Compositions Comprising Exon Skipping Oligonucleotide Conjugates for Treating Muscular Dystrophies - Patent application

Abstract

Provided herein is a composition comprising: (a) an antisense oligonucleotide conjugate comprising a cell membrane-permeable peptide covalently attached to a nucleic acid analog, or a pharma- ceutically acceptable salt thereof, wherein the cell membrane-permeable peptide comprises at least two positively charged amino acids; (b) one or more surfactants; (c) one or more sugars; and (d) one or more buffering agents.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 129,752, filed December 23, 2020. The entire teachings of the above applications are incorporated by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on December 21, 2021, is named 8169_51_WO00_SL.txt, and is 16,618 bytes in size.

[0003] The present disclosure relates to compositions comprising antisense oligonucleotides, or pharma- ceutically acceptable salts thereof, suitable for exon skipping in the human dystrophin gene. [Background technology]

[0004] Duchenne muscular dystrophy (DMD) is caused by defective expression of protein dystrophin. The gene that codes for the protein contains 79 exons spread over more than 2 million nucleotides of DNA. Any mutation in an exon, which changes the reading frame of an exon, introduces a stop codon, or is characterized by the removal of an entire out-of-frame exon or the duplication of one or more exons, can prevent the production of functional dystrophin and result in DMD.

[0005] Mutations, typically deletions of one or more exons, have been shown to result in a less severe form of muscular dystrophy, Becker muscular dystrophy (BMD), if they result in the correct reading frame along the entire dystrophin transcript, and thus prevent premature termination of translation of the mRNA into protein. If the joining 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 codes for a protein with a short internal deletion that retains some activity, resulting in the Becker phenotype.

[0006] There is a need for pharmaceutical compositions comprising antisense oligonucleotide conjugates suitable for exon skipping that are useful in therapeutic methods for producing dystrophin and treating DMD. Summary of the Invention [Means for solving the problem]

[0007] A composition comprising: (a) an antisense oligonucleotide conjugate comprising a cell membrane-permeable peptide covalently attached to a nucleic acid analog, or a pharma- ceutically acceptable salt thereof, wherein the cell membrane-permeable peptide comprises at least two positively charged amino acids; (b) one or more surfactants; (c) one or more sugars; and (d) one or more buffering agents.

[0008] In some embodiments, the composition comprises about 50 mg to about 500 mg of the antisense oligonucleotide conjugate, or a pharma- ceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide conjugate in the composition is capable of binding to a selected target and inducing exon skipping in the human dystrophin gene.

[0009] In some embodiments, the antisense oligonucleotide conjugate in the composition comprises a cell membrane penetrating peptide that is an arginine-rich peptide. In some embodiments, the antisense oligonucleotide conjugate in the composition comprises a -(RXR)4-R a (SEQ ID NO:54), R-(FFR)3-R a (SEQ ID NO:55), -BX-(RXR)4-R a (SEQ ID NO:56), -BXR-(FFR)3-R a (SEQ ID NO:57), -GLY-R-(FFR)3-R a (SEQ ID NO:58), -GLY-R5-R a (SEQ ID NO:59), -R5-R a , (SEQ ID NO: 60), -GLY-R6-R a( SEQ ID NO: 52), and -R6-R a (SEQ ID NO: 53), a is selected from H, acyl, benzoyl, and stearoyl, R is arginine, X is 6-aminohexanoic acid, B is β-alanine, F is phenylalanine, and GLY (or G) is glycine. In some embodiments, the arginine-rich peptide is -GLY-R5-R a (SEQ ID NO:59), -R5-R a (SEQ ID NO: 60), -GLY-R6-R a (SEQ ID NO:52), or -R6-R a (SEQ ID NO:53), R is arginine, and R a is hydrogen or an acyl group.

[0010] In some embodiments, the antisense oligonucleotide conjugate in the composition comprises a sequence complementary to 15-35 nucleobases of a target region of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of the dystrophin pre-mRNA. [ka] or a pharma- ceutically acceptable salt thereof in a composition, wherein each Nu is a nucleobase that together form a targeting sequence, T, [ka] and [ka] is a moiety selected from R 100 is a cell membrane penetrating peptide, Each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 1-1] [Table 1-2] A is [ka] and C is [ka] and G is [ka] and T is [ka] and U is [ka] where Gm is a methylated guanine, Am is a methylated adenine, and mC is [ka] It is.

[0011] In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (I) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, or SEQ ID NO: 51. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (I) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 1. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (I) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 7. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (I) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 17. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (I) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 51.

[0012] In some embodiments, T' of the antisense oligonucleotide conjugate of Formula (I) in the composition is [ka] It is.

[0013] In some embodiments, the antisense oligonucleotide conjugate in the composition has Formula (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu in the range 1 to (n+1) corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 2-1] [Table 2-2] A is [ka] and C is [ka] and G is [ka] and T is [ka] and U is [ka] where Gm is a methylated guanine, Am is a methylated adenine, and mC is [ka] It is.

[0014] In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, or SEQ ID NO: 51. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (II) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 1. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (II) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 7. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (II) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 17. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (II) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 51.

[0015] In some embodiments, the antisense oligonucleotide conjugate in the composition has Formula (III): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu in the range 1 to (n+1) corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 3-1] [Table 3-2] A is [ka] and C is [ka] and G is [ka] and T is [ka] and U is [ka] where Gm is a methylated guanine, Am is a methylated adenine, and mC is [ka] It is.

[0016] In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, or SEQ ID NO: 51. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (III) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 1. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (III) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 7. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (III) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 17. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide of formula (III) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 51.

[0017] In some embodiments, the antisense oligonucleotide conjugate in the composition is PPMO#1, PPMO#2, PPMO#3, or PPMO#4, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide conjugate in the composition is PPMO#1, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide conjugate in the composition is PPMO#2, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide conjugate in the composition is PPMO#3, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide conjugate in the composition is PPMO#4, or a pharmaceutically acceptable salt thereof.

[0018] In some embodiments, the antisense oligonucleotide conjugate in the composition is in free base form.In some embodiments, the antisense oligonucleotide conjugate in the composition is a pharma- ceutically acceptable salt.In some embodiments, the antisense oligonucleotide conjugate in the composition is a halogen salt.In some embodiments, the antisense oligonucleotide conjugate is an HCl salt.In some embodiments, the HCl salt of the antisense oligonucleotide conjugate is 1HCl, 2HCl, 3HCl, 4HCl, 5HCl or 6HCl salt.

[0019] In some embodiments, the antisense oligonucleotide conjugate in the composition is the 6HCl salt form of PPMO#1, PPMO#2, PPMO#3, or PPMO#4. In some embodiments, the antisense oligonucleotide conjugate in the composition is the 6HCl salt form of PPMO#1. In some embodiments, the antisense oligonucleotide conjugate in the composition is the 6HCl salt form of PPMO#2. In some embodiments, the antisense oligonucleotide conjugate in the composition is the 6HCl salt form of PPMO#3. In some embodiments, the antisense oligonucleotide conjugate in the composition is the 6HCl salt form of PPMO#4.

[0020] In some embodiments, the one or more surfactants are present in the composition in an amount of about 0.001% to about 2% by weight of the composition. In some embodiments, the one or more surfactants are present in the composition in an amount of about 0.002% to about 1% by weight of the composition. In some embodiments, the one or more surfactants in the composition are selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80, polysorbate 20), polyvinylpyrrolidone (PVP, povidone, poloxamer, poloxamer F188), and mixtures thereof.

