Methods for treating muscular dystrophy with casimersen

Abstract

To provide improved compositions and methods for treating muscular dystrophy.SOLUTION: For example, the disclosure provides methods for treating Duchenne muscular dystrophy patients having a mutation in the DMD gene that is suited to exon 45 skipping by administering an effective amount of casimersen. The present disclosure is based on clinical evidence showing that treatment with casimersen, which is an exon 45 skipping antisense oligonucleotide, significantly increased dystrophin protein in patients over baseline. Furthermore, a positive correlation between exon skipping and de novo dystrophin protein was observed.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 825,573, filed Mar. 28, 2019, and U.S. Provisional Application No. 62 / 902,518, filed Sep. 19, 2019. The entire teachings of the above applications are incorporated herein by reference in their entirety.

[0002] The present invention relates to improved methods for treating muscular dystrophy in patients. Also provided are compositions suitable for promoting exon 45 skipping in the human dystrophin gene.

Background Art

[0003] Background of the Invention In various genetic diseases, the effect of mutations on the final expression of a gene can be regulated through a target exon skipping process during the splicing process. When a normal functioning protein is prematurely terminated due to a mutation therein, it has been shown that a means for restoring some functional protein production by antisense technology is possible through intervention during the splicing process, and also that when an exon associated with a disease-causing mutation can be specifically deleted from some genes, a shortened protein product having the biological properties of the same native protein or having sufficient biological activity to ameliorate the disease caused by the mutation in the exon can sometimes be produced (see, for example, Sierakowska, Sambade et al. 1996; Wilton, Lloyd et al. 1999; van Deutekom, Bremmer-Bout et al. 2001; Lu, Mann et al. 2003; Aartsma-Rus, Janson et al. 2004).

[0004] Duchenne muscular dystrophy (DMD) is caused by a defect in the expression of the protein dystrophin. Dystrophin is a rod-shaped cytoplasmic protein and an important part of a protein complex that links the cytoskeleton of muscle fibers through the cell membrane to the surrounding extracellular matrix. Dystrophin plays an important structural role in muscle fibers, connecting the extracellular matrix and the cytoskeleton. The N-terminal region binds actin, while the C-terminal is part of the dystrophin glycoprotein complex (DGC) that extends into the muscle sheath. Dystrophin-deficient muscle fibers in mdx mice have been shown to exhibit increased sensitivity to contraction-induced muscle sheath rupture (see Petrof et al. 1993; Cirak et al. 2012).

[0005] The gene encoding the dystrophin protein contains 79 exons spread over more than 2 million nucleotides of DNA. Any exon mutations that change the reading frame of the exon, introduce a stop codon, or remove all out-of-frame exon(s) or duplicate one or more exons can prevent the production of functional dystrophin and potentially lead to DMD.

[0006] The onset of the disease can be demonstrated at birth by an increase in creatine kinase levels, and significant motor impairment may appear by the first year of life. By the age of 7 or 8, most DMD patients experience increasing difficulty walking, lose the ability to rise from the floor and climb stairs, and by the age of 10 to 14, most become wheelchair-dependent. DMD is uniformly fatal, and affected individuals typically die of respiratory failure and / or heart failure in their late teens or early twenties. Regarding the continuous progression of DMD, therapeutic intervention is possible at all stages of the disease, but until recently, treatment has been limited to glucocorticoids. This glucocorticoid is associated with numerous side effects such as weight gain, behavioral changes, pubertal changes, osteoporosis, Cushingoid appearance, growth inhibition, and cataracts. Therefore, the development of better treatment methods to treat the root cause of this disease is essential.

[0007] Mutations, typically deletions of one or more exons, that result in a correct reading frame along the entire dystrophin transcript, and thus do not prematurely terminate translation of the mRNA into protein, result in a less severe form of muscular dystrophy, Becker muscular dystrophy (BMD). When the splicing of upstream and downstream exons in the processing of the mutated dystrophin pre-mRNA maintains the correct reading frame of the gene, the result is an mRNA that encodes a protein with a short internal deletion that retains some activity, resulting in the Becker phenotype.

[0008] Over the years, deletions of exon(s) that do not change the reading frame of the dystrophin protein have been known to cause the BMD phenotype, while exon deletions that cause a frameshift result in DMD (Monaco, Bertelson et al. 1988). In general, dystrophin mutations that change the reading frame and thus disrupt proper protein translation, including point mutations and exon deletions, result in DMD. It should also be noted that some BMD and DMD patients have exon deletions that span multiple exons.

[0009] Recent clinical trials testing the safety and efficacy of splice-switching oligonucleotides (SSOs) for the treatment of DMD are based on SSO technology to induce alternative splicing of pre-mRNA by steric blockade of the spliceosome (Cirak et al., 2011; Goemans et al., 2011; Kinali et al., 2009; van Deutekom et al., 2007). However, despite these successes, the pharmacological options available for the treatment of DMD are limited.

[0010] Accordingly, there remains a need for improved compositions and methods for producing dystrophin and treating muscular dystrophies such as DMD and BMD in patients.

SUMMARY OF THE INVENTION

MEANS FOR SOLVING THE PROBLEM

[0011] The present disclosure is based, at least in part, on clinical evidence showing that treatment with casimersen, an exon 45 skipping antisense oligonucleotide, significantly increased dystrophin protein in patients above baseline. Further, a positive correlation was observed between exon skipping and de novo dystrophin protein.

[0012] Accordingly, in some aspects, the present disclosure provides a method of treating Duchenne muscular dystrophy (DMD) in a patient having a mutation in the DMD gene suitable for exon 45 skipping and in need thereof, the method comprising administering to the patient a defined dose of casimersen or a pharmaceutically acceptable salt thereof.

[0013] In some aspects, the present disclosure provides a method of restoring the mRNA reading frame and inducing exon skipping in a patient having DMD with a mutation in the Duchenne muscular dystrophy (DMD) gene suitable for exon 45 skipping and in need thereof, the method comprising administering to the patient a defined dose of casimersen or a pharmaceutically acceptable salt thereof.

[0014] In some aspects, the present disclosure provides a method of increasing dystrophin production in a patient having DMD with a mutation in the Duchenne muscular dystrophy (DMD) gene suitable for exon 45 skipping and in need thereof, the method comprising administering to the patient a defined dose of casimersen or a pharmaceutically acceptable salt thereof.

[0015] In some embodiments, the dosage is administered at a dosage of about 4 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg relative to the patient's body weight.

[0016] In some embodiments, the dosage is administered as a single dose. In some embodiments, the dosage is administered once a week. In some embodiments, the dosage is administered intravenously. In some embodiments, the dosage is administered intravenously by infusion. In some embodiments, the dosage is administered intravenously by infusion over 35 - 60 minutes. In some embodiments, the dosage is administered intravenously by subcutaneous injection.

[0017] In some embodiments, the patient is up to 40 years old, up to 30 years old, or up to 21 years old. In some embodiments, the patient is 1 - 21 years old. In some embodiments, the patient is 5 - 21 years old. In some embodiments, the patient is 7 - 13 years old.

[0018] In some embodiments, the present disclosure provides a method according to any of the foregoing or related embodiments, wherein the patient has a mutation in the DMD gene selected from the group consisting of exons 7 - 42, 12 - 42, 18 - 42, 44 - 46, 44 - 47, 44 - 48, 44 - 49, 44 - 51, 44 - 53, 44 - 55, 44 - 57, or 44 - 59, or exon 44.

[0019] In some embodiments, the present disclosure provides a method according to any of the foregoing or related embodiments, wherein the patient is chronically administered casimersen. In some embodiments, the patient is administered casimersen for at least 48 weeks. In some embodiments, the patient is administered casimersen for more than 1 year, more than 2 years, more than 3 years, more than 4 years, more than 5 years, more than 10 years, more than 20 years, or more than 30 years.

[0020] In some embodiments, the present disclosure provides a method according to any of the foregoing or related embodiments, wherein the patient receives a stable dose of corticosteroid administration for at least 6 months prior to the administration of casimersen. In some embodiments, the patient has received a stable dose of corticosteroid administration for at least 6 months prior to the administration of casimersen and continues to receive corticosteroid administration during the administration of casimersen.

[0021] In some embodiments, the present disclosure provides a method according to any of the foregoing or related embodiments, wherein casimersen or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition. In some embodiments, casimersen or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition having a concentration of 50 mg / mL. In some embodiments, casimersen or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition having a concentration of 50 mg / mL and is presented in a dosage form of 100 mg / 2 mL. In some embodiments, casimersen or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition having a concentration of 50 mg / mL and is presented in a dosage form of 500 mg / 2 mL. In some embodiments, the dosage form is contained in a single-use vial.

[0022] In some embodiments, casimersen or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition comprising casimersen or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a phosphate buffer.

[0023] In some embodiments, the present disclosure provides a method according to any of the foregoing or related embodiments, wherein exon skipping is measured by reverse transcription polymerase chain reaction (RT-PCR).

[0024] In some aspects, the present disclosure provides a method according to any of the foregoing or related aspects, the method increasing dystrophin production in a patient. In some aspects, dystrophin production is measured by Western blot analysis. In some aspects, dystrophin production is measured by immunohistochemistry (IHC).

[0025] In some aspects, the present disclosure provides a method according to any of the foregoing or related aspects, the method further comprising confirming whether the patient has a mutation within the DMD gene suitable for exon 45 skipping before administering casimersen.

[0026] In some aspects, the present disclosure provides casimersen or a pharmaceutically acceptable salt thereof for use in the treatment of Duchenne muscular dystrophy (DMD) in a patient in need thereof, the patient having a mutation in the DMD gene suitable for exon 45 skipping, the treatment comprising administering to the patient a single intravenous dose of about 30 mg / kg of casimersen once a week.

[0027] In some aspects, the present disclosure provides casimersen or a pharmaceutically acceptable salt thereof for use in restoring the mRNA reading frame and inducing exon skipping in a patient in need thereof having Duchenne muscular dystrophy (DMD), the patient having a mutation in the DMD gene suitable for exon 45 skipping, the treatment comprising administering to the patient a single intravenous dose of about 30 mg / kg of casimersen once a week.

