioate-based oligonucleotides. Given that AON1 is also a phosphorothioate-based oligonucleotide, microsomes may not be representative of the in vivo situation.

[0284] To this end, further studies were performed using cryopreserved human hepatocytes and demonstrated that AON1 is not a direct or time-dependent inhibitor of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 / 5 and that for all CYPs evaluated, the IC 50 Values ​​>100 μM.

[0285] The potential to induce CYP enzymes was assessed in hepatocytes incubated with 100 μM AON1. The validity of the assay was confirmed by positive controls (i.e., reference compounds) for each isozyme. In vitro assays of CYP induction showed that AON1 did not induce CYP1A2, CYP2B6, and CYP3A4 at the transcriptional level.

[0286] Embodiment 13

[0287] Evaluation of AON1 in male cynomolgus monkeys treated for 13 weeks

[0288] Male cynomolgus monkeys (3 / group (primary toxicity study); 2 / group, 0, 12 and 18 mg / kg QW (4-week recovery)) were dosed with 0 (vehicle: 0.9% NaCl injection, USP), 6, 12 or 18 mg / kg QW AON1 by IV infusion (60 minutes) for 13 weeks. Blood was collected for bioanalysis on days 1 and 85 before dosing and 0.5, 1, 2, 3, 6, 8 and 24 hours after dosing, and for complement activation analysis before dosing and 1, 3, 6 and 24 hours after dosing. A pre-dose sample was also collected on day 78. The following parameters were evaluated: mortality, clinical signs, body weight, food consumption, 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 indicators (Bb, C3a, and sC5b-9), ophthalmology, ECG, organ weights, and histopathology.

[0289] Exon skipping in tissues was measured by RT-ddPCR. This method quantifies the number of non-skipped DMD (dystrophin) transcripts, as well as the number of DMD transcripts that skip exon 51; and calculates the exon skipping percentage (% skipping). The exon 51 skipping percentage ranged from 1.7% to 14.4% and generally increased with increasing doses.

[0290] The scope of the present disclosure is not limited by the embodiments disclosed in the examples, which are intended as single illustrations of various aspects, and any equivalents are within the scope of the present disclosure. In addition to those modifications shown and described herein, various modifications will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims.

[0291] Various references such as patents, patent applications, and publications are cited herein, the disclosures of which are hereby incorporated by reference in their entireties.

Claims

1. A method for treating a subject with DMD or delaying the onset of DMD in a subject, comprising administering AON1 to the subject 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 in a QW manner.

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

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

4. The method of any one of claims 1-3, wherein the AON1 is administered in a QWx25W manner.

5. The method of any one of claims 1-3, wherein the AON1 is administered in a QWx24W manner.

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

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

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

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

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

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

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

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

14. The method of claim 13, wherein the second active agent is idepsen, casimersen, golodisen, viltolasen, SRP-5051 (Sarepta Therapeutics), a corticosteroid such as deflazacort, a gene therapy (e.g., SRP-9001, GALGT2, or GNT 0004 (Sarepta Therapeutics)), a gene editing (e.g., CRISPR / CAS9 (Sarepta Therapeutics)), or a cell therapy (e.g., CAP-1002 (Capricor Therapeutics / Nippon Shinyaku Co. Ltd.)).

Images