[0021] In some embodiments, the one or more sugars are present in an amount of about 0.1% to about 11% by weight of the composition. In some embodiments, the one or more sugars are present in an amount of about 6% by weight. In some embodiments, the one or more sugars are selected from the group consisting of trehalose, mannitol, sucrose, and mixtures thereof. In some embodiments, the sugar is a mixture of mannitol and sucrose. In some embodiments, the sugar is mannitol. In some embodiments, the sugar is sucrose.

[0022] In some embodiments, the one or more buffering agents are present in an amount sufficient to provide a pH of about 6.0 to about 7.0 when the composition is dissolved or dispersed in an aqueous medium. In some embodiments, the one or more buffering agents are present in an amount to provide a pH of about 6.3 to about 6.5. In some embodiments, the one or more buffering agents are present in a range of about 10 mM to about 50 mM when the composition is dissolved or dispersed in an aqueous medium. In some embodiments, the one or more buffering agents are selected from the group consisting of histidine, citrate, maleate, arginine, and mixtures thereof. In some embodiments, the buffering agent is citrate.

[0023] In some embodiments, the composition has an osmolality of about 250 mOsm / kg to about 650 mOsm / kg. In some embodiments, the composition has an osmolality of about 300 mOsm / kg to about 500 mOsm / kg. In some embodiments, the composition has an osmolality of about 400 mOsm / kg to about 600 mOsm / kg.

[0024] In some embodiments, the composition is in lyophilized form.In some embodiments, the composition dissolves within 30 minutes after adding aqueous solvent.In some embodiments, the composition dissolves within 15 minutes after adding aqueous solvent.In some embodiments, the composition dissolves within 3 minutes after adding aqueous solvent.

[0025] In some aspects, the aqueous solvent used to dissolve the composition is sterile water.

[0026] Also disclosed herein is a method of treating a human patient with Duchenne muscular dystrophy comprising dissolving or dispersing a composition disclosed herein in an aqueous solvent.

[0027] In some embodiments, the amount of aqueous solvent used to dissolve or disperse the composition is sufficient to provide a concentration of antisense oligonucleotide conjugate of about 20 mg / mL to about 200 mg / mL. In some embodiments, the amount of aqueous solvent used to dissolve or disperse the composition is sufficient to provide a concentration of antisense oligonucleotide conjugate of about 20 mg / mL to about 100 mg / mL. In some embodiments, the amount of aqueous solvent used to dissolve or disperse the composition is sufficient to provide a concentration of antisense oligonucleotide conjugate of about 50 mg / mL. In some embodiments, the amount of aqueous solvent used to dissolve or disperse the composition is sufficient to provide a concentration of antisense oligonucleotide conjugate of about 100 mg / mL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Aspects of the present disclosure generally relate to pharmaceutical compositions comprising antisense oligonucleotide conjugates specifically designed to induce exon skipping in the human dystrophin gene, and methods of use thereof. Dystrophin plays a key role in muscle function, and various muscle-related diseases are characterized by mutant forms of this gene, such as the mutant dystrophin gene found in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

[0029] Due to the abnormal mRNA splicing events caused by mutation, these mutant human dystrophin genes either express defective dystrophin protein or do not express any measurable dystrophin, which is a condition that causes various forms of muscular dystrophy.To treat this condition, the antisense oligonucleotide conjugate in the pharmaceutical composition of the present disclosure hybridizes to the selected region of the pre-processed mRNA of mutant human dystrophin gene, and induces exon skipping and differential splicing in otherwise abnormally spliced ​​dystrophin mRNA, thereby allowing muscle cells to produce the mRNA transcript that codes for functional dystrophin protein.In certain embodiments, the resulting dystrophin protein is not necessarily the "wild type" form of dystrophin, but rather a truncated but functional form of dystrophin.

[0030] By increasing the level of functional dystrophin protein in muscle cells, these and related embodiments are useful for the prevention and treatment of muscular dystrophies, particularly those forms of muscular dystrophies, such as DMD and BMD, that are characterized by defective expression of dystrophin protein due to aberrant mRNA splicing.The specific antisense oligonucleotide conjugates contained in the pharmaceutical compositions described herein further provide dystrophin-exon specific targeting over other oligonucleotides.

[0031] Accordingly, the present disclosure relates to a pharmaceutical composition comprising: (a) an antisense oligonucleotide conjugate comprising a cell membrane penetrating peptide covalently attached to a nucleic acid analog, or a pharma- ceutically acceptable salt thereof, wherein the cell membrane penetrating peptide ("CPP") comprises at least two positively charged amino acids; (b) one or more surfactants; (c) one or more sugars; and (d) one or more buffering agents.

[0032] In some embodiments, the CPP is an arginine-rich peptide. The term "arginine-rich" refers to a CPP having at least two, preferably 2, 3, 4, 5, 6, 7, or 8 arginine residues, each optionally separated by one or more uncharged hydrophobic residues, and optionally containing about 6 to 14 amino acid residues. As described below, the CPP is linked at its carboxy terminus to the 3' end of the antisense oligonucleotide via a linker, which may also be one or more amino acids, and is preferably selected from H, acyl, acetyl, benzoyl, or stearoyl. a Substituent R containing a In some embodiments, R a is acetyl.

[0033] As seen in the table below, non-limiting examples of CPPs for use herein include -(RXR)4-R a( SEQ ID NO:54), R-(FFR)3-R a (SEQ ID NO:55), -BX-(RXR)4-R a( SEQ ID NO: 56), -BXR-(FFR)3-R a (SEQ ID NO:57), -GLY-R-(FFR)3-R a (SEQ ID NO:58), -GLY-R5-R a (SEQ ID NO:59), -R5-R a( SEQ ID NO: 60), -GLY-R6-R a( SEQ ID NO: 52) and -R6-R a( SEQ ID NO: 53), in which R a is selected from H, acyl, benzoyl, and stearoyl, R is arginine, X is 6-aminohexanoic acid, B is β-alanine, F is phenylalanine, and GLY (or G) is glycine. CPP "R5" is meant to represent a peptide of five arginine residues linked together via amide bonds (a single substituent, e.g., R 5("R5" is disclosed as SEQ ID NO: 60). CPP "R6" is meant to represent a peptide of six arginine residues linked together via amide bonds (not a single substituent, e.g., R 6 ("R6" is not SEQ ID NO:53). In some embodiments, R a is acetyl.

[0034] Exemplary CPPs are shown in Table 1 (SEQ ID NOs:52-60). [Table 4]

[0035] CPPs, their synthesis, and methods of conjugating to oligonucleotides are further described in U.S. Application Publication No. US2012 / 0289457, and International Patent Application Publication Nos. WO2004 / 097017, WO2009 / 005793, and WO2012 / 150960, the disclosures of which are incorporated by reference in their entireties.

[0036] In some embodiments, the antisense oligonucleotide comprises a substituent "Z" that is defined as a combination of a CPP and a linker. The linker bridges the CPP at its carboxy terminus to the 3' terminus of the oligonucleotide.

[0037] The linker in Z can comprise, for example, 1, 2, 3, 4, or 5 amino acids.

[0038] In certain embodiments, Z is -C(O)(CH2)5NH-CPP, -C(O)(CH2)2NH-CPP, -C(O)(CH2)2NHC(O)(CH2)5NH-CPP, -C(O)CH2NH-CPP, and is selected from the following formula: [ka] wherein the CPP is attached to the linker moiety via an amide bond at the CPP carboxy terminus.