[0028] In some aspects, the present disclosure provides casimersen or a pharmaceutically acceptable salt thereof for use in increasing dystrophin production in a patient in need thereof having Duchenne muscular dystrophy (DMD), the patient having a mutation in the DMD gene suitable for exon 45 skipping, the treatment comprising administering to the patient a single intravenous dose of about 30 mg / kg of casimersen once a week. In an embodiment of the present invention, for example, the following items are provided. (Item 1) A method for treating Duchenne muscular dystrophy (DMD) in a patient having a mutation in the DMD gene suitable for exon 45 skipping, and in a patient in need thereof, the method comprising administering to the patient a dose of casimersen or a pharmaceutically acceptable salt thereof. (Item 2) The method according to item 1, wherein the dose is administered at a dose of about 30 mg / kg based on the body weight of the patient. (Item 3) The method according to items 1 to 2, wherein the dose is administered as a single administration. (Item 4) The method according to items 1 to 3, wherein the dose is administered once a week. (Item 5) The method according to any one of items 1 to 4, wherein the patient has a mutation in the DMD gene selected from the group consisting of exons 7-42, 12-42, 18-42, 44-46, 44-47, 44-48, 44-49, 44-51, 44-53, 44-55, 44-57, or 44-59, or exon 44. (Item 6) The method according to any one of items 1 to 5, wherein the patient is chronically administered casimersen. (Item 7) The method according to any one of items 1 to 6, wherein the patient is administered casimersen for at least 48 weeks. (Item 8) The method according to any one of items 1 to 7, wherein the patient receives a stable dose of corticosteroid administration for at least 6 months prior to the administration of casimersen. (Item 9) The method according to any one of items 1 to 8, wherein casimersen or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition. (Item 10) The method according to any one of items 1 to 9, wherein casimersen or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition having a concentration of about 50 mg / mL. (Item 11) The method according to item 10, wherein casimersen or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition having a concentration of about 50 mg / mL and presented in a dosage form of about 100 mg / 2 mL. (Item 12) The method according to item 11, wherein the dosage form is contained in a single-use vial. (Item 13) The method according to items 10 to 12, wherein casimersen or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition comprising casimersen or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. (Item 14) The method according to item 13, wherein the pharmaceutically acceptable carrier is a phosphate buffer solution. (Item 15) A method for a patient with Duchenne muscular dystrophy (DMD) having a mutation in the DMD gene suitable for exon 45 skipping, and in a patient in need thereof, for restoring the mRNA reading frame to induce exon skipping, the method comprising administering a certain dose of casimersen or a pharmaceutically acceptable salt thereof to the patient. (Item 16) The method according to item 15, wherein the dose is administered at a dose of about 30 mg / kg based on the body weight of the patient. (Item 17) The method according to items 15 to 16, wherein the dose is administered once a week. (Item 18) The method according to items 15 to 17, wherein the patient is administered casimersen for at least 48 weeks. (Item 19) A method for a patient with Duchenne muscular dystrophy (DMD) having a mutation in the DMD gene suitable for exon 45 skipping, and in a patient in need thereof, for increasing dystrophin production, the method comprising administering a certain dose of casimersen or a pharmaceutically acceptable salt thereof to the patient. (Item 20) The method according to item 19, wherein the dosage is administered at a dosage of about 30 mg / kg based on the weight of the patient. (Item 21) The method according to items 19 to 20, wherein the dosage is administered once a week. (Item 22) The method according to items 19 to 21, wherein the patient is administered casimersen for at least 48 weeks. (Item 23) The method according to any one of items 1 to 22, further comprising confirming whether the patient has a mutation in the DMD gene suitable for exon 45 skipping before administering casimersen. (Item 24) Casimersen or a pharmaceutically acceptable salt thereof for use in the treatment of Duchenne muscular dystrophy (DMD) in patients in need thereof, wherein the patient has a mutation in the DMD gene suitable for exon 45 skipping, and the treatment comprises administering a single intravenous dose of about 30 mg / kg of casimersen to the patient once a week. (Item 25) Casimersen or a pharmaceutically acceptable salt thereof for use in restoring the mRNA reading frame and inducing exon skipping in patients with Duchenne muscular dystrophy (DMD) in need thereof, wherein the patient has a mutation in the DMD gene suitable for exon 45 skipping, and the treatment comprises administering a single intravenous dose of about 30 mg / kg of casimersen to the patient once a week. (Item 26) Casimersen or a pharmaceutically acceptable salt thereof for use in increasing dystrophin production in patients with Duchenne muscular dystrophy (DMD) in need thereof, wherein the patient has a mutation in the DMD gene suitable for exon 45 skipping, and the treatment comprises administering a single intravenous dose of about 30 mg / kg of casimersen to the patient once a week.

BRIEF DESCRIPTION OF THE INVENTION

[0029] Embodiments of the present disclosure relate to a method of treating muscular dystrophy such as DMD by administering casimersen, an antisense oligonucleotide specifically designed to induce exon 45 skipping within the human dystrophin gene. Dystrophin plays an important role in muscle function, and various muscle-related diseases are characterized by mutant forms of this gene. Thus, in certain embodiments, the methods described herein can be used to induce exon 45 skipping in mutant forms of the human dystrophin gene, such as the mutant dystrophin gene found in DMD.

[0030] Accordingly, the present disclosure relates to a method of treating muscular dystrophy such as DMD by inducing exon 45 skipping in a patient. Further, the present disclosure relates to a method of restoring the mRNA reading frame and inducing exon skipping in DMD patients. The present disclosure also relates to a method of increasing dystrophin production in DMD patients.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0032] I. Definitions "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0033] As used herein with respect to a subject or patient, "suitable for exon 45 skipping" is intended to include subjects and patients having one or more mutations in the dystrophin gene such that the absence of exon 45 skipping of the dystrophin gene causes the reading frame to be out of frame, thereby preventing translation of the pre-mRNA and preventing the subject or patient from being able to produce dystrophin. Non-limiting examples of mutations in the following exons of the dystrophin gene are suitable for exon 45 skipping, such as exons 7-42, 12-42, 18-42, 44-46, 44-47, 44-48, 44-49, 44-51, 44-53, 44-55, 44-57, or 44-59, or a deletion of exon 44. Determining whether a patient has a mutation in the dystrophin gene suitable for exon skipping is well within the understanding of one of ordinary skill in the art (see, for example, Aartsma-Rus et al. (2009) Hum Mutat. 30:293-299, Gurvich et al., Hum Mutat. 2009;30(4) 633-640, and Fletcher et al. (2010) Molecular Therapy 18(6)1218-1223).

[0034] The terms "antisense oligomer", "antisense compound", "antisense oligonucleotide", "oligomer", and "oligonucleotide" are used interchangeably in this disclosure and refer to a sequence of cyclic subunits linked by subunit linkages, each cyclic subunit consisting of (i) a ribose sugar or derivative thereof, and (ii) a base pair moiety attached thereto, such that the order of the base pair moieties forms a base sequence complementary to a target sequence of a nucleic acid (typically RNA) by Watson-Crick base pairing, forming a nucleic acid:oligomer heteroduplex within the target sequence. In certain embodiments, the oligomer is a phosphorodiamidate morpholino oligomer ("PMO"). In other embodiments, the antisense oligonucleotide is a 2'-O-methyl phosphorothioate. In other embodiments, the antisense oligonucleotides of this disclosure are cross-linked nucleic acids (BNAs) such as peptide nucleic acids (PNAs), locked nucleic acids (LNAs), or 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs).

[0035] The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each containing a nucleic acid base sequence) that are related to each other by the Watson-Crick base pairing rules. For example, the nucleic acid base sequence "T-G-A (5'→3')" is complementary to the nucleic acid base sequence "A-C-T (3'→5')". Complementarity can be "partial", with nucleic acid bases that do not cover all of a given nucleic acid base sequence being matched to another nucleic acid base sequence according to the base pairing rules. For example, in some embodiments, the complementarity between a given nucleic acid base sequence and another nucleic acid base sequence can be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Alternatively, continuing the example, "complete" or "perfect" (100%) complementarity can exist between a given nucleic acid base sequence and another nucleic acid base sequence. The degree of complementarity between nucleic acid base sequences significantly affects the efficiency and strength of hybridization between the sequences.

[0036] "Dystrophin" is a rod-shaped cytoplasmic protein and an important part of a protein complex that links the cytoskeleton of muscle fibers through the cell membrane to the surrounding extracellular matrix. Dystrophin contains multiple functional domains. For example, dystrophin contains an actin-binding domain at approximately amino acids 14 - 240 and a central rod domain at approximately amino acids 253 - 3040. This large central domain is formed by 24 spectrin-like triple-helical elements of approximately 109 amino acids that have homology with α-actinin and spectrin. The repeats are typically interrupted by four proline-rich non-repeat segments, also called hinge regions. Repeats 15 and 16 are separated by an 18-amino acid extension that appears to provide a major site for protein cleavage of dystrophin. The sequence identity between most repeats ranges from 10 - 25%. One repeat contains three α-helices: 1, 2, and 3. α-Helices 1 and 3 are each formed by seven helical turns and interact as coiled-coils, probably via a hydrophobic interface. α-Helix 2 has a more complex structure and is formed by segments of four and three helical turns separated by glycine or proline residues. Each repeat is encoded by two exons and is typically interrupted by an intron between amino acids 47 and 48 in the first part of α-helix 2. Other introns are usually scattered on helix-3 and are found at different positions within the repeat. Dystrophin also contains a cysteine-rich segment (i.e., 15 cysteines out of 280 amino acids) and contains a cysteine-rich domain at approximately amino acids 3080 - 3360, showing homology with the C-terminal domain of slime mold (Physarum polycephalum) α-actinin. The carboxy-terminal domain is at approximately amino acids 3361 - 3685.

[0037] The amino terminus of dystrophin binds to F-actin, and the carboxy terminus binds to the dystrophin-associated protein complex (DAPC) in the muscle sheath. The DAPC includes dystroglycan, sarcoglycan, integrin, and caveolin, and mutations in any of these components cause autosomal recessive muscular dystrophy. The DAPC is destabilized in the absence of dystrophin, resulting in a decrease in the level of member proteins and thus leading to progressive fiber damage and membrane leakage. In various forms of muscular dystrophy such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), muscle cells produce altered and functionally defective forms of dystrophin or no dystrophin at all due to mutations in gene sequences that mainly result in inaccurate splicing. The dominant expression of defective dystrophin protein, or the complete absence of dystrophin or dystrophin-like proteins, leads to a rapid progression of muscle degeneration as described above. In this regard, a "defective" dystrophin protein can be characterized by the form of dystrophin produced in a particular subject with DMD or BMD, as is known in the art, or by the absence of detectable dystrophin.

[0038] An "exon" refers to a defined section of nucleic acid encoding a protein or a nucleic acid sequence represented in the mature form of an RNA molecule after any portion of the pre-processed (or precursor) RNA has been removed by splicing. The mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA such as rRNA or tRNA. The human dystrophin gene has approximately 79 exons.

[0039] An "intron" refers to a nucleic acid region within a gene that is not translated into protein. Introns are non-coding sections that are transcribed into precursor mRNA (pre-mRNA) and then removed by splicing during the formation of mature RNA.

[0040] "Effective amount" or "therapeutically effective amount" refers to the amount of a therapeutic compound, such as an antisense oligomer including casimersen, that is administered to a mammalian subject, either as a single dose or as part of a series of doses, and that is effective to produce the desired therapeutic effect. For antisense oligomers, this effect can be brought about by inhibiting translation or natural splicing processing of the selected target sequence, or by exon skipping to increase dystrophin production.

[0041] In some embodiments, the effective amount of an antisense oligomer, or a composition comprising an antisense oligomer, is at least 4 mg / kg, at least 10 mg / kg, or at least 20 mg / kg over a period of time for treating a subject. In some embodiments, the effective amount of an antisense oligomer, or a composition comprising an antisense oligomer, for increasing the number of dystrophin-positive fibers in a subject is at least 4 mg / kg, at least 10 mg / kg, or at least 20 mg / kg. In various embodiments, the effective amount is at least 4 mg / kg, at least 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 20 mg / kg to about 30 mg / kg, about 25 mg / kg to about 30 mg / kg, or about 30 mg / kg to about 50 mg / kg. In some embodiments, the effective amount is about 30 mg / kg, or about 50 mg / kg.

[0042] In various embodiments, the effective amount of an antisense oligomer, or a composition comprising an antisense oligomer, for increasing dystrophin production in a subject is at least 4 mg / kg, at least 10 mg / kg, or at least 20 mg / kg. In various embodiments, the effective amount is at least 4 mg / kg, at least 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 20 mg / kg to about 30 mg / kg, about 25 mg / kg to about 30 mg / kg, or about 30 mg / kg to about 50 mg / kg. In some embodiments, the effective amount is about 30 mg / kg, or about 50 mg / kg.

[0043] In certain embodiments, the effective amount of an antisense oligomer, or a composition comprising an antisense oligomer, for a patient to stabilize, maintain, or improve walking distance from a 20% deficit, for example, in the 6MWT, compared to healthy equivalents, is at least 4 mg / kg, at least 10 mg / kg, or at least 20 mg / kg. In various embodiments, the effective amount is at least 4 mg / kg, at least 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 20 mg / kg to about 30 mg / kg, about 25 mg / kg to about 30 mg / kg, or about 30 mg / kg to about 50 mg / kg. In some embodiments, the effective amount is about 30 mg / kg, or about 50 mg / kg.

[0044] In certain embodiments, the effective amount is at least 24 weeks, at least 36 weeks, or at least 48 weeks, at least 4 mg / kg, at least 10 mg / kg, about 10 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, or about 30 mg / kg to about 50 mg / kg to increase the number of dystrophin-positive fibers in a subject. In certain embodiments, the increase in dystrophin-positive fibers in a subject is up to at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of normal. In some embodiments, the treatment increases the number of dystrophin-positive fibers in a patient to 20 - 60% or 30 - 50% of normal.

[0045] In certain embodiments, the effective amount for a patient to stabilize or improve walking distance from a 20% deficit, for example, in the 6MWT, compared to healthy equivalents, is at least 24 weeks, at least 36 weeks, or at least 48 weeks, at least 4 mg / kg, at least 10 mg / kg, about 10 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, or about 30 mg / kg to about 50 mg / kg.