[0039] In various embodiments, the CPP is an arginine-rich peptide as described herein and shown in Table 1. In certain embodiments, the arginine-rich CPP is -R6-R a (SEQ ID NO:53) (i.e., six arginine residues, SEQ ID NO:53), where R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. a is acetyl. In various embodiments, the CPP is selected from SEQ ID NO: 53, 54, or 55, and the linker is -C(O)(CH2)5NH-, -C(O)(CH2)2NH-, -C(O)(CH2)2NHC(O)(CH2)5NH-, -C(O)CHNH-, and [ka] In some embodiments, the linker comprises 1, 2, 3, 4, or 5 amino acids.

[0040] In some embodiments, the CPP is SEQ ID NO:53 and the linker is Gly. In some embodiments, the CPP is SEQ ID NO:52.

[0041] In certain embodiments, Z is —C(O)CHNH—R—R, which is covalently attached to the antisense oligonucleotide at the 3′ end of the oligonucleotide. a (SEQ ID NO: 52), R a is H, acyl, acetyl, benzoyl, or stearoyl and caps the amino terminus of R6 (SEQ ID NO:53). a is acetyl. In these non-limiting examples, the CPP is -R-R a (SEQ ID NO:53), and the linker is -C(O)CH2NH-, (i.e., GLY). Z=-C(O)CH2NH-R6-R a This particular example of (SEQ ID NO:52) is also exemplified by the following structure: [ka] In the formula, R a is selected from H, acyl, acetyl, benzoyl, and stearoyl.

[0042] In various embodiments, the CPP is -R-R a (SEQ ID NO:53), also exemplified by the formula: [ka] In the formula, R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. In certain embodiments, the CPP is SEQ ID NO: 52. In some embodiments, R a is acetyl.

[0043] In some embodiments, the CPP is -(RXR)-R a (SEQ ID NO:54), and is also exemplified by the formula: [ka]

[0044] In various embodiments, the CPP is -R-(FFR)3-R a (SEQ ID NO:55), and is also exemplified as the formula: [ka]

[0045] In various embodiments, Z is -C(O)(CH2)5NH-CPP, -C(O)(CH2)2NH-CPP, -C(O)(CH2)2NHC(O)(CH2)5NH-CPP, -C(O)CH2NH-CPP, and is selected from the following formula: [ka] wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, and the CPP is [ka] (-R-(FFR)3-R a ) (SEQ ID NO: 55), [ka] (-(RXR)4-R a ) (SEQ ID NO: 54), [ka] or (-R6-R a ) (SEQ ID NO:53).

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Although any method and material similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are described.For the purpose of this disclosure, the following terms are defined below.

[0047] I. definition As used herein, the term "alkyl" refers to a saturated straight chain or branched hydrocarbon, unless otherwise specified. In certain embodiments, an alkyl group is a primary, secondary, or tertiary hydrocarbon. In certain embodiments, an alkyl group is a group having 1 to 10 carbon atoms, i.e., C1 to C6. 10In certain embodiments, the alkyl group includes 1-6 carbon atoms, i.e., C1-C6 alkyl. The term includes both substituted and unsubstituted alkyl groups, including halogenated alkyl groups. In certain embodiments, the alkyl group is a fluorinated alkyl group. Non-limiting examples of moieties with which the alkyl group may be substituted are selected from the group consisting of halogen (fluoro, chloro, bromo, or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected or optionally protected, as known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, which is incorporated herein by reference. In certain aspects, the alkyl group is selected from the group consisting of methyl, CF3, CCl3, CFCl2, CF2Cl, ethyl, CH2CF3, CF2CF3, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

[0048] "Suitable for exon skipping", as used herein with respect to a subject or patient, is intended to include subjects and patients with one or more mutations in the dystrophin gene, in which the absence of skipping of certain exons or introns in the dystrophin pre-mRNA results in out-of-frame reading frame, thereby preventing translation of the pre-mRNA and rendering the subject or patient unable to produce functional or semi-functional dystrophin.It is well within the understanding of those skilled in the art to determine whether a patient has a mutation in the dystrophin gene that is suitable for exon skipping (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).

[0049] As used herein, the term "nucleic acid analog" or "polynucleic acid analog" refers to a compound that is structurally similar to natural RNA. A nucleic acid analog or polynucleic acid analog is a chain of nucleotides that has modified phosphate backbone, pentose sugar, and / or nucleobase compared to natural RNA. Exemplary nucleic acid analogs include peptide nucleic acid (PNA), morpholino, and locked nucleic acid (LNA).

[0050] As used herein, the term "oligonucleotide" refers to a sequence of subunits linked by intersubunit linkages. In certain instances, the term "oligonucleotide" is used in connection with "antisense oligonucleotides". For "antisense oligonucleotides", each subunit consists of (i) a ribose sugar or derivative thereof and (ii) a nucleic acid base linked thereto, whereby the order of base-pairing moieties forms a base sequence that is complementary to a target sequence in a nucleic acid (typically RNA) by Watson-Crick base pairing to form a nucleic acid:oligonucleotide heteroduplex within the target sequence, provided that either the subunits, the intersubunit linkages, or both are not naturally occurring. In certain embodiments, the "antisense oligonucleotide conjugate" is a phosphorodiamidate morpholino antisense oligonucleotide ("PPMO") in which a CPP is attached to the 3' end of the antisense oligonucleotide.

[0051] The terms "complementary" and "complementarity" refer to two or more oligonucleotides (i.e., each containing a nucleobase sequence) that are related to each other by Watson-Crick base pairing rules. For example, the nucleobase sequence "TGA(5'→3')" is complementary to the nucleobase sequence "ACT(3'→5')". Complementarity can be "partial", where less than all of the nucleobases of a given nucleobase sequence are matched with another nucleobase sequence according to the base pairing rules. For example, in some embodiments, the complementarity between a given nucleobase sequence and another nucleobase sequence can be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Alternatively, continuing the example, there can be "complete" or "perfect" (100%) complementarity between a given nucleobase sequence and another nucleobase sequence. The degree of complementarity between nucleobase sequences has a significant effect on the efficiency and strength of hybridization between the sequences.

[0052] "Enhance" or "enhancing", or "increase" or "increasing", or "stimulate" or "stimulating" generally refers to the ability of one or more of the foregoing antisense oligonucleotide conjugates or pharmaceutical compositions to produce or cause a greater physiological response (i.e., downstream effect) in a cell or subject compared to the response caused by either no antisense oligonucleotide conjugate, or a control compound. A greater physiological response can include increased expression of a functional form of dystrophin protein, or increased dystrophin-related biological activity in muscle tissue, among other responses apparent from understanding in the art and the description herein.

[0053] As used herein, terms such as "function" and "functionality" refer to biological, enzymatic, or therapeutic functions.

[0054] A "functional" dystrophin protein generally refers to a dystrophin protein that has sufficient biological activity to reduce the progressive degradation of muscle tissue otherwise characteristic of muscular dystrophies, as compared to the altered or "defective" form of dystrophin protein typically present in certain subjects with DMD or BMD.As an example, dystrophin-related activity in in vitro muscle cultures can be measured according to myotube size, myofibril organization (or disintegration), contractile activity, and spontaneous clustering of acetylcholine receptors (see, for example, Brown et al., Journal of Cell Science. 112:209-216, 1999).In addition, animal models are valuable resources for studying disease pathogenesis and provide a means to test dystrophin-related activity. Two of the most widely used animal models for DMD research are the mdx mouse and the Golden Retriever Muscular Dystrophy (GRMD) dog, both of which are dystrophin negative (see, for example, 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. These include truncated forms of dystrophin, such as those produced after administration of certain exon skipping antisense oligonucleotide conjugates of the present disclosure.

[0055] The term "mismatch" refers to one or more nucleobases (whether contiguous or separated) in the oligonucleotide nucleobase sequence that do not match the target pre-mRNA according to base pairing rules. In many cases, perfect complementarity is desired, but some embodiments may include one or more, but preferably 6, 5, 4, 3, 2, or 1 mismatches to the target pre-mRNA. Variations at any position within the oligonucleotide are included. In certain embodiments, the antisense oligonucleotide conjugates of the present disclosure include nucleobase sequence variations near the ends, internal variations, and when present, typically within about 6, 5, 4, 3, 2, or 1 subunit at the 5' and / or 3' ends. In certain embodiments, one, two, or three nucleobases can be removed and still provide binding on the target.

[0056] In certain embodiments, the morpholino is conjugated to a "tail" moiety at the 5' end of the oligonucleotide to enhance its stability and / or solubility. Exemplary tails include: [ka] and [ka] Among the exemplary tail moieties above, "TEG" or "EG3" refers to the following tail moieties: [ka]

[0057] Of the exemplary tail moieties above, "GT" refers to the following tail moieties: [ka]

[0058] As used herein, the terms "-G-R5 (SEQ ID NO: 59)" and "-G-R5-Ac (SEQ ID NO: 59)" are used interchangeably and refer to a cell membrane permeable peptide moiety conjugated to an antisense oligonucleotide. In various embodiments, "G" represents a glycine residue conjugated to "R5" by an amide bond, and each "R" represents an arginine residue conjugated together by an amide bond, whereby "R5" means five arginine residues conjugated together by amide bonds ("R5" is disclosed as SEQ ID NO: 60). The arginine residues can have any configuration, for example, the arginine residues can be L-arginine residues, D-arginine residues, or a mixture of D-arginine residues and L-arginine residues. In some embodiments, "-G-R5 (SEQ ID NO: 59)" or "-G-R5-Ac (SEQ ID NO: 59)" is attached to the 3' end of the antisense oligonucleotide and has the following formula: [ka] or a pharma- ceutically acceptable salt thereof, or of the formula: [ka]

[0059] As used herein, the terms "-G-R6 (SEQ ID NO: 52)" and "-G-R6-Ac (SEQ ID NO: 52)" and "R6G (SEQ ID NO: 52)" are used interchangeably and refer to a cell membrane permeable peptide moiety conjugated to an antisense oligonucleotide. In various embodiments, "G" represents a glycine residue conjugated to "R6" by an amide bond, and each "R" represents an arginine residue conjugated together by an amide bond, whereby "R6" refers to six arginine residues conjugated together by amide bonds ("R6" is disclosed as SEQ ID NO: 53). The arginine residues can have any configuration, for example, the arginine residues can be L-arginine residues, D-arginine residues, or a mixture of D-arginine residues and L-arginine residues. In some embodiments, "-G-R6 (SEQ ID NO:52)" or "-G-R6-Ac (SEQ ID NO:52)" is conjugated to the 3' end of the antisense oligonucleotide and has the following formula: [ka] or [ka]

[0060] The terms "nucleobase" (Nu), "base-pairing moiety," or "base" are used interchangeably and refer to the purine or pyrimidine bases found in naturally occurring or "natural" DNA or RNA (e.g., uracil, thymine, adenine, cytosine, and guanine), as well as analogs of these naturally occurring purines and pyrimidines. These analogs may confer improved properties, such as binding affinity, to oligonucleotides. Exemplary analogs include hypoxanthine (the base component of inosine), 2,6-diaminopurine, 5-methylcytosine, C5-propynyl-modified pyrimidines, 10-(9-(aminoethoxy)phenoxazinyl) (G-clamp), and the like.

[0061] Further examples of base pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine, and hypoxanthine (inosine) (with their respective amino groups protected by acyl protecting groups), pyrimidine analogs such as 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pseudoisocytosine and pseudouracil, and other modified nucleobases such as 8-substituted purines, xanthines, or hypoxanthines (the latter two are natural decomposition products). Also contemplated are modified nucleobases disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048; Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196, and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313, the contents of which are incorporated herein by reference.

[0062] Further examples of base pairing moieties include, but are not limited to, extended size nucleobases with one or more additional benzene rings. Nucleobase substitutions described in the Glen Research catalog (www.glenresearch.com), Krueger AT et al., Acc. Chem. Res., 2007, 40, 141-150; Kool, ET, Acc. Chem. Res., 2002, 35, 936-943; Benner SA, et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, FE, et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; and Hirao, I., Curr. Opin. Chem. Biol., 2006, 10, 622-627, the contents of which are incorporated herein by reference, are contemplated as useful in the antisense oligonucleotide conjugates described herein. Examples of extended size nucleobases include those shown below, as well as tautomers thereof. [ka]

[0063] As used herein, a set of brackets used within a structural formula indicates that the structural feature between the brackets is repeated. In some embodiments, the brackets used can be "[" and "]", and in certain embodiments, the brackets used to indicate a repeating structural feature can be "(" and ")". In some embodiments, the number of repeats of the structural feature between the brackets is the number shown outside the brackets, e.g., 2, 3, 4, 5, 6, 7, etc. In various embodiments, the number of repeats of the structural feature between the brackets is indicated by a variable shown outside the brackets, such as "Z".

[0064] As used herein, a straight or squiggly bond drawn to a chiral carbon or phosphorus atom in a 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 and / or mixtures thereof. Examples of such diagrams are shown below. [ka]

[0065] The phrase "pharmacologically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the subject being treated therewith.

[0066] The term "repair" with respect to dystrophin synthesis or production generally refers to the production of dystrophin protein, including truncated forms of dystrophin, in muscular dystrophy patients after treatment with antisense oligonucleotides as described herein.The proportion of dystrophin-positive fibers in patients after treatment can be determined by muscle biopsy using known techniques.For example, muscle biopsy can be taken from a suitable muscle, such as the biceps brachii muscle, of a patient.

[0067] The analysis of the percentage of positive dystrophin fibers can be performed before and / or after treatment, or at points throughout the course of treatment.In some embodiments, the post-treatment biopsy is taken from the muscle contralateral to the pre-treatment biopsy.The pre-treatment and post-treatment dystrophin expression analysis can be performed using any suitable assay for dystrophin.In some embodiments, immunohistochemical detection is performed on tissue sections from 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 highly sensitive marker for dystrophin, can be used.Any suitable secondary antibody can be used.

[0068] 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. A normal muscle sample has 100% dystrophin-positive fibers. Thus, the percentage of dystrophin-positive fibers can be expressed as a percentage of normal. When counting dystrophin-positive fibers in post-treatment muscle to control for the presence of trace levels of dystrophin in pre-treatment muscle as well as revertant fibers, a baseline can be set using pre-treatment muscle sections from the patient. This can be used as a threshold for counting dystrophin-positive fibers in post-treatment muscle sections of that patient. In other embodiments, antibody-stained tissue sections can also be used for dystrophin quantification using Bioquant image analysis software (Bioquant Image Analysis Corporation, Nashville, TN). Total dystrophin fluorescent signal intensity can be reported as a percentage of normal. Additionally, Western blot analysis using monoclonal or polyclonal anti-dystrophin antibodies can be used to determine the percentage of dystrophin-positive fibers. For example, anti-dystrophin antibody NCL-Dys1 from Leica Biosystems can be used. The percentage of dystrophin-positive fibers can also be analyzed by determining the expression of components of the sarcoglycan complex (β, γ) and / or neuronal NOS.