[0046] In various embodiments, the effective amount is at least 24 weeks, at least 36 weeks, or at least 48 weeks, at least 4 mg / kg, at least 10 mg / kg, about 10 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, or about 30 mg / kg to about 50 mg / kg to increase dystrophin production in a patient. In some embodiments, the increased dystrophin production is about 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% compared to a healthy equivalent. In certain embodiments, the increase in dystrophin production is about 0.1% to 0.5%, 0.5% to 0.9%, 0.8% to 1%, 0.9% to 1.2%, 0.9% to 1.0%, 0.9% to 1.01%, 1% to 1.01%, 1% to 1.5%, 1.5% to 2%, 1.9% to 2.0%, 1.9% to 2.01%, 2% to 2.01%, 2% to 2.5%, 2.5% to 3%, 2.9% to 3.0%, 2.9% to 3.01%, 2% to 3.01%, 3% to 3.5%, 3.5% to 4%, 4% to 4.5%, 4.5% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 1% to 2%, 1% to 3%, 1% to 5%, 2% to 4%, 2% to 5%, 4% to 6%, 5% to 8%, 8% to 10%, 1% to 5%, 2% to 6%, 3% to 7%, 4% to 8%, 5% to 10%, 10% to 12%, 12% to 15%, 15% to 20%, 17% to 20%, 20% to 22%, 20% to 25%, 25% to 30%, or 30% to 35% compared to a healthy equivalent.

[0047] "Enhance" or "enhancement", or "increase" or "increasing", or "stimulate" or "stimulation" generally refers to the ability of one or more antisense oligonucleotides, including, for example, casimersen, or a pharmaceutical composition thereof, to produce or cause a greater physiological response (i.e., downstream effect) in a cell or subject as compared to the response caused by either no antisense oligonucleotide or a control compound. A measurable physiological response can include, among other responses apparent from the understanding in the art and the description herein, an increase in the expression (or production) of dystrophin protein in a functional form, or an increase in dystrophin-related biological activity in muscle tissue. An increase in muscle function can also be measured and can include an increase or improvement in muscle function of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The percentage of muscle fibers expressing functional dystrophin can also be measured and can include an increase in dystrophin expression of about 1%, 2%, 5%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the muscle fibers. For example, it has been shown that when 25-30% of the fibers express dystrophin, an improvement in muscle function of about 40% can occur (e.g., DelloRusso et al, Proc Natl Acad Sci USA (see 99:12979-12984, 2002). In some embodiments, the increased dystrophin production is about 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% compared to healthy counterparts. In certain embodiments, the increase in dystrophin production is about 0.1% - 0.5%, 0.5% - 0.9%, 0.8% - 1%, 0.9% - 1.2%, 0.9% - 1.0%, 0.9% - 1.01%, 1% - 1.01%, 1% - 1.5%, 1.5% - 2%, 1.9% - 2.0%, 1.9% - 2.01%, 2% - 2.01%, 2% - 2.5%, 2.5% - 3%, 2.9% - 3.0%, 2.9% - 3.01%, 2% - 3.01%, 3% - 3.5%, 3.5% - 4%, 4% - 4.5%, 4.5% - 5%, 5% - 6%, 6% - 7%, 7% - 8%, 8% - 9%, 9% - 10%, 1% - 2%, 1% - 3%, 1% - 5%, 2% - 4%, 2% - 5%, 4% - 6%, 5% - 8%, 8% - 10%, 1% - 5%, 2% - 6%, 3% - 7%, 4% - 8%, 5% - 10%, 10% - 12%, 12% - 15%, 15% - 20%, 17% - 20%, 20% - 22%, 20% - 25%, 25% - 30%, or 30% - 35% compared to healthy counterparts. As used herein, "increased dystrophin production", "increase in dystrophin production", etc. refer to an increase in the production of at least one of dystrophin, dystrophin-like protein, or functional dystrophin protein in a subject.

[0048] An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 times or more (e.g., 500, 1000 times) (including all integers and decimal points between and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the amount produced without the antisense oligonucleotide (absence of the agent) or by a control compound.

[0049] The terms "reduce" or "inhibit" generally relate to the ability of one or more antisense compounds of the invention to "decrease" a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic arts. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and can include a decrease in the symptoms or pathology of muscular dystrophy, or a decrease in the expression of a defective form of dystrophin, such as a modified form of dystrophin expressed in an individual having DMD or BMD. A "decrease" in the response can be statistically significant as compared to the response produced without the antisense compound or by a control composition and can include a decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all integers therebetween.

[0050] As used herein, terms such as "function" and "functional" refer to biological, enzymatic, or therapeutic functions.

[0051] A "functional" dystrophin protein generally refers to a dystrophin protein that has sufficient biological activity to typically reduce the progressive breakdown of muscle tissue, which is characteristic of muscular dystrophy, in a different manner when compared to a "modified" or "deficient" form of the dystrophin protein present in a particular subject having DMD or BMD. In certain embodiments, a functional dystrophin protein can have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers therebetween) of the in vitro or in vivo biological activity of wild-type dystrophin, as measured according to the ordinary skill in the art. As an example, dystrophin-related activity in in vitro muscle culture can be measured according to myotube size, organization (or disorganization) of myofibrils, contractile activity, and spontaneous clustering of acetylcholine receptors (see, e.g., Brown et al., Journal of Cell Science. 112:209-216, 1999). Also, animal models are a valuable resource for studying the etiology of diseases and provide a means for testing dystrophin-related activity. Two of the most widely used animal models in DMD research are the mdx mouse and the Golden Retriever muscular dystrophy (GRMD) dog, both of which are dystrophin-negative (see, e.g., Collins & Morgan, Int J Exp Pathol 84:165-172, 2003). These and other animal models can be used to measure the functional activity of various dystrophin proteins. Cleaved forms of dystrophin are included, such as those produced by certain exon-skipping antisense oligonucleotides of the present disclosure.

[0052] The terms "morpholino", "morpholino oligomer", or "PMO" have the following general structure:

Chemical formula

[0053] "Casilmesen", also known by its code name "SRP-4045", is a PMO having the base sequence 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 1). Casilmesen is registered with the CAS Registry Number 1422959-91-8. The chemical name is all-P-ambo-[P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-seco](2'a→5')(C-A-A-T-G-C-C-A-T-C-C-T-G-G-A-G-T-T-C-C-T-G)5'-[4-({2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}carbonyl)-N,N-dimethylpiperazine-1-phosphonamidate] (SEQ ID NO: 1).

[0054] Casilmesen has the following structure: [Chemical Formula] It is also represented by the following chemical structure: [Chemical Formula]

[0055] For clarity, the structures of the present disclosure, such as the structure of the above-mentioned casimersen, are continuous from 5' to 3', and for the convenience of depicting the entire structure in a compact form, various figure interruptions labeled "Interruption A" and "Interruption B" are included. As will be understood by those skilled in the art, for example, each indication of "Interruption A" shows the continuation of the structure diagram at these points. Those skilled in the art will understand that the same applies to each case of "Interruption B" in the above structure. However, none of the figure interruptions are intended to indicate an actual discontinuity in the above structure, and those skilled in the art will not understand them to mean so.

[0056] As used herein, a set of parentheses used within a structural formula indicates that the structural features between the parentheses are repeated. In some embodiments, the parentheses used may be "[" and "]", and in certain embodiments, the parentheses used to indicate repeating structural features may be "(" and ")". In some embodiments, the number of repetitions of the structural features between the parentheses is the number shown outside the parentheses, for example, 2, 3, 4, 5, 6, 7, etc. In various embodiments, the number of repetitions of the structural features between the parentheses is indicated by a variable shown outside the parentheses, such as "Z".

[0057] As used herein, a bond drawn to a chiral carbon or phosphorus atom in a straight-chain or squiggly bond structural formula indicates that the stereochemistry of the chiral carbon or phosphorus is undefined and is intended to include all forms of the chiral center. Examples of such figures are shown below. [Chemical Formula]

[0058] As used herein, the terms "parenteral administration" and "administered parenterally" mean a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intracavitary, intrasynovial, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0059] The term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation, including those used in therapy, and the subject being treated.

[0060] As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free distilled water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate; and coloring agents, release agents, coating agents, sweetening agents, flavoring agents, and perfuming agents, preservatives, and antioxidants may also be present in the composition, as determined by the judgment of the formulator.

[0061] The term "restoration" of dystrophin synthesis or production generally refers to the production of dystrophin protein, including truncated dystrophin, in muscular dystrophy patients after treatment with the antisense oligomers described herein. In some embodiments, the treatment results in an increase in dystrophin production in the patient of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers therebetween). In some embodiments, the treatment increases the number of dystrophin-positive fibers in the subject to at least 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% - 100% of normal. In other embodiments, the treatment increases the number of dystrophin-positive fibers in the subject to about 20% - about 60% or about 30% - about 50% of normal. The percentage of dystrophin-positive fibers in the treated patient can be determined by muscle biopsy using known techniques. For example, the muscle biopsy can be taken from a suitable muscle such as the biceps brachii of the patient.

[0062] Analysis of the percentage of positive dystrophin fibers can be performed before and / or after treatment, or at time points throughout the course of treatment. In some embodiments, the post-treatment biopsy is taken from the contralateral muscle as the pre-treatment biopsy. Dystrophin expression studies before and after treatment can be performed using any suitable assay for dystrophin. In some embodiments, immunohistochemical detection is performed on tissue sections from the muscle biopsy using an antibody that is a marker for dystrophin, such as a monoclonal or polyclonal antibody. For example, the MANDYS106 antibody, which is a sensitive marker for dystrophin, can be used. Any suitable secondary antibody can be used.

[0063] In some embodiments, the percentage of dystrophin-positive fibers is calculated by dividing the number of positive fibers by the total number of fibers counted. Normal muscle samples have 100% dystrophin-positive fibers. Thus, the percentage of dystrophin-positive fibers can be expressed as a normal percentage. To control for the presence of trace levels of dystrophin in pre-treatment muscle as well as revertant mutant fibers, when counting dystrophin-positive fibers in post-treatment muscle, the baseline can be set using pre-treatment muscle sections from each patient. This can be used as a threshold for counting dystrophin-positive fibers in post-treatment muscle sections from that patient. In other embodiments, tissue sections stained with antibodies can also be used for dystrophin quantification using Bioquant image analysis software (Bioquant Image Analysis Corporation, Nashville, TN). The total dystrophin fluorescence signal intensity can be reported as a normal percentage. Additionally, the percentage of dystrophin-positive fibers can be determined using Western blot analysis with monoclonal or polyclonal anti-dystrophin antibodies. For example, the anti-dystrophin antibody NCL-Dys1 from Novacastra can be used. Also, the percentage of dystrophin-positive fibers can be analyzed by determining the expression of components of the sarcoglycan complex (β, γ) and / or neuronal NOS.

[0064] In some embodiments, treatment with an antisense oligomer of the present disclosure, such as casimersen, delays or reduces progressive respiratory muscle dysfunction and / or failure in DMD patients expected without treatment. In some embodiments, treatment with an antisense oligomer of the present disclosure can reduce or eliminate the need for ventilatory support expected without treatment. In some embodiments, measurements of respiratory function to track the course of the disease, as well as the evaluation of possible therapeutic interventions, include maximal inspiratory pressure (MIP), maximal expiratory pressure (MEP), and forced vital capacity (FVC). MIP and MEP measure, respectively, the pressure levels that a person can generate during inhalation and exhalation, and are sensitivity measures of respiratory muscle strength. MIP is a measure of the diaphragmatic muscle strength decline.

[0065] In some embodiments, MEP can decline before changes in other pulmonary function tests including MIP and FVC. In certain embodiments, MEP can be an early indicator of respiratory dysfunction. In certain embodiments, FVC can be used to measure the total volume of air exhaled during forced exhalation after maximal inhalation. In DMD patients, FVC increases with physical growth until the mid-teens. However, as growth slows or fails due to disease progression and muscle strength decline progresses, vital capacity enters a decline phase and declines at an average rate of about 8-8.5 percent per year after 10-12 years of age. In certain embodiments, predicted MIP percent (MIP adjusted for body weight), predicted MEP percent (MEP adjusted for age), and predicted FVC percent (FVC adjusted for age and height) are supportive analyses.

[0066] As used herein, "subject" or "patient" includes any animal that has or is at risk of having DMD or BMD, or any symptom associated with these conditions (e.g., muscle fiber loss), or a symptom that can be treated with the antisense oligonucleotides of the present disclosure, or is at risk of exhibiting a symptom. Suitable subjects (patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock, and domestic or pet animals (such as cats or dogs). Non-human primates, and preferably human patients, are included. Also included is a method of producing dystrophin in a subject having a mutation in the dystrophin gene suitable for exon 45 skipping.