[0069] In some embodiments, treatment with pharmaceutical compositions comprising antisense oligonucleotide conjugates slows or reduces the progressive respiratory muscle dysfunction and / or failure in DMD patients that would be expected without treatment. In some embodiments, treatment with pharmaceutical compositions comprising antisense oligonucleotide conjugates can reduce or eliminate the need for ventilatory support that would be expected without treatment. In some embodiments, measurements of respiratory function to track disease progression and evaluate potential therapeutic interventions include maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), and forced vital capacity (FVC). MIP and MEP measure the pressure levels that a person can generate during inhalation and exhalation, respectively, and are sensitive measures of respiratory muscle strength. MIP is a measure of diaphragm muscle weakness.

[0070] In some embodiments, MEP may decline before changes in other pulmonary function tests, including MIP and FVC. In certain embodiments, MEP may be an early indicator of respiratory dysfunction. In certain embodiments, FVC may be used to measure the total volume of air expelled during forced expiration after maximal inspiration. In DMD patients, FVC increases in parallel with physical growth until the early teens. However, as growth slows or becomes poorer with disease progression and muscle weakness progresses, vital capacity enters a decline phase, declining at an average rate of about 8-8.5 percent per year after age 10-12. In certain embodiments, percent predicted MIP (MIP adjusted for weight), percent predicted MEP (MEP adjusted for age), and percent predicted FVC (FVC adjusted for age and height) are supportive analyses.

[0071] As used herein, the terms "subject" and "patient" include any animal that exhibits or is at risk of exhibiting a condition that can be treated with a pharmaceutical composition comprising an antisense oligonucleotide conjugate, such as a subject (or patient) that has or is at risk of having DMD or BMD, or any of the symptoms associated with these conditions (e.g., muscle fiber loss). Suitable subjects (or patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock, and household animals or pets (such as cats or dogs). Non-human primates, and preferably human patients (or subjects) are included. Also included are methods of producing dystrophin in subjects (or patients) that have a mutation in the dystrophin gene that is suitable for exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping.

[0072] The phrase "targeting sequence" or "base sequence" refers to the sequence of the nucleic acid base of the oligonucleotide that is complementary to the sequence of the nucleotide in the target pre-mRNA.In some embodiments of the present disclosure, the sequence of the nucleotide in the target pre-mRNA is the target region of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of dystrophin pre-mRNA.

[0073] "PPMO#1" has the following structure: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, PPMO#1 is in the form of a halogen salt. In some embodiments, PPMO#1 is in the form of a hexahalogen salt. In some embodiments, PPMO#1 is in the form of an HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a 6HCl salt.

[0074] "PPMO#2" has the following structure: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, PPMO#2 is in the form of a halogen salt. In some embodiments, PPMO#2 is in the form of a hexahalogen salt. In some embodiments, PPMO#2 is in the form of an HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a 6HCl salt.

[0075] "PPMO#3" has the following structure: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, PPMO#3 is in the form of a halogen salt. In some embodiments, PPMO#3 is in the form of a hexahalogen salt. In some embodiments, PPMO#3 is in the form of an HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a 6HCl salt.

[0076] "PPMO#4" has the following structure: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, PPMO#4 is in the form of a halogen salt. In some embodiments, PPMO#4 is in the form of a hexahalogen salt. In some embodiments, PPMO#4 is in the form of an HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a 6HCl salt.

[0077] "Treatment" of a subject (e.g., a mammal, such as a human) or cell is any type of intervention used in an attempt to change the natural course of the subject or cell. Treatment includes, but is not limited to, administration of an oligonucleotide or pharmaceutical composition thereof, and can be performed either prophylactically or after the onset of a pathological event or contact with a pathogen. Treatment includes any desired effect on the symptoms or pathology of a disease or condition associated with 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 are "prophylactic" treatments, which can be aimed at slowing the progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate a complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0078] In some embodiments, treatment with a pharmaceutical composition comprising an antisense oligonucleotide conjugate increases new dystrophin production, delays disease progression, delays or reduces lameness, reduces muscle inflammation, reduces muscle damage, improves muscle function, reduces loss of lung function, and / or enhances muscle regeneration that would be expected without treatment. In some embodiments, treatment maintains, delays, or slows disease progression. In some embodiments, treatment maintains ambulation or reduces lameness. In some embodiments, treatment maintains lung function or reduces loss of lung function. In some embodiments, treatment maintains or increases the patient's stable walking distance, for example, as measured by the 6-minute walk test (6MWT). In some embodiments, treatment maintains or reduces the time to walk / run 10 meters (i.e., 10-meter walk / run test). In some embodiments, treatment maintains or reduces the time to stand up from a supine position (i.e., time to stand test). In some embodiments, the treatment maintains or reduces the time to climb four standard stairs (i.e., the four-stair climb test). In some embodiments, the treatment maintains or reduces the inflammation of the patient's muscles, for example, as measured 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 muscle structure and composition caused by inflammation, edema, muscle damage, and fat infiltration.

[0079] In some embodiments, treatment with a pharmaceutical composition comprising an antisense oligonucleotide conjugate increases new dystrophin production and delays or reduces the loss of ambulation that would be expected without treatment. For example, treatment can stabilize, maintain, improve, or increase the walking ability of a subject (e.g., stabilization of ambulation). In some embodiments, treatment maintains or increases the patient's stable walking distance, for example, as measured by the 6-minute walk test (6MWT) described by McDonald, et al. (Muscle Nerve, 2010; 42: 966-74, incorporated herein by reference). The change in 6-minute walk distance (6MWD) can be expressed as an absolute value, a percentage change, or a change in % predicted value. The performance of a DMD patient in the 6MWT compared to the typical performance of a healthy counterpart can be determined by calculating the % predicted value. For example, the % predicted 6MWD can be calculated for men using the following equation: 196.72 + (39.81 x age) - (1.36 x age) 2 ) + (132.28 × height in meters). For women, the % 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).

[0080] Loss of muscle function in DMD patients may occur against the background of normal childhood growth and development. In fact, children with DMD may show an increase in walking distance during 6MWT over the course of about one year, despite progressive muscle impairment. In some embodiments, 6MWD from DMD patients is compared to existing normative data from typically developing control subjects and age- and sex-matched subjects. In some embodiments, normal growth and development may be accounted for using an equation based on age and height fitted to normative data. Using such an equation, 6MWD in subjects with DMD may be converted to percent predicted (% predicted) values. In some embodiments, analysis of % predicted 6MWD data represents a way to account for normal growth and development, and may show that increases in function at younger ages (e.g., 7 years old or younger) represent a stable rather than improved performance in DMD patients (Henricson et al. PLoS Curr., 2012, version 2, incorporated herein by reference).

[0081] In order 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 system has become particularly suitable when testing several slightly different antisense molecules all directed against the same target region, as shown below.

[0082] H#A / D(x:y)

[0083] The first 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 beginning and end of the exon, respectively. (xy) 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. The closest splice site will be the acceptor, so these coordinates are preceded by "A". The description of the annealing coordinates at the donor splice site can 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 coordinate is represented by A(+65+85), ie, the site from 65th to 85th nucleotides from the start of the exon.

[0084] II. composition The present disclosure relates to a composition comprising: (a) an antisense oligonucleotide conjugate comprising a cell membrane-permeable peptide covalently attached to a nucleic acid analog, or a pharma- ceutically acceptable salt thereof, wherein the cell membrane-permeable peptide comprises at least two positively charged amino acids; (b) one or more surfactants; (c) one or more sugars; and (d) one or more buffering agents.