[0067] As used herein, "pediatric patient" is a patient from 1 year to 21 years of age, inclusive.

[0068] As used herein, the phrases "systemic administration", "administered systemically", "peripheral administration", and "administered peripherally" mean the administration of a compound, drug, or other material other than direct administration to the central nervous system, whereby it enters the patient's system and thus is subject to metabolism and other similar processes, such as subcutaneous administration.

[0069] As used herein, "chronic administration" refers to continuous, regular, long-term therapeutic administration, i.e., regular administration without substantial interruption. For example, daily for a period of at least several weeks or months or years for the purpose of treating a patient's muscular dystrophy. For example, weekly for a period of at least several months or years for the purpose of treating a patient's muscular dystrophy (e.g., weekly for at least 6 weeks, at least 12 weeks, at least 24 weeks, at least 48 weeks, at least 72 weeks, at least 96 weeks, at least 120 weeks, at least 144 weeks, at least 168 weeks, at least 180 weeks, at least 192 weeks, at least 216 weeks, or at least 240 weeks).

[0070] As used herein, "regular administration" refers to administration with an interval between doses. For example, regular administration includes administration at fixed intervals that can be repeated (e.g., weekly, monthly).

[0071] As used herein, "placebo" refers to a substance that has no therapeutic effect and can be used as a control.

[0072] As used herein, "placebo control" refers to a subject or patient who receives a placebo, rather than a combination therapy, an antisense oligonucleotide, a non-steroidal anti-inflammatory compound, and / or another pharmaceutical composition. The placebo control may have the same mutation status as the subject or patient, be of the same age, have similar walking ability, and may receive the same concomitant medications (including steroids, etc.).

[0073] The terms "targeting sequence", "base sequence", or "nucleotide base sequence" refer to the sequence of nucleobases of an oligomer that is complementary to the sequence of nucleotides in the target pre-mRNA. In some embodiments of the present disclosure, the sequence of nucleotides in the target pre-mRNA is the exon 45 annealing site in the dystrophin pre-mRNA designated as H45A(-03+19).

[0074] "Treatment" of a subject (e.g., a mammal such as a human) or a cell is any kind of intervention used in an attempt to alter the natural course of the subject or cell. Treatment includes, but is not limited to, administration of an oligomer or a pharmaceutical composition thereof, and can be carried out either prophylactically or either after the onset of a pathological event or contact with a pathogen. Treatment includes any desirable effect on the symptoms or pathology of a disease or condition associated with the dystrophin protein, such as in certain forms of muscular dystrophy, and can include, for example, a minimal change or improvement in one or more measurable markers of the disease or condition being treated. Also included is "prophylactic" treatment that can be aimed at reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of the disease or condition or its attendant symptoms.

[0075] In some embodiments, treatment with the antisense oligomers of the present disclosure increases dystrophin production, delays disease progression, delays or reduces loss of ambulation, reduces muscle inflammation, reduces muscle damage, improves muscle function, reduces loss of lung function, and / or enhances muscle regeneration, which may be expected with or without treatment. In some embodiments, the treatment maintains, delays, or slows disease progression. In some embodiments, the treatment maintains ambulation or reduces non-ambulation. In some embodiments, the treatment maintains lung function or reduces loss of lung function. In some embodiments, the treatment maintains or increases the patient's stable walking distance, as measured, for example, by the 6 Minute Walk Test (6MWT). In some embodiments, the treatment maintains or decreases the time to walk / run 10 meters (i.e., the 10 Meter Walk / Run Test). In some embodiments, the treatment maintains or reduces the time from supine position to standing up (i.e., the rise time test). In some embodiments, the treatment maintains or reduces the time to climb four standard stairs (i.e., the 4-Stair Climb Test). In some embodiments, the treatment maintains or reduces the patient's muscle inflammation, as measured, for example, by MRI (e.g., MRI of leg muscles). In some embodiments, the MRI measures T2 and / or fat fraction to identify muscle degeneration. MRI can identify changes in the structure and composition of muscle caused by inflammation, edema, muscle damage, and fat infiltration.

[0076] In some embodiments, treatment with the antisense oligomers of the present disclosure increases dystrophin production. In some embodiments, the increased dystrophin production is about 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% compared to healthy counterparts. In certain embodiments, the increase in dystrophin production is about 0.1% - 0.5%, 0.5% - 0.9%, 0.8% - 1%, 0.9% - 1.2%, 0.9% - 1.0%, 0.9% - 1.01%, 1% - 1.01%, 1% - 1.5%, 1.5% - 2%, 1.9% - 2.0%, 1.9% - 2.01%, 2% - 2.01%, 2% - 2.5%, 2.5% - 3%, 2.9% - 3.0%, 2.9% - 3.01%, 2% - 3.01%, 3% - 3.5%, 3.5% - 4%, 4% - 4.5%, 4.5% - 5%, 5% - 6%, 6% - 7%, 7% - 8%, 8% - 9%, 9% - 10%, 1% - 2%, 1% - 3%, 1% - 5%, 2% - 4%, 2% - 5%, 4% - 6%, 5%, - 8%, 8% - 10%, 1% - 5%, 2% - 6%, 3% - 7%, 4% - 8%, 5% - 10%, 10% - 12%, 12% - 15%, 15% - 20%, 17% - 20%, 20% - 22%, 20% - 25%, 25% - 30%, or 30% - 35% compared to healthy counterparts.

[0077] In certain embodiments, treatment with the antisense oligomers of the present disclosure increases dystrophin production and delays or reduces the predicted paralysis without treatment. For example, treatment can stabilize, maintain, improve, or increase the walking ability of the subject (e.g., stabilization of walking). In some embodiments, treatment maintains or increases the stable walking distance of the patient, as measured by, for example, the 6-minute walk test (6MWT) as described by McDonald, et al. (Muscle Nerve, 2010; 42:966-74, incorporated herein by reference). Changes in the 6-minute walk distance (6MWD) can be expressed as an absolute value, rate of change, or change in % predicted value. In some embodiments, treatment maintains or improves the stable walking distance in the 6MWT from a 20% deficit in the subject compared to healthy peers. The performance of DMD patients in the 6MWT compared to the typical performance of healthy peers can be determined by calculating the % predicted value. For example, % predicted 6MWD can be calculated using the following equation for males: 196.72+(39.81×age)-(1.36×age 2 )+(132.28×height (meters)). For females, % predicted 6MWD can be calculated using the following equation: 188.61+(51.50×age)-(1.86×age 2 )+(86.10×height (meters)) (Henricson et al. PLoS Curr., 2012, version 2, incorporated herein by reference).

[0078] In some embodiments, treatment with the antisense oligomer increases the stable walking distance in a patient from baseline by 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 50 meters (including all integers therebetween). In some embodiments, the increased dystrophin production is about 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% compared to healthy equivalents. In certain embodiments, the increase in dystrophin production is about 0.1% - 0.5%, 0.5% - 0.9%, 0.8% - 1%, 0.9% - 1.2%, 0.9% - 1.0%, 0.9% - 1.01%, 1% - 1.01%, 1% - 1.5%, 1.5% - 2%, 1.9% - 2.0%, 1.9% - 2.01%, 2% - 2.01%, 2% - 2.5%, 2.5% - 3%, 2.9% - 3.0%, 2.9% - 3.01%, 2% - 3.01%, 3% - 3.5%, 3.5% - 4%, 4% - 4.5%, 4.5% - 5%, 5% - 6%, 6% - 7%, 7% - 8%, 8% - 9%, 9% - 10%, 1% - 2%, 1% - 3%, 1% - 5%, 2% - 4%, 2% - 5%, 4% - 6%, 5% - 8%, 8% - 10%, 1% - 5%, 2% - 6%, 3% - 7%, 4% - 8%, 5% - 10%, 10% - 12%, 12% - 15%, 15% - 20%, 17% - 20%, 20% - 22%, 20% - 25%, 25% - 30%, or 30% - 35% compared to healthy equivalents.

[0079] The loss of muscle function in DMD patients can occur against the backdrop of normal pediatric growth and development. Indeed, pediatric patients with DMD can show an increase in walking distance during the 6MWT over the course of about one year, despite having a progressive muscular disorder. In some embodiments, the 6MWD from DMD patients is compared to normative data from typically developing controls, as well as from existing standards data from age- and sex-matched subjects. In some embodiments, normal growth and development can be accounted for using equations based on age and height fit to the normative data. Such equations can be used to convert the 6MWD in subjects with DMD to a percent predicted (% predicted) value. In certain embodiments, analysis of the % predicted 6MWD data represents a way to account for normal growth and development, and can show that the increase in function at younger ages (e.g., below 7 years old) represents stability rather than improvement in ability in DMD patients (see Henricson et al. PLoS Curr., 2012, version 2, incorporated herein by reference).

[0080] To distinguish between different antisense molecules, a nomenclature system for antisense molecules has been proposed and published (see Mann et al., (2002) J Gen Med 4, 644-654). This nomenclature has proven particularly appropriate when testing several slightly different antisense molecules all directed to the same target region, as shown below. H#A / D(x:y)

[0081] The initial letter indicates the species (e.g., H: human, M: mouse, C: dog). "#" indicates the target dystrophin exon number. "A / D" indicates the acceptor or donor splice site at the start and end of the exon, respectively. (x y) represents the annealing coordinates, and "-" or "+" indicates the intron or exon sequence, respectively. For example, A(-6+18) indicates the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. Since the nearest splice site is the acceptor, "A" is appended before these coordinates. The description of the annealing coordinates at the donor splice site could be D(+2-18), where the last 2 exon bases and the first 18 intron bases correspond to the annealing site of the antisense molecule. The entire exon annealing coordinates are represented by A(+65+85), i.e., the site of nucleotides 65 to 85 from the start of that exon.

[0082] II. Antisense oligonucleotides Antisense oligonucleotides targeting the pre-mRNA of the dystrophin gene that result in skipping of exon 45 are used according to the methods of the present disclosure.

[0083] Such antisense oligonucleotides can be designed to block or inhibit mRNA translation or to inhibit the natural pre-mRNA splicing process, and the target sequence to which it hybridizes can be said to be "targeted" or "targeted". Target sequences typically include the AUG start codon of mRNA, translation inhibitory oligomers, or regions containing splicing sites of pre-processed mRNA or splicing inhibitory oligomers (SSOs). The target sequence of the splicing site may include an mRNA sequence having 1 to about 25 base pairs at its 5' end downstream of the normal splicing acceptor junction of the pre-processed mRNA. In some embodiments, the target sequence can be any region of the pre-processed mRNA that includes a splicing site, or is entirely within an exon coding sequence, or spans a splicing acceptor or donor site. When an oligomer targets a target nucleic acid by the methods described above, it is more generally said to "target" a biological-related target such as a protein, virus, or bacterium.

[0084] In certain embodiments, the antisense oligonucleotide specifically hybridizes to the exon 45 target region of dystrophin pre-mRNA and induces exon 45 skipping. In certain embodiments, the antisense oligonucleotide that hybridizes to the exon 45 target region of dystrophin pre-mRNA and induces exon 45 skipping is a phosphorodiamidate morpholino oligomer (PMO).

[0085] In certain embodiments, the antisense oligonucleotide is casimersen.

[0086] Casimersen belongs to a distinct class of novel synthetic antisense RNA therapeutics called phosphorodiamidate morpholino oligomers (PMOs), which are redesigns of natural nucleic acid structures. Casimersen is a PMO that hybridizes to the exon 45 target region of dystrophin pre-mRNA and induces exon 45 skipping. Casimersen can be prepared by stepwise solid-phase synthesis using the methods detailed in the references cited above and, further, in International Patent Application No. PCT / US2017 / 040017, which is hereby expressly incorporated by reference in its entirety.

[0087] PMOs offer potential clinical advantages based on in vivo non-clinical observations. PMOs incorporate modifications into the sugar ring of RNA to protect it from enzymatic degradation by nucleases in order to ensure stability in vivo. PMOs are distinguished from natural nucleic acids and other antisense oligonucleotide classes, in part, by the use of a six-membered synthetic morpholino ring that replaces the five-membered ribofuranosyl ring found in RNA, DNA, and many other synthetic antisense RNA oligonucleotides.

[0088] The uncharged phosphorodiamidate linkages specific to PMOs are thought to potentially result in reduced off-target binding to proteins. PMOs have uncharged phosphorodiamidate linkages that connect each morpholino ring, instead of the negatively charged phosphorothioate linkages used in other synthetic antisense RNA oligonucleotides in clinical development.

[0089] Potential therapeutic approaches for the treatment of DMD caused by out-of-frame mutations in the DMD gene are suggested by a milder form of dystrophinopathy known as Becker muscular dystrophy (BMD), which is caused by in-frame mutations. The ability to convert an out-of-frame mutation to an in-frame mutation is hypothesized to preserve the mRNA reading frame and produce an internally truncated but still functional dystrophin protein. Casimersen is designed to achieve this.

[0090] Casimersen is excluded or skipped from the mature spliced mRNA transcript in order to target dystrophin pre-mRNA and induce skipping of exon 45. By skipping exon 45, the disrupted reading frame is restored to an in-frame mutation. DMD consists of various genetic subtypes, but casimersen is specifically designed to skip exon 45 of dystrophin pre-mRNA.

[0091] The sequence of 22 nucleobases of casimersen is designed to be complementary to a specific annealing site of dystrophin pre-mRNA and induces skipping of exon 45 during treatment. Each morpholino ring in casimersen is linked to one of the four heterocyclic nucleobases (adenine, cytosine, guanine, and thymine) found in DNA.

[0092] Hybridization of casimersen with the target pre-mRNA sequence prevents the formation of the pre-mRNA splicing complex and deletes exon 45 from the mature mRNA. The structure and conformation of casimersen enable sequence-specific base pairing to complementary sequences. For example, eteplirsen, a PMO designed to skip exon 51 of dystrophin pre-mRNA, enables sequence-specific base pairing to the complementary sequence contained in exon 51 of dystrophin pre-mRNA.

[0093] Restoration of the dystrophin reading frame using exon skipping Normal dystrophin mRNA containing all 79 exons produces normal dystrophin protein.

[0094] Dystrophin mRNA that has lost all exons from the dystrophin gene typically results in DMD.

[0095] Eteplirsen, another exon-skipping PMO, skips exon 51 to restore the mRNA reading frame. Since exon 49 ends with a complete codon and exon 52 starts with the first nucleotide of a codon, deletion of exon 51 by exon skipping restores the reading frame and results in the production of an internally truncated dystrophin protein with an intact dystroglycan binding site.

[0096] The feasibility of using exon skipping to improve the DMD phenotype by restoring the dystrophin mRNA open reading frame has been supported in preclinical studies. Many studies in dystrophic animal models of DMD have shown that restoration of dystrophin by exon skipping results in a consistent improvement in muscle strength and function (Sharp 2011, Yokota 2009, Wu 2008, Wu 2011, Barton-Davis 1999, Goyenvalle 2004, Gregorevic 2006, Yue 2006, Welch 2007, Kawano 2008, Reay 2008, van Putten 2012). This compelling example is obtained from studies comparing dystrophin levels with muscle function in the same tissue after exon skipping (PMO use) therapy. In dystrophic mdx mice, the tibialis anterior (TA) muscle treated with a mouse-specific PMO maintained approximately 75% of its maximum tolerable force after stress-induced contraction, while the untreated contralateral TA muscle maintained only approximately 25% of its maximum tolerable force (p<0.05) (Sharp 2011). In another study, three dystrophic CXMD dogs (2- to 5-month-old) received exon skipping therapy using PMO specificity for their genetic mutation once a week for 5-7 weeks or every other week for 22 weeks. After exon skipping therapy, all three dogs showed widespread systemic expression of dystrophin in skeletal muscle and maintenance or improvement of walking (15 m running test) compared to baseline. In contrast, untreated age-matched CXMD dogs showed a marked decline in walking over the course of the study (Yokota 2009).

[0097] In both mdx mice and humanized DMD (hDMD) mouse models that express the entire human DMD transcript, PMO has been shown to have more exon skipping activity at equimolar concentrations than phosphorothioate (Heemskirk 2009).

[0098] Clinical outcomes for analyzing the effect of an antisense oligonucleotide that specifically hybridizes to the exon 45 target region of dystrophin pre-mRNA and induces exon 45 skipping include the percentage of dystrophin-positive fibers (PDPF), 6-minute walk test (6MWT), loss of ambulation (LOA), North Star Ambulatory Assessment (NSAA), pulmonary function tests (PFT), ability to rise (from the supine position) without external support, de novo dystrophin production, and increases from baseline in other functional measures.

[0099] III. Formulations and Modes of Administration In certain embodiments, the disclosure provides formulations or pharmaceutical compositions suitable for the therapeutic delivery of the antisense oligonucleotides described herein. Thus, in certain embodiments, the disclosure provides a pharmaceutically acceptable composition comprising one or more of the antisense oligonucleotides described herein in a therapeutically effective amount formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. While it is possible to administer the antisense oligonucleotides of the disclosure alone, it is preferred to administer the compounds as a pharmaceutical formulation (composition).

[0100] Methods for the delivery of nucleic acid molecules are described, for example, in Akhtar et al., 1992, Trends Cell Bio., 2:139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar; Sullivan et al., PCT WO It is described in 94 / 02595. These and other protocols can be utilized for the delivery of substantially any nucleic acid molecule, including the antisense oligonucleotides of the present disclosure.

[0101] As detailed below, the pharmaceutical compositions of the present disclosure can be specially formulated for administration in solid or liquid form, including the following: (1) oral administration, e.g., a soak (aqueous or non-aqueous solution or suspension), tablets, e.g., tablets targeted for oral, sublingual, or systemic absorption, boluses, powders, granules, a paste for application to the tongue, (2) parenteral administration, e.g., by subcutaneous injection, intramuscular injection, intravenous injection, or epidural injection, as a sterile solution or suspension or a sustained release formulation, (3) topical application, e.g., as a cream, ointment, or controlled release patch or spray for application to the skin, (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam, (5) sublingual administration, (6) intraocular administration, (7) transdermal administration, or (8) administration adapted for nasal administration.

[0102] Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) saccharides such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free distilled water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical formulations, but are not limited thereto.

[0103] Additional non-limiting examples of agents suitable for formulations using the antisense oligonucleotides of the present disclosure include PEG-conjugated nucleic acids, phospholipid-conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) that can enhance the penetration of drugs into various tissues; biodegradable polymers such as poly(DL-lactide-co-glycolide) microspheres for sustained release delivery after implantation (Emerich, D F et al., 1999, Cell Transplant, 8, 47-58) Alkermes, Inc., Cambridge, Mass.; and loaded nanoparticles such as those composed of polybutylcyanoacrylate that can deliver drugs across the blood-brain barrier and can alter the nerve uptake mechanism (Prog (Neuropsychopharmacol Biol Psychiatry, 23, 941 - 949, 1999) can be cited.

[0104] The present disclosure also features the use of a composition comprising surface - modified liposomes containing poly(ethylene glycol) lipids (PEG - modified, branched, and unbranched, or combinations thereof, or long - circulating liposomes or stealth liposomes). The antisense oligonucleotides of the present disclosure can also include covalently - bound PEG molecules of various molecular weights. These formulations provide a method for increasing the accumulation of drugs in target tissues. This class of drug carriers is resistant to opsonization and elimination by the mononuclear phagocyte system (MPS or RES), thereby enabling an extended blood - circulation time and enhanced tissue exposure of the encapsulated drug (Lasic et al., Chem. Rev. 1995, 95, 2601 - 2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005 - 1011). Such liposomes have been shown to accumulate selectively in tumors, perhaps by sequestration in hemorrhagic and angiogenesis - target tissues (Lasic et al., Science 1995, 267, 1275 - 1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86 - 90). Long - circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA compared to conventional cationic liposomes, which are known to accumulate particularly in MPS tissues (Liu et al., J. Biol. Chem. 1995, 42, 24864 - 24870; Choi et al., International PCT Publication No. WO96 / 10391; Ansell et al., International PCT Publication No. WO96 / 10390; Holland et al., International PCT Publication No. WO96 / 10392). Also, long - circulating liposomes are likely to protect drugs to a greater extent from nuclease degradation compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

[0105] In a further embodiment, the disclosure includes antisense oligonucleotide pharmaceutical compositions prepared for delivery as described in U.S. Patent Nos. 6,692,911, 7,163,695, and 7,070,807. In this regard, in one embodiment, the disclosure provides the oligomers of the disclosure (described in U.S. Patent Nos. 7,163,695, 7,070,807, and 6,692,911) in a composition comprising a copolymer of lysine and histidine (HK), alone, or in combination with PEG (e.g., branched or unbranched PEG, or a mixture of both), or in combination with any of the foregoing in combination with a targeting moiety or crosslinking agent. In certain embodiments, the disclosure provides antisense oligonucleotides in a pharmaceutical composition comprising gluconic acid-modified polyhistidine or glucosylated polyhistidine / transferrin-polylysine. Those skilled in the art will also recognize that amino acids having properties similar to His and Lys can be substituted within the composition.

[0106] Certain embodiments of the antisense oligonucleotides described herein may contain basic functional groups such as amino or alkylamino, and thus be able to form pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this context refers to relatively non-toxic inorganic and organic acid addition salts of the compounds of the present disclosure. These salts can be prepared in situ in the administration vehicle or dosage form manufacturing process, or by reacting the purified compounds of the present disclosure, in their free base form, separately with a suitable organic or inorganic acid and isolating the salt so formed during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate, among others. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0107] Pharmaceutically acceptable salts of the subject antisense oligonucleotides include, for example, conventional non-toxic salts or quaternary ammonium salts of the compounds from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicyclic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothionic acid, etc.

[0108] In certain embodiments, the antisense oligonucleotides of the disclosure may contain one or more acidic functional groups and, thus, be capable of forming pharmaceutically acceptable base and pharmaceutically acceptable salts. In these instances, the term “pharmaceutically acceptable salts” refers to relatively non-toxic inorganic and organic base addition salts of the compounds of the disclosure. These salts may likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, among others. Representative organic amines useful in the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like. (See, e.g., Berge et al. supra).

[0109] Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents, and perfuming agents, preservatives, and antioxidants may also be present in the composition.

[0110] Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like, (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like, and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0111] Formulations of the present disclosure include formulations suitable for oral, nasal, topical (including oral and sublingual), rectal, vaginal, and / or parenteral administration. The formulations can be conveniently presented in unit dosage form and can be prepared by any method well known in the art of pharmacy. The amount of the active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending on the host to be treated and the particular mode of administration. The amount of the active ingredient that can be combined with the carrier materials to produce a single dosage form is generally the amount of the compound that produces a therapeutic effect. Generally, out of 100 percent, this amount ranges from about 0.1 percent to about 99 percent, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent of the active ingredient.

[0112] In certain embodiments, the formulations of the present disclosure include an excipient selected from cyclodextrin, cellulose, liposomes, micelle formers such as bile acids, and polymeric carriers such as polyesters and polyanhydrides, and the oligomers of the present disclosure. In certain embodiments, the above formulations make the oligomers of the present disclosure orally bioavailable.

[0113] The method of preparing these formulations or pharmaceutical compositions includes the step of associating the antisense oligonucleotides of the present disclosure with a carrier and optionally one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately associating the compounds of the present disclosure with a liquid carrier, or a micronized solid carrier, or both, and then shaping the product, if necessary.

[0114] The formulations of the present disclosure suitable for oral administration can be in the form of capsules, cachets, pills, tablets, troches (using flavored bases, usually sucrose and acacia or tragacanth), powders, granules, or solutions or suspensions of aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as lozenges (using inert bases such as gelatin and glycerin, or sucrose and acacia), and / or as mouthwashes, etc., each containing a predetermined amount of the compound of the present disclosure as an active ingredient. The antisense oligonucleotides of the present disclosure can also be administered as a bolus, a liniment, or a paste.