[0085] A. Antisense Oligonucleotide Conjugates 1. Antisense oligonucleotide conjugates designed to induce exon skipping In certain embodiments, the antisense oligonucleotide conjugate of the disclosed compositions, or a pharma- ceutically acceptable salt thereof, is complementary to a target region of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of a dystrophin pre-mRNA. In some embodiments, the antisense oligonucleotide conjugate of the disclosed compositions, or a pharma- ceutically acceptable salt thereof, is complementary to 15-35 nucleobases of a target region of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of a dystrophin pre-mRNA. In some embodiments, the antisense oligonucleotide conjugate of the disclosed composition, or a pharma- ceutically acceptable salt thereof, is complementary to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases of a target region of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of a dystrophin pre-mRNA.

[0086] The antisense oligonucleotide conjugates of the present disclosure target dystrophin pre-mRNA and induce skipping of the targeted exon, so that it is excluded or skipped from the mature spliced ​​mRNA transcript. By skipping the targeted exon, the disrupted reading frame is restored to an in-frame mutation.

[0087] The nucleic acid base sequence of the antisense oligonucleotide conjugate that induces the skipping of target exon is designed to be complementary to a specific target sequence in dystrophin pre-mRNA.Antisense oligonucleotide conjugate is a PPMO, and each morpholino ring of PMO is linked to the nucleic acid base that includes, for example, the nucleic acid base found in DNA (adenine, cytosine, guanine, and thymine) and cell membrane permeable peptide.

[0088] Exemplary aspects of the present disclosure include compounds of the following general structure: [ka] And as described in Figure 2 of Summerton, J., et al., Antisense & Nucleic Acid Drug Development, 7:187-195 (1997), phosphorodiamidate morpholino oligonucleotides.The morpholino described herein is intended to include all stereoisomers and tautomers of the above general structure.The synthesis, structure and binding properties of morpholino oligonucleotides are described in detail in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337, 8,076,476 and 8,299,206, all of which are incorporated herein by reference.

[0089] In certain embodiments, the morpholino is conjugated to a "tail" moiety at the 5' end of the oligonucleotide to enhance its stability and / or solubility. Exemplary tails include: [ka] and [ka] Includes.

[0090] In some embodiments, the antisense oligonucleotide conjugate in the composition has Formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu is a nucleobase that together form a targeting sequence, T, [ka] and [ka] is a moiety selected from R 100 is a cell membrane penetrating peptide, Each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 5-1] [Table 5-2] A is [ka] and C is [ka] and G is [ka] and T is [ka] and U is [ka] where Gm is a methylated guanine, Am is a methylated adenine, and mC is [ka] It is.

[0091] In some embodiments, each of the 1 to n Nu of the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, or SEQ ID NO: 51. In some embodiments, each of the 1 to n Nu of the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 1. In some embodiments, each of the 1 to n Nu of the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 7. In some embodiments, each of the 1 to n Nu of the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 17. In some embodiments, each of the 1 to n Nu of the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 51.

[0092] In some embodiments, T' of the antisense oligonucleotide conjugate of Formula (I) or a pharma- ceutically acceptable salt thereof in the composition is [ka] It is.

[0093] The antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof comprises a cell membrane penetrating peptide that is an arginine-rich peptide. In some embodiments, the antisense oligonucleotide conjugate comprises -(RXR)4-R a (SEQ ID NO:54), R-(FFR)3-R a (SEQ ID NO:55), -BX-(RXR)4-R a (SEQ ID NO:56), -BXR-(FFR)3-R a (SEQ ID NO:57), -GLY-R-(FFR)3-R a (SEQ ID NO:58), -GLY-R5-R a (SEQ ID NO:59), -R5-R a , (SEQ ID NO: 60), -GLY-R6-R a(SEQ ID NO:52), and -R6-R a (SEQ ID NO: 53), a is selected from H, acyl, benzoyl, and stearoyl, R is arginine, X is 6-aminohexanoic acid, B is β-alanine, F is phenylalanine, and GLY (or G) is glycine. In some embodiments, the arginine-rich peptide is -GLY-R5-R a (SEQ ID NO:59), -R5-R a (SEQ ID NO: 60), -GLY-R6-R a (SEQ ID NO:52), or -R6-R a (SEQ ID NO:53), R is arginine, and R a is hydrogen or an acyl group. In some aspects, it is an arginine-rich peptide.

[0094] In some embodiments, the antisense oligonucleotide conjugate in the composition has Formula (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu in the range 1 to (n+1) corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 6-1] [Table 6-2] A is [ka] and C is [ka] and G is [ka] and T is [ka] and U is [ka] where Gm is a methylated guanine, Am is a methylated adenine, and mC is [ka] It is.

[0095] In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof in the composition corresponds from 5' to 3' to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, or SEQ ID NO: 51. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof in the composition corresponds from 5' to 3' to SEQ ID NO: 1. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof in the composition corresponds from 5' to 3' to SEQ ID NO: 7. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof in the composition corresponds from 5' to 3' to SEQ ID NO: 17. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof in the composition corresponds from 5' to 3' to SEQ ID NO: 51.

[0096] In some embodiments, the antisense oligonucleotide conjugate in the composition has Formula (III): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu in the range 1 to (n+1) corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 7-1] [Table 7-2] A is [ka] and C is [ka] and G is [ka] and T is [ka] and U is [ka] where Gm is a methylated guanine, Am is a methylated adenine, and mC is [ka] It is.

[0097] In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, or SEQ ID NO: 51. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 1. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 7. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 17. In some embodiments, each of 1 to n Nu of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof in the composition corresponds, from 5' to 3', to SEQ ID NO: 51.

[0098] In some embodiments, the antisense oligonucleotide conjugate in the composition is PPMO#1, PPMO#2, PPMO#3, or PPMO#4, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide conjugate in the composition is PPMO#1, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide conjugate in the composition is PPMO#2, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide conjugate in the composition is PPMO#3, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide conjugate in the composition is PPMO#4, or a pharmaceutically acceptable salt thereof.