[0115] In the solid dosage forms (capsules, tablets, pills, dragees, powders, granules, troches, etc.) of the present disclosure for oral administration, the active ingredient is one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid, (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia, (3) humectants, such as glycerol, (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (5) dissolution retardants, such as paraffin, (6) absorption promoters, such as quaternary ammonium compounds and surfactants, such as poloxamer and sodium lauryl sulfate, (7) wetting agents, such as cetyl alcohol, glycerol monostearate, and nonionic surfactants, (8) absorbents, such as kaolin and bentonite clay, (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof, (10) coloring agents, and (11) release control agents, such as crospovidone or ethyl cellulose, and can be mixed with any of them. In the case of capsules, tablets, and pills, the pharmaceutical composition may also contain a buffering agent. Similar types of solid pharmaceutical compositions can also be used as fillers in soft and hard shell gelatin capsules using excipients such as lactose or lactulose, and high molecular weight polyethylene glycol.

[0116] Tablets can be made, optionally, by compression or molding with one or more accessory components. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant or a dispersant. Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent using a suitable machine.

[0117] Tablets and other solid dosage forms of the pharmaceutical compositions of the present disclosure, such as lozenges, capsules, pills, and granules, can be obtained or prepared, optionally, using coatings and shells, such as enteric coatings, and other coatings well-known in pharmaceutical formulation technology. They can also be formulated to provide a desired release profile, other polymer matrices, liposomes and / or microspheres, for example, using hydroxypropylmethylcellulose in various ratios to provide sustained or controlled release of the active ingredient(s) therein. They can be formulated, for example, for rapid release, such as by lyophilization. They can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid pharmaceutical composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These pharmaceutical compositions can also optionally contain an opacifying agent and can be compositions that release the active ingredient(s), optionally in a delayed manner, only in or preferentially in certain parts of the digestive tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also, where appropriate, be in the form of microcapsules having one or more of the excipients described above.

[0118] Liquid dosage forms for oral administration of the compounds of the present disclosure include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol of sorbitan and fatty acid esters, and mixtures thereof.

[0119] In addition to the inert diluent, the oral pharmaceutical composition can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives.

[0120] In addition to the active compound, the suspension may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum hydroxide metas, bentonite, agar, and tragacanth, and mixtures thereof.

[0121] Preparations for rectal or vaginal administration may be presented as suppositories, which can be prepared by mixing one or more compounds of the present disclosure with one or more suitable non - irritating excipients or carriers, such as, for example, cocoa butter, polyethylene glycol, suppository wax, or salicylates, which are solid at room temperature but liquid at body temperature and thus dissolve in the rectal or vaginal cavity and release the active compound.

[0122] Formulations or dosage forms for the topical or transdermal administration of the oligomers provided herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active antisense oligonucleotides can be mixed, under sterile conditions, with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required. Ointments, pastes, creams, and gels can contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof, in addition to the active compounds of the present disclosure.

[0123] Powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances, in addition to the oligomers of the present disclosure. Sprays can further contain conventional propellants such as chlorofluorocarbons, as well as volatile unsubstituted hydrocarbons such as butane and propane.

[0124] Transdermal patches have the additional advantage of providing controlled delivery of the oligomers of the present disclosure into the body. Such dosage forms can be made by dissolving or dispersing the oligomers in a suitable medium. Penetration enhancers can also be used to increase the flow of the drug across the skin. The rate of such flow can be controlled by, among other methods known in the art, either providing a rate-controlling membrane or dispersing the drug in a polymer matrix or gel.

[0125] Pharmaceutical compositions suitable for parenteral administration may comprise one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, in combination with one or more antisense oligonucleotides of the present disclosure, which may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0126] These pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms on the antisense oligonucleotides of the subject matter can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenolsorbic acid, etc. It may also be desirable to include in the composition isotonic agents such as sugars, sodium chloride, etc. Furthermore, sustained absorption of injectable dosage forms can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0127] In some cases, it is desirable to delay the absorption of the drug from subcutaneous or intramuscular injection in order to prolong the effect of the drug. This can be achieved, among other methods known in the art, by the use of liquid suspensions of crystalline or amorphous materials with poor water solubility. The absorption rate of the drug then depends on its dissolution rate, which in turn can depend on the crystal size and crystal form. Alternatively, the delay in absorption of parenterally administered drug forms is achieved by dissolving or suspending the drug in an oily vehicle.

[0128] The depot form for injection can be prepared by forming a microcapsule matrix of the subject antisense oligonucleotide in a biodegradable polymer such as polylactide - polyglycolide. Depending on the ratio of the oligomer to the polymer and the nature of the specific polymer used, the rate of oligomer release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injection formulations can also be prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

[0129] When the antisense oligonucleotides of the present disclosure are administered as pharmaceuticals to humans and animals, they can be administered as a pharmaceutical composition containing 0.1 - 99% (more preferably 10 - 30%) of the active ingredient, either alone or in combination with a pharmaceutically acceptable carrier, for example.

[0130] As described above, the formulations or preparations of the present disclosure can be administered orally, parenterally, topically, or rectally. They are typically administered in a form suitable for each route of administration. For example, they can be administered in the form of tablets or capsules, by injection, inhalation, eye drops, ointments, suppositories, etc., by administration by injection, drip, or inhalation, by topical administration with lotions or ointments, and by rectal administration with suppositories.

[0131] Regardless of the route of administration selected, the antisense oligonucleotides of the present disclosure, and / or the pharmaceutical compositions of the present disclosure, which can be used in a suitable hydrated form, can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. The actual dosage level of the active ingredient in the pharmaceutical compositions of the present disclosure can vary to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, without being so toxic as to be unacceptable to the patient.

[0132] The selected dosage level depends on a variety of factors, including the activity of the particular oligomer of the present disclosure, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion or metabolism of the particular oligomer used, the rate and extent of absorption, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular oligomer being treated, the age, sex, weight, condition, general health, and medical history of the patient being treated, as well as similar factors well known in the medical arts.

[0133] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician or veterinarian can initiate the dosage of the compound of the present disclosure used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, a suitable daily dosage of the compound of the present disclosure is the amount of the compound that is the lowest dosage effective to produce a therapeutic effect. Such effective amounts generally depend on the factors described above. Generally, the oral, intravenous, intraventricular, and subcutaneous dosages of the compound of the present disclosure for a patient are in the range of about 0.0001 to about 100 mg per kilogram of body weight per day when used for the indicated effects.

[0134] In some embodiments, the antisense oligonucleotides of the present disclosure are generally administered at a dose of about 4 to 100 mg / kg, about 10 to 100 mg / kg, or 20 to 100 mg / kg. In some embodiments, for example, the parenteral dose such as intravenous administration is about 0.5 mg to 100 mg / kg. In some embodiments, the antisense oligonucleotides are administered at a dose of about 4 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 14 mg / kg, 15 mg / kg, 17 mg / kg, 20 mg / kg, 21 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, 50 mg / kg, 51 mg / kg, 52 mg / kg, 53 mg / kg, 54 mg / kg, 55 mg / kg, 56 mg / kg, 57 mg / kg, 58 mg / kg, 59 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, or 100 mg / kg, including all integers therebetween. In some embodiments, the oligomer is administered at about 4 mg / kg. In some embodiments, the oligomer is administered at about 10 mg / kg. In some embodiments, the oligomer is administered at about 20 mg / kg. In some embodiments, the oligomer is administered at about 30 mg / kg. In some embodiments, the oligomer is administered at about 40 mg / kg. In some embodiments, the oligomer is administered at about 50 mg / kg.

[0135] Optionally, the effective daily dose of the active compound may be administered as two, three, four, five, six or more partial doses, which are optionally administered separately in unit dosage forms at appropriate intervals throughout the day. In certain situations, the dosage is once-daily administration. In certain embodiments, the dosage is one or more administrations once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or once every one or two weeks, as necessary, to maintain the desired expression of the functional dystrophin protein. In certain embodiments, the administration is once-weekly administration. In certain embodiments, the dosage is one or more administrations once every two weeks. In some embodiments, the dosage is one administration once every two weeks.

[0136] In various embodiments, the antisense oligonucleotide is administered weekly at about 4 mg / kg. In various embodiments, the antisense oligonucleotide is administered weekly at about 10 mg / kg. In various embodiments, the antisense oligonucleotide is administered weekly at about 20 mg / kg. In various embodiments, the antisense oligonucleotide is administered weekly at about 30 mg / kg. In some embodiments, the antisense oligonucleotide is administered weekly at about 40 mg / kg. In some embodiments, the antisense oligonucleotide is administered weekly at about 50 mg / kg. In some embodiments, the antisense oligonucleotide is administered weekly at about 60 mg / kg. In some embodiments, the antisense oligonucleotide is administered weekly at 80 mg / kg. As used herein, weekly is understood to have its generally recognized meaning in the art as once every week.

[0137] In various embodiments, the antisense oligonucleotide is administered biweekly at about 4 mg / kg. In various embodiments, the antisense oligonucleotide is administered biweekly at about 10 mg / kg. In various embodiments, the antisense oligonucleotide is administered biweekly at about 20 mg / kg. In various embodiments, the antisense oligonucleotide is administered biweekly at about 30 mg / kg. In some embodiments, the antisense oligonucleotide is administered biweekly at about 40 mg / kg. In some embodiments, the antisense oligonucleotide is administered biweekly at about 50 mg / kg. In some embodiments, the antisense oligonucleotide is administered biweekly at about 60 mg / kg. In some embodiments, the antisense oligonucleotide is administered biweekly at about 80 mg / kg. As used herein, biweekly is understood to have the meaning generally recognized in the art as every two weeks.

[0138] Nucleic acid molecules are administered to cells by a variety of methods known to those of skill in the art, such methods including, but not limited to, encapsulation in liposomes, electroporation, or incorporation into other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, as described herein and known in the art. In certain embodiments, microemulsion techniques can be utilized to improve the bioavailability of lipophilic (water-insoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo, S.K., et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991, and REV 5901 (Sheen, P.C., et al., J Pharm Sci 80(7), 712-714, 1991). Among the advantages, microemulsion preferentially induces absorption into the lymphatic system rather than the circulatory system, thereby bypassing the liver and preventing destruction of the compound in the enterohepatic circulation, providing enhanced bioavailability.

[0139] In one aspect of the disclosure, the formulation contains micelles formed from the oligomers provided herein and at least one amphiphilic carrier having an average diameter of less than about 100 nm. More preferred embodiments provide micelles having an average diameter of less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter of less than about 30 nm or even less than about 20 nm.

[0140] All suitable amphiphilic carriers are contemplated, but currently preferred carriers generally have a generally recognized as safe (GRAS) status and are capable of both solubilizing the compounds of the present disclosure and microemulsifying at stages after the solution contacts a complex aqueous phase (such as those found in the human gastrointestinal tract). Amphiphilic components that meet these requirements typically have an HLB (hydrophilic-lipophilic balance) value of 2-20, and their structures contain linear aliphatic radicals in the range of C-6 to C-20. Examples include polyethylene glycolated fatty acid glycerides and polyethylene glycol.

[0141] Examples of amphiphilic carriers include saturated and mono-unsaturated polyethylene glycolated fatty acid glycerides, such as those obtained from various vegetable oils that are fully or partially hydrogenated. Such oils can advantageously consist of tri-, di-, and mono-fatty acid glycerides, as well as di- and mono-polyethylene glycol esters of the corresponding fatty acids. Particularly preferred fatty acid compositions include 4-10 capric acid, 3-9 capric acid, 40-50 lauric acid, 14-24 myristic acid, 4-14 palmitic acid, and 5-15 stearic acid. Another useful class of amphiphilic carriers includes partially esterified sorbitan and / or sorbitol having saturated or mono-unsaturated fatty acids (SPAN® series) or corresponding ethoxylated analogs (TWEEN® series).

[0142] Commercially available amphiphilic carriers, including Gelucire series, Labrafil, Labrasol, or Lauroglycol (all manufactured and distributed by Gattefosse Corporation, Saint Priest, France), PEG-mono-oleic acid, PEG-di-oleic acid, PEG-mono-lauric acid and di-lauric acid, lecithin, polysorbate 80, etc. (manufactured and distributed by many companies in the United States and around the world), may be particularly useful.

[0143] In certain embodiments, delivery can occur by using liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. to introduce the pharmaceutical composition of the present disclosure into a suitable host cell. In particular, the pharmaceutical composition of the present disclosure can be encapsulated in any of lipid particles, liposomes, vesicles, nanospheres, nanoparticles, or the like and formulated for delivery. The formulation and use of such delivery vehicles can be carried out using known and conventional techniques.

[0144] Hydrophilic polymers suitable for use in the present disclosure are readily water-soluble, can covalently bind to vesicle-forming lipids, and are tolerated in vivo without toxic effects (i.e., are biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic acid (also called polylactide), polyglycolic acid (also called polyglycolide), polylactic acid-polyglycolic acid copolymers, and polyvinyl alcohol. In certain embodiments, the polymer has a molecular weight of about 100 or 120 Daltons to a maximum of about 5,000 or 10,000 Daltons, or about 300 Daltons to about 5,000 Daltons. In other embodiments, the polymer is polyethylene glycol having a molecular weight of about 100 to about 5,000 Daltons, or about 300 to about 5,000 Daltons. In certain embodiments, the polymer is polyethylene glycol of 750 Daltons (PEG(750)). The polymer can also be defined by the number of monomers therein, and preferred embodiments of the present disclosure utilize polymers of at least about three monomers, such PEG polymers consisting of three monomers (about 150 Daltons).

[0145] Other hydrophilic polymers that may be suitable for use in the present disclosure include polyvinyl pyrrolidone, poly(meth)oxazolines, poly(ethyl)oxazolines, poly(hydroxypropyl)methacrylamide, poly(meth)acrylamide, poly(dimethyl)acrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0146] In certain embodiments, the formulations of the present disclosure comprise a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and their copolymers, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acids, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylate, and blends, mixtures, or copolymers thereof.

[0147] Cyclodextrins are cyclic oligosaccharides consisting of 6, 7, or 8 glucose units, and are denoted by the Greek letters α, β, or γ, respectively. The glucose units are linked by α-1,4-glucoside bonds. As a result of the chair conformation of the sugar units, all secondary hydroxyl groups (at C-2, C-3) are located on one side of the ring, while all primary hydroxyl groups at C-6 are located on the other side. As a result, the outer surface is hydrophilic, making cyclodextrin water-soluble. In contrast, the cavities of cyclodextrins are hydrophobic because they are covered by the hydrogens of atoms C-3 and C-5, as well as by ether-like oxygens. These matrices allow for complex formation with various relatively hydrophobic compounds, including steroid compounds such as 17α-estradiol (see, for example, van Uden et al. Plant Cell Tiss. Org. Cult. 38:1-3-113 (1994)). Complex formation occurs by van der Waals interactions and hydrogen bond formation. For a general review of the chemical properties of cyclodextrins, see Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822 (1994).

[0148] The physicochemical properties of cyclodextrin derivatives greatly depend on the type and degree of substitution. For example, their water solubility ranges from insoluble (e.g., triacetyl-beta-cyclodextrin) to 147% soluble (w / v) (G-2-beta-cyclodextrin). Furthermore, they are soluble in many organic solvents. The properties of cyclodextrins enable the control of the solubility of various formulation components by increasing or decreasing their solubility.

[0149] Numerous cyclodextrins and methods for their preparation have been described. For example, Parmeter (I) et al. (U.S. Patent No. 3,453,259) and Gramera et al. (U.S. Patent No. 3,459,731) described electrically neutral cyclodextrins. Other derivatives include cyclodextrins having cationic properties [Parmeter (II), U.S. Patent No. 3,453,257], insoluble cross-linked cyclodextrins (Solms, U.S. Patent No. 3,420,788), and cyclodextrins having anionic properties [Parmeter (III), U.S. Patent No. 3,426,011]. Among the cyclodextrin derivatives having anionic properties, carboxylic acids, phosphorous acids, phosphinic acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfinic acids, and sulfonic acids have been added to the parent cyclodextrin [Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrin derivatives have been described by Stella et al. (U.S. Patent No. 5,134,127).

[0150] Liposomes consist of at least one lipid bilayer membrane surrounding an aqueous internal compartment. Liposomes can be characterized by the type and size of the membrane. Small unilamellar vesicles (SUVs) have a single membrane and typically range in diameter from 0.02 to 0.05 μm, while large unilamellar vesicles (LUVs) are typically larger than 0.05 μm. Large oligolamellar vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 μm. Liposomes with some non-concentric membranes, i.e., some smaller vesicles contained within larger vesicles, are called polyplex vesicles.

[0151] One aspect of the present disclosure relates to a formulation comprising liposomes containing the antisense oligonucleotides of the present disclosure, wherein the liposome membrane is formulated to provide liposomes with increased loading capacity. Alternatively, or in addition, the compounds of the present disclosure can be contained within or adsorbed onto the liposome bilayer of the liposome. The antisense oligonucleotides of the present disclosure can be aggregated with a lipid surfactant and carried within the internal space of the liposome. In these cases, the liposome membrane is formulated to resist the disintegrating effect of the active agent-surfactant aggregate.

[0152] According to one embodiment of the present disclosure, the lipid bilayer of the liposome contains a lipid derivatized with polyethylene glycol (PEG), whereby the PEG chains extend from the inner surface of the lipid bilayer into the internal space encapsulated by the liposome and from the outside of the lipid bilayer into the surrounding environment.

[0153] The active agent contained within the liposomes of the present disclosure is in solubilized form. Aggregates of surfactant and active agent (such as emulsions or micelles containing the active agent of interest) can be confined within the internal space of the liposomes according to the present disclosure. The surfactant acts to disperse and solubilize the active agent and can be selected from any suitable aliphatic, alicyclic, or aromatic surfactant, including but not limited to biocompatible lysophosphatidylcholine (LPG) of various chain lengths (e.g., from about C14 to about C20). Polymer-derivatized lipids such as PEG-lipids can also be utilized in micelle formation because they act to inhibit micelle / membrane fusion and because the addition of the polymer to the surfactant molecule decreases the critical micelle concentration ("CMC") of the surfactant and aids in micelle formation. Surfactants having a CMC in the micromolar range are preferred, and higher CMC surfactants can be utilized to prepare micelles confined within the liposomes of the present disclosure.

[0154] The liposomes according to the present disclosure can be prepared by any of a variety of techniques known in the art. See, for example, U.S. Patent No. 4,235,871, Published PCT Application No. WO96 / 14057, New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104; Lasic DD, Liposomes from physics to applications, Elsevier Science Publishers BV, Amsterdam, 1993. For example, the liposomes of the present disclosure can be prepared by diffusing a lipid derivatized with a hydrophilic polymer into pre-formed liposomes at a lipid concentration corresponding to the final molar percentage of the desired derivatized lipid in the liposomes, e.g., by exposing pre-formed liposomes to micelles composed of lipid-grafted polymers. Liposomes containing hydrophilic polymers can also be formed by homogenization, lipid field hydration, or extrusion techniques, as known in the art.

[0155] In another exemplary formulation procedure, the active agent is first dispersed by sonication in lysophosphatidylcholine or other low CMC surfactants (including polymeric-grafted lipids) that readily solubilize hydrophobic molecules. The resulting micellar suspension of the active agent is then used to rehydrate a dry lipid sample containing a suitable molar percent of polymeric-grafted lipid or cholesterol. The lipid and active agent suspension is then formed into liposomes using extrusion techniques known in the art, and the resulting liposomes are separated from the unencapsulated solution by standard column separation.

[0156] In one aspect of the disclosure, liposomes are prepared to have a substantially uniform size within a selected size range. One effective sizing method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes having a selected uniform pore size, where the pore size of the membrane generally corresponds to the maximum size of the liposomes effected by extrusion through that membrane. See, e.g., U.S. Patent No. 4,737,323 (Apr. 12, 1988). In certain embodiments, reagents such as DharmaFECT® and Lipofectamine® can be utilized to introduce polynucleotides or proteins into cells.

[0157] The release characteristics of the formulations of the present disclosure depend on the encapsulating material, the concentration of the encapsulated drug, and the presence of a release regulator. For example, the release can be made pH-dependent using a pH-sensitive coating that releases only at a low pH, such as in the stomach, or a high pH, such as in the intestine. An enteric coating can be used so that release does not occur until after passing through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach followed by a subsequent release in the intestine. The release can also be manipulated by including salts or pore-forming agents, which can increase water uptake or drug release by diffusion from the capsule. Excipients that modify the solubility of the drug can also be used to control the release rate. Agents that enhance the degradation of the matrix or release from the matrix can also be incorporated. They can be added to the drug, added as a separate phase (i.e., as microparticles), or co-dissolved in the polymer phase depending on the compound. In most cases, the amount should be from 0.1 to 30 percent (w / w polymer). Types of degradation accelerators include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine, and surfactants such as Tween® and Pluronic®. Pore-forming agents (i.e., water-soluble compounds such as inorganic salts and saccharides) that add a microstructure to the matrix are added as microparticles. The range is typically from 1 to 30 percent (w / w polymer).

[0158] Moreover, uptake can also be manipulated by changing the residence time of the particles in the intestine. This can be achieved, for example, by coating the particles with a mucoadhesive polymer or by selecting it as an encapsulating material. Examples include most polymers having free carboxyl groups, such as chitosan, cellulose, and especially polyacrylates (as used herein, polyacrylates refer to polymers containing acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates).

[0159] Antisense oligonucleotides can be formulated to be contained within or adapted to be released by a surgical or medical device or implant. In certain embodiments, the implant can be coated with or otherwise treated with an antisense oligonucleotide. For example, hydrogels, or other polymers such as biocompatible and / or biodegradable polymers, can be used to coat the implant with the pharmaceutical composition of the present disclosure (i.e., the composition can be adapted to be used with a medical device by using a hydrogel or other polymer). Polymers and copolymers for coating medical devices with drugs are well known in the art. Examples of implants include, but are not limited to, stents, drug-eluting stents, sutures, artificial organs, vascular catheters, dialysis catheters, vascular grafts, artificial heart valves, cardiac pacemakers, implantable defibrillators, IV needles, devices for bone fixation and bone formation, such as pins, screws, plates, and other devices, as well as artificial tissue matrices for wound healing.

[0160] In addition to the methods provided herein, antisense oligonucleotides for use according to the present disclosure can be formulated for administration by any convenient method for use in human or veterinary medicine, by analogy with other pharmaceuticals. Antisense antisense oligonucleotides and their corresponding formulations can be administered alone or in combination with other therapeutic strategies in the treatment of muscular dystrophy, such as myoblast transfer, stem cell therapy, administration of aminoglycoside antibiotics, proteasome inhibitors, and upregulation therapies (e.g., upregulation of utrophin, an autosomal paralog of dystrophin).

[0161] In some embodiments, the additional therapeutic agent can be administered before, simultaneously with, or after administration of the antisense oligonucleotides of the present disclosure. For example, the antisense oligonucleotides can be administered in combination with steroids and / or antibiotics. In certain embodiments, the antisense oligonucleotides are administered to patients receiving background steroid theory (e.g., intermittent or long-term / continuous background steroid therapy). For example, in some embodiments, the patient has been treated with corticosteroids prior to administration of the antisense oligomer and continues to receive steroid therapy. In some embodiments, the steroid is a glucocorticoid or prednisone.

[0162] The described routes of administration are intended only as a guide, since one of ordinary skill in the art can readily determine the optimal route of administration and any dosage for any particular animal and condition. Multiple approaches have been attempted to introduce functional new genetic material into cells both in vitro and in vivo (Friedmann (1989) Science, 244:1275-1280). These approaches include incorporation into modified retroviruses that express the gene (Friedmann (1989), supra, Rosenberg (1991) Cancer Research 51(18), suppl.:5074S-5079S), incorporation into non-retroviral vectors (e.g., adeno-associated virus vectors) (Rosenfeld, et al. (1992) Cell, 68:143-155, Rosenfeld, et al. (1991) Science, 252:431-434), or delivery of the transgene linked to a heterologous promoter-enhancer element via liposomes (Friedmann (1989), supra, Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281, Nabel, et al. (1990) Science, 249:1285-1288, Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209, and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855), binding to a ligand-specific, cationic transport system (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624), or use of naked DNA, expression vectors (Nabel et al. (1990), supra), Wolff et al. (1990) Science, 247:1465-1468). Direct injection of the transgene into tissue results in only local expression (Rosenfeld (1992), supra, Rosenfeld et al. (1991), supra, Brigham et al. (1989), supra, Nabel (1990), supra, and Hazinski et al. (1991), supra).The group of Brigham et al. (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) reported only in vivo transfection of the lungs of mice after either intravenous or intratracheal administration of DNA-liposome complexes. An example of a review article on human gene therapy procedures is Anderson, Science (1992) 256:808-813.