[0099] In some embodiments, the composition comprises about 50 mg to about 500 mg of the antisense oligonucleotide conjugate, or a pharma- ceutically acceptable salt thereof. In some embodiments, the composition comprises about 50 mg to about 450 mg of the antisense oligonucleotide conjugate, or a pharma- ceutically acceptable salt thereof. In some embodiments, the composition comprises about 50 mg to about 400 mg, about 50 mg to about 350 mg, about 50 mg to about 300 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 75 mg to about 500 mg, about 75 mg to about 450 mg, about 75 mg to about 400 mg, about 75 mg to about 350 mg, about 75 mg to about 300 mg, about 75 mg to about 250 mg, about 75 mg to about 200 mg, about 75 mg to about 150 mg, about 100 mg g ~ approx. 500 mg, approx. 100 mg ~ approx. 450 mg, approx. 100 mg ~ approx. 400 mg, approx. 100 mg ~ approx. 350 mg, approx. 100 mg ~ approx. 300 mg, approx. , about 125mg to about 450mg, about 125mg to about 400mg, about 125mg to about 350mg, about 125mg to about 300mg, about 125mg to about 250mg, about 125mg to about 200mg, about 150mg to about 500mg, about 150mg to about 450mg, about 150mg to about 400mg, about 150mg to about 350mg, about 150mg to about 300mg, about 150mg to about 250mg, about 150mg to about 200mg, about 175mg to about 500mg, about 175mg to about 450mg, about 1 75mg to about 400mg, about 175mg to about 350mg, about 175mg to about 300mg, about 175mg to about 250mg, about 200mg to about 500mg, about 200mg to about 450mg, about 200mg to about 400mg, about 200mg to about 350 mg, about 200mg to about 300mg, about 200mg to about 250mg, about 225mg to about 500mg, about 225mg to about 450mg, about 225mg to about 400mg, about 225mg to about 350mg, about 225mg to about 300mg, about 250m g ~ about 500mg, about 250mg - about 450mg, about 250mg - about 400mg, about 250mg - about 350mg, about 275mg - about 500mg, about 275mg - about 450mg, about 275mg - about 400mg, about 275mg - about 350mg,about 300 mg to about 500 mg, about 300 mg to about 450 mg, about 300 mg to about 400 mg, about 300 mg to about 350 mg, about 325 mg to about 500 mg, about 325 mg to about 450 mg, about 325 mg to about 400 mg, about 350 mg to about 500 mg, about 350 mg to about 450 mg, about 350 mg to about 400 mg, about 400 mg to about 500 mg, about 400 mg to about 450 mg, or about 450 mg to about 500 mg of the antisense oligonucleotide conjugate, or a pharma- ceutically acceptable salt thereof. In some embodiments, the composition comprises about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, or about 500 mg of antisense oligonucleotide conjugate based on the weight of the free base. The weight of the antisense oligonucleotide conjugate as a particular pharma- ceutically acceptable salt contained in the composition will be adjusted. For example, 102 mg of PPMO#1 as its hexachloride (6HCl) salt form corresponds to 100 mg of PPMO#1 as its free base.

[0100] B. Pharmaceutically acceptable salts of the antisense oligonucleotide conjugates Certain aspects of the antisense oligonucleotides described herein may contain basic functional groups such as amino or alkylamino, and thus can form pharmaceutically acceptable salts with pharmaceutically acceptable acids.The term " pharmaceutically acceptable salts " in this respect refers to the relatively non-toxic inorganic and organic acid addition salts of the antisense oligonucleotides of the present disclosure.These salts can be prepared in situ during administration vehicle or dosage form manufacturing process, or can be prepared by separately reacting the purified antisense oligonucleotides of the present disclosure in their free base form with suitable organic or inorganic acid, and then isolating the salts thus formed during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulfonate, and the like (see, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0101] The pharma- ceutically acceptable salts of the subject antisense oligonucleotides include, for example, the conventional non-toxic salts or quaternary ammonium salts of antisense oligonucleotides from non-toxic organic or inorganic acids.For example, such conventional non-toxic salts include those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; and the 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, salicylic acid (salicyclic), sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothionic acid, etc.

[0102] In certain embodiments, the antisense oligonucleotides of the present disclosure may contain one or more acidic functional groups, and thus can form pharma- ceutically acceptable salts with pharma-ceutically acceptable bases.In these cases, the term "pharma-ceutically acceptable salts" refers to the relatively non-toxic inorganic and organic base addition salts of the antisense oligonucleotides of the present disclosure.These salts can also be prepared in situ during the administration vehicle or dosage form manufacturing process, or can be prepared by separately reacting purified antisense oligonucleotides in their free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharma-ceutically acceptable metal cation, with ammonia, or with a pharma-ceutically acceptable organic primary, secondary, or tertiary amine.Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like.Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like.(See, for example, Berge et al., supra).

[0103] The salt form may be a complex of multiple cations or anions with the antisense oligonucleotide conjugate. For example, the salt form may be a monohalide (1HCl), dihalide (2HCl), trihalide (3HCl), tetrahalide (4HCl), pentahalide (5HCl), or hexahalide (6HCl).

[0104] B. Surfactants The composition of the present disclosure includes one or more surfactants. In some embodiments, the one or more surfactants are present in the composition in an amount of about 0.001% to about 2% by weight of the composition. In some embodiments, the one or more surfactants are present in the composition in an amount of about 0.002% to about 1% by weight of the composition. In some embodiments, the one or more surfactants are present in the composition in an amount of about 0.01% to about 1% by weight of the composition.

[0105] In some embodiments, the one or more surfactants in the composition are selected from the group consisting of polyoxyethylene sorbitan monooleate (e.g., polysorbate 80, polysorbate 20), polyvinylpyrrolidone (PVP, povidone, poloxamer, poloxamer F188), and mixtures thereof. In some embodiments, the surfactant is polyoxyethylene sorbitan monooleate (e.g., polysorbate 80, polysorbate 20). In some embodiments, the surfactant is polyvinylpyrrolidone.

[0106] In some embodiments, the composition contains polyoxyethylene sorbitan monooleate (e.g., polysorbate 80, polysorbate 20) in an amount of about 0.001% to about 2% by weight of the composition. In some embodiments, the composition contains polyoxyethylene sorbitan monooleate (e.g., polysorbate 80, polysorbate 20) in an amount of about 0.001% to about 2% by weight of the composition. In some embodiments, the composition contains polyoxyethylene sorbitan monooleate (e.g., polysorbate 80, polysorbate 20) in an amount of about 0.002% to about 1% by weight of the composition. In some embodiments, the composition contains polyoxyethylene sorbitan monooleate (e.g., polysorbate 80, polysorbate 20) in an amount of about 0.01% to about 1% by weight of the composition.

[0107] In some embodiments, the composition contains polyvinylpyrrolidone in an amount of about 0.001% to about 2% by weight of the composition. In some embodiments, the composition contains polyvinylpyrrolidone in an amount of about 0.001% to about 2% by weight of the composition. In some embodiments, the composition contains polyvinylpyrrolidone in an amount of about 0.002% to about 1% by weight of the composition. In some embodiments, the composition contains polyvinylpyrrolidone in an amount of about 0.01% to about 1% by weight of the composition.

[0108] C. Sugars The compositions of the present disclosure include one or more sugars. In some embodiments, the one or more sugars are present in the composition in an amount of about 0.1% to about 11% by weight of the composition. In some embodiments, the one or more sugars are present in the composition in an amount of about 0.5% to about 7% by weight of the composition. In some embodiments, the one or more sugars are present in the composition in an amount of up to 6% by weight of the composition. In some embodiments, the one or more sugars are present in the composition in an amount of about 6% by weight of the composition.

[0109] In some aspects, the one or more sugars are selected from the group consisting of trehalose, mannitol, sucrose, and mixtures thereof. In some aspects, the sugar is a mixture of mannitol and sucrose. In some aspects, the sugar is mannitol. In some aspects, the sugar is sucrose.

[0110] In some embodiments, the composition contains a mixture of mannitol and sucrose in an amount of about 0.1% to about 11% by weight of the composition. In some embodiments, the composition contains a mixture of mannitol and sucrose in an amount of about 0.5% to about 7% by weight of the composition. In some embodiments, the composition contains a mixture of mannitol and sucrose in an amount up to 6% by weight of the composition. In some embodiments, the one or more sugars are present in the composition in an amount of about 6% by weight of the composition.

[0111] In some embodiments, the sugar is mannitol and is present in the composition in an amount of about 0.1% to about 11% by weight of the composition. In some embodiments, the sugar is mannitol and is present in the composition in an amount of about 0.5% to about 7% by weight of the composition. In some embodiments, the sugar is mannitol and is present in the composition in an amount of up to 6% by weight of the composition. In some embodiments, the sugar is mannitol and is present in the composition in an amount of about 6% by weight of the composition.