[0163] In further embodiments, the pharmaceutical compositions of the present disclosure may additionally include carbohydrates as provided in Han et al., Nat. Comms. 7, 10981 (2016) (which is hereby incorporated by reference in its entirety). In some embodiments, the pharmaceutical compositions of the present disclosure may include 5% hexose carbohydrates. For example, the pharmaceutical compositions of the present disclosure may include 5% glucose, 5% fructose, or 5% mannose. In certain embodiments, the pharmaceutical compositions of the present disclosure may include 2.5% glucose and 2.5% fructose. In some embodiments, the pharmaceutical compositions of the present disclosure may include carbohydrates selected from arabinose present in an amount of 5% by volume, glucose present in an amount of 5% by volume, sorbitol present in an amount of 5% by volume, galactose present in an amount of 5% by volume, fructose present in an amount of 5% by volume, xylitol present in an amount of 5% by volume, mannose present in an amount of 5% by volume, a combination of glucose and fructose each present in an amount of 2.5% by volume, and a combination of glucose present in an amount of 5.7% by volume, fructose present in an amount of 2.86% by volume, and xylitol present in an amount of 1.4% by volume.

[0164] IV. Kit The present disclosure also provides a kit for the treatment of patients with genetic diseases (such as DMD), the kit comprising at least an antisense molecule (such as casimersen) packaged in a suitable container, together with instructions for its use. The kit may also contain peripheral reagents such as buffers, stabilizers, etc. Those skilled in the art should understand that the use of the above method has a wide range of uses for identifying antisense molecules suitable for use in the treatment of many other diseases.

Example

[0165] Example 1: Essence ClinicalTrials.gov ID: NCT02500381 The primary objective of this study was to evaluate the efficacy of casimersen (SRP-4045) and golodirsen (SRP-4053) compared to placebo in patients with Duchenne muscular dystrophy (DMD) having out-of-frame deletion mutations suitable for skipping of exon 45 and exon 53, respectively.

Table 1

[0166] Materials and Methods Casimersen (also known as SRP-4045) is a PMO of the chemical structure described herein and was supplied by Sarepta Therapeutics, Inc. The casimersen formulation was formulated as a sterile, isotonic, phosphate-buffered aqueous solution supplied in single-use vials at a concentration of 50 mg / mL. The formulation was diluted with physiological saline (0.9% sodium chloride injection solution) prior to administration via IV infusion at the clinical site.

[0167] Patients: Eligibility Eligible patients were 7 to 13 years old and had an out-of-frame deletion of the DMD gene suitable for skipping of exon 45. Selection criteria: · Genotypically confirmed and diagnosed with DMD. · Stable oral corticosteroid dosage for at least 24 weeks. · Intact left and right biceps or two alternative upper muscle groups. · Average 6MWT is 300 meters or more and 450 meters or less. · Stable lung and heart function: Predicted forced vital capacity (FVC) of 50% or more, left ventricular ejection fraction (LVEF) of more than 50%. Exclusion criteria: · Past treatment with SMT C1100 (BMN - 195) at any time point · Treatment with gene therapy at any time point · Past treatment with PRO045 or PRO053 within the first 24 weeks of week 1 · Current or past treatment with any other experimental treatment (other than deflazacort) within the first 12 weeks of week 1 · Participation in any other DMD - intervention clinical trial within the first 12 weeks of week 1 · Major surgery within the first 3 months of week 1 · Presence of other clinically significant diseases · Substantial change in physical therapy regimen within the first 3 months of week 1.

[0168] Trial design Patients suitable for exon 45 skipping received casimersen (SRP - 4045) by intravenous (IV) infusion at 30 mg / kg for up to 96 weeks during the double - blind period. Subsequently, during the open - label continuation period, all patients received open - label active treatment with casimersen (SRP - 4045) by IV infusion at 30 mg / kg / week for 48 weeks (up to week 144 of the maximum trial). Primary endpoint: Change from baseline in total walking distance during the 6 - Minute Walk Test (6MWT) at week 96 [Timeframe: Baseline and week 96]. Secondary evaluation items: · Change from baseline in total walking distance during the 6-minute walk test (6MWT) at the 144th week (the 48th week of the non-blind period) [Time frame: Baseline, 144th week]. · Change from baseline in dystrophin protein levels as determined by Western blot at the 48th or 96th week [Time frame: Baseline and 48th or 96th week]. · Change from baseline in dystrophin intensity levels as determined by immunohistochemistry (IHC) at the 48th or 96th week [Time frame: Baseline and 48th or 96th week]. · Ability to stand up independently from the floor [Time frame: 96th week, 144th week]. · Time to loss of ambulation (LOA) [Time frame: Baseline, 96th week, 144th week]. · Change from baseline in the North Star Ambulatory Assessment (NSAA) total score at the 96th and 144th weeks [Time frame: Baseline, 96th week, 144th week]. The NSAA is a functional scale developed from the Hammersmith Scale of Motor Ability, specifically for use in children with Duchenne muscular dystrophy (DMD) who are ambulant. It consists of 17 activities rated 0 (not possible), 1 (perform with modification), 2 (normal movement). The scale assesses activities necessary to maintain a functionally ambulatory state (e.g., rising from the floor), activities that can be difficult even early in the disease (e.g., standing on tiptoe), and activities known to deteriorate progressively over time (rising from a chair, walking). The NSAA total score ranges from 0 to 34 points, with 34 points indicating normal function. · Change from baseline in predicted forced vital capacity percent (FVC%) at the 96th and 144th weeks [Time frame: Baseline, 96th week, 144th week].

[0169] Interim analysis Patients suitable for exon 45 skipping were randomized to receive 30 mg / kg of casimersen (N = 27) or placebo (N = 16) by intravenous (IV) infusion once a week for 96 weeks. An interim analysis was performed on data from baseline and biceps biopsies at week 48 during treatment. The main findings from the interim analysis included the following. · The mean dystrophin protein (% of normal dystrophin measured by Western blot) increased to 1.736% of normal, compared with a mean baseline of 0.925% of normal (p < 0.001). · A statistically significant difference in the mean change in dystrophin protein from baseline to week 48 was observed between the casimersen-treated group compared with the placebo group (p = 0.009 (sensitivity analysis), p = 0.004 (main method analysis)). · In an analysis at baseline using reverse transcription polymerase chain reaction (RT-PCR) to test for an increase in exon-skipping mRNA, all 22 patients treated with casimersen showed an increase in exon 45 skipping (p < 0.001) above baseline levels, indicating a 100% response rate. · A statistically significant positive correlation was observed between exon 45 skipping and dystrophin production at baseline analysis (Spearman's rank correlation = 0.635, p < 0.001). · In an analysis of 27 patients treated with casimersen, tested for an increase in exon-skipping mRNA using reverse transcription polymerase chain reaction (RT-PCR), all patients showed an increase in exon 45 skipping (p < 0.001) above baseline levels, indicating a 100% response rate. · A statistically significant positive correlation was observed between exon 45 skipping and dystrophin production in the analysis of all 27 patients (Spearman's rank correlation = 0.627, p < 0.001). This trial is ongoing and remains blinded to collect additional efficacy and safety data.

[0170] Example 2: The primary objective of this trial is to evaluate the safety and tolerability of casimersen and to assess the pharmacokinetics (PK) of casimersen in ambulatory DMD patients with a confirmed mutation suitable for exon 45 skipping. Methods

[0171] Ambulatory DMD patients with a confirmed mutation suitable for exon 45 skipping were enrolled in a multi - center, randomized, double - blind, placebo - controlled, dose - escalation, Phase 1 / 2 trial.

[0172] During the double - blind dose - escalation period, patients were randomized (2:1) to receive casimersen or placebo for approximately 12 weeks. Patients randomized to casimersen were administered escalating doses in four steps (4, 10, 20, and 30 mg / kg) once weekly by intravenous (IV) infusion for at least 2 weeks per dose level. After the double - blind dose - escalation period, the safety and efficacy of weekly casimersen 30 mg / kg were evaluated in an open - label continuation period of up to 132 weeks.

[0173] Patients: Eligibility Eligible patients were male, 7 - 21 years of age, with a clinical diagnosis of DMD, having a confirmed gene mutation suitable for exon 45 skipping, stable cardiac and pulmonary function, on a stable dose of oral corticosteroids for at least 24 weeks prior to study start or not receiving oral corticosteroids, and unable to walk or having a 6 - minute walk test distance of less than 300 meters.

[0174] Trial Assessments Safety assessments included treatment - emergent adverse events (TEAEs), abnormal clinical laboratory values, abnormalities in vital signs and physical examinations, and clinically significant worsening on electrocardiogram and echocardiogram.

[0175] For PK evaluation, area under the concentration-time curve (AUC), total body clearance (CL), maximum plasma concentration (C max ), terminal half-life (t 1 / 2 ), time to maximum plasma concentration (t max ), and volume of distribution at steady state (V ss ) were included.

[0176] Data were evaluated and presented using summary and descriptive statistics.

[0177] PK parameters of casimersen were calculated using non-compartmental analysis of visits when PK measurements were collected continuously.

[0178] Results Of the 12 registered patients, 11 (91.7%) completed the trial. Patients randomized to casimersen treatment were generally slightly older and had more advanced disease than those randomized to placebo (Table 1).

Table 2

[0179] The mean (SD) total duration of the trial was 144.7 (3.45) weeks. The mean (SD) duration of casimersen treatment during the combined trial period was 139.6 (9.26) weeks.

[0180] Safety All patients experienced one or more treatment-emergent adverse events (TEAEs) that occurred under the treatments described in Table 2.

Table 3

[0181] No patients discontinued the investigational drug or had a dose reduction of the investigational drug due to TEAE. The severity of the majority of TEAEs was mild in both the double-blind treatment period (88.7%) and the open-label treatment period (90.9%). Treatment-emergent pain and pharyngitis were the most frequently reported TEAEs during the double-blind and open-label treatment periods, respectively (Tables 3 and 4). [Table 4] a Only TEAEs with onset dates from the first dose of the investigational drug through the first dose during the open-label period are included. [Table 5] a Only TEAEs with onset dates after the first dose of casimersen are included.

[0182] Treatment-related TEAEs were one case of moderate iron deficiency, one case of mild flushing in two patients treated with casimersen, and one case of mild contact dermatitis in one patient receiving placebo. TEAEs related to casimersen resolved during the study period. TEAEs related to placebo persisted at the end of the study.

[0183] During the combined treatment period, five serious TEAEs occurred in patients receiving casimersen 30 mg / kg (one patient had a tibial fracture, one patient had bacteremia, septic embolism, and a superior vena cava thrombosis, and one patient had a femoral fracture). All five events were considered not related to treatment, resolved during the study, and did not recur with further dosing.

[0184] No patterns, trends, or abnormalities were observed in hematological tests, coagulation abnormalities, chemistry, or other clinical laboratory parameters. Cardiac signals were not seen on conduction time or functional evaluation by echocardiogram. One case of transient ventricular tachycardia was reported, but the event was considered unrelated to casimersen treatment, and the electrocardiogram normalized without sequelae.

[0185] Pharmacokinetics For the 30 mg / kg dose level, at week 7 and week 60, the mean plasma casimersen concentration-time profiles were similar. All PK parameters were similar at week 7 and week 60 for casimersen at the 30 mg / kg dose level (Table 5). [Table 6] Values are presented as geometric mean (geometric coefficient of variation) for all parameters except time to peak plasma concentration and half-life in plasma, time to peak plasma concentration is presented as median (minimum - maximum), and half-life in plasma is presented as mean (standard deviation). a At week 3, for AUC ∞ , steady-state volume of distribution, clearance, and half-life in plasma, n = 7. AUC ∞ , area under the concentration-time curve extrapolated from 0 hours to infinite time.

[0186] Summary In this study, casimersen 30 mg / kg was well tolerated in patients with advanced DMD in whom mutations suitable for exon 45 skipping were identified. Most of the reported TEAEs were mild in severity, few treatment-related TEAEs were reported, and no patients discontinued the investigational drug or had a dose reduction of the investigational drug due to TEAEs. Clinically significant abnormal clinical laboratory values or worsening of electrocardiogram and echocardiogram were not observed. PK analysis of casimersen suggests little or no accumulation after once-weekly administration at 30 mg / kg. *********************

[0187] All publications and patent applications cited in this specification are hereby incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

[Claim 1] The invention described in this specification.

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