[0112] In some embodiments, the sugar is sucrose and is present in the composition in an amount of about 0.1% to about 11% by weight of the composition. In some embodiments, the sugar is sucrose and is present in the composition in an amount of about 0.5% to about 7% by weight of the composition. In some embodiments, the sugar is sucrose and is present in the composition in an amount of up to 6% by weight of the composition. In some embodiments, the sugar is sucrose and is present in the composition in an amount of about 6% by weight of the composition.

[0113] D. Buffer The composition of the present disclosure includes one or more buffering agents. In some embodiments, the one or more buffering agents are present in an amount sufficient to provide a pH of about 6.0 to about 7.0 when the composition is dissolved or dispersed in an aqueous medium. In some embodiments, the one or more buffering agents are present in an amount sufficient to provide a pH of about 6.3 to about 6.5. In some embodiments, the one or more buffering agents are present in an amount sufficient to provide a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. In some embodiments, the one or more buffering agents are present in an amount sufficient to provide a pH of about 6.3. In some embodiments, the one or more buffering agents are present in an amount sufficient to provide a pH of about 6.4. In some embodiments, the one or more buffering agents are present in an amount sufficient to provide a pH of about 6.5.

[0114] In some embodiments, the one or more buffering agents are present in a range of about 10 mM to about 50 mM when the composition is dissolved or dispersed in an aqueous solvent. In some embodiments, the one or more buffering agents are present in a range of about 10 mM to about 40 mM when the composition is dissolved or dispersed in an aqueous solvent. In some embodiments, the one or more buffering agents are present in a range of about 10 mM to about 30 mM when the composition is dissolved or dispersed in an aqueous solvent. In some embodiments, the one or more buffering agents are present in a range of about 20 mM to about 50 mM when the composition is dissolved or dispersed in an aqueous solvent. In some embodiments, the one or more buffering agents are present in a range of about 20 mM to about 40 mM when the composition is dissolved or dispersed in an aqueous solvent. In some embodiments, when the composition is dissolved or dispersed in an aqueous solvent, the one or more buffering agents are present at about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0115] In some aspects, the one or more buffering agents are selected from the group consisting of histidine, citrate, maleate, arginine, and mixtures thereof, hi some aspects, the buffering agent is citrate.

[0116] In some embodiments, the composition has an osmolality of about 250 mOsm / kg to about 650 mOsm / kg. In some embodiments, the composition has an osmolality of about 300 mOsm / kg to about 500 mOsm / kg. In some embodiments, the composition has an osmolality of about 300 mOsm / kg to about 600 mOsm / kg. In some embodiments, the composition has an osmolality of about 400 mOsm / kg to about 600 mOsm / kg.

[0117] In some embodiments, the composition is in a lyophilized form. In some embodiments, the composition dissolves within 30 minutes after adding aqueous solvent. In some embodiments, the composition dissolves within 20 minutes after adding aqueous solvent. In some embodiments, the composition dissolves within 15 minutes after adding aqueous solvent. In some embodiments, the composition dissolves within 10 minutes after adding aqueous solvent. In some embodiments, the composition dissolves within 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, or about 3 minutes after adding aqueous solvent.

[0118] In some aspects, the aqueous solvent used to dissolve the composition is sterile water.

[0119] III.How to use Restoring the dystrophin reading frame using exon skipping Potential therapeutic approach for the treatment of DMD caused by out-of-frame mutation of dystrophin gene is suggested by milder form of dystrophinopathy known as BMD caused by in-frame mutation.The ability to convert out-of-frame mutation into in-frame mutation hypothetically maintains mRNA reading frame and produces internally truncated but functional dystrophin protein.The antisense oligonucleotide of the present disclosure is designed to achieve this.

[0120] Hybridization of an antisense oligonucleotide conjugate (e.g., an antisense oligonucleotide conjugate of Formula (I), Formula (II), or Formula (III)) with the targeted pre-mRNA sequence inhibits formation of the pre-mRNA splicing complex and deletes exon 50 from the mature mRNA. The structure and conformation of the antisense oligonucleotides of the present disclosure allow for sequence-specific base pairing to complementary sequences.

[0121] Normal dystrophin mRNA containing all 79 exons will produce normal dystrophin protein. The shape of each exon indicates how the codon is split between exons. Notably, a codon consists of three nucleotides. Rectangular exons begin and end with a complete codon. Arrow-shaped exons begin with a complete codon but end with a split codon that contains only nucleotide #1 of the codon. Nucleotides #2 and #3 of this codon are contained in the subsequent exon that begins with a chevron shape.

[0122] Clinical outcomes for analyzing the effect of antisense oligonucleotide conjugates complementary to target regions of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of human dystrophin pre-mRNA and inducing exon skipping include percentage of dystrophin positive fibers (PDPF), six-minute walk test (6MWT), loss of ambulation (LOA), North Star Ambulation Assessment (NSAA), pulmonary function tests (PFTs), ability to rise (from supine position) without external support, de novo dystrophin production, and other functional measures.

[0123] In some aspects, the present disclosure provides a method for producing dystrophin in a subject having a mutation in the dystrophin gene that is suitable for skipping exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53, comprising administering to the subject a composition as described herein. In certain aspects, the present disclosure provides a method for restoring the mRNA reading frame to induce dystrophin protein production in a subject having Duchenne muscular dystrophy (DMD) having a mutation in the dystrophin gene that is suitable for skipping exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 by administering a composition as described herein. Protein production can be measured by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, or immunohistochemistry (IHC).

[0124] In some aspects, the disclosure provides a method for treating DMD in a subject in need thereof, the subject having a mutation in the dystrophin gene that is amenable to skipping of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53, the method comprising administering to the subject a composition described herein. In some aspects, the subject in need thereof is a human patient with DMD.

[0125] In various embodiments, the treatment of the subject is measured by delaying disease progression.In some embodiments, the treatment of the subject is measured by maintaining the walking of the subject or reducing the walking disability of the subject.In some embodiments, the walking is measured using 6-minute walk test (6MWT).In certain embodiments, the walking is measured using North Start Walking Assessment (NSAA).

[0126] In various aspects, the present disclosure provides a method for maintaining or reducing the loss of lung function in a subject with DMD, the subject has a mutation in the DMD gene that is suitable for skipping exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53, and the method comprises administering to the subject a composition as described herein. In some aspects, the lung function is measured as maximum expiratory pressure (MEP). In certain aspects, the lung function is measured as maximum inspiratory pressure (MIP). In some aspects, the lung function is measured as forced vital capacity (FVC).

[0127] In various embodiments, the composition of the present disclosure is co-administered with a therapeutically effective amount of a nonsteroidal anti-inflammatory compound. In some embodiments, the nonsteroidal anti-inflammatory compound is an NF-kB inhibitor. For example, in some embodiments, the NF-kB inhibitor can be CAT-1004 or a pharmaceutically acceptable salt thereof. In various embodiments, the NF-kB inhibitor can be a conjugate of salicylate and DHA. In some embodiments, the NF-kB inhibitor is CAT-1041 or a pharmaceutically acceptable salt thereof. In certain embodiments, the NF-kB inhibitor is a conjugate of salicylate and EPA. In various embodiments, the NF-kB inhibitor is [ka] or a pharma- ceutically acceptable salt thereof.

[0128] In some embodiments, the nonsteroidal anti-inflammatory compound is a TGF-b inhibitor. For example, in certain embodiments, the TGF-b inhibitor is HT-100.

[0129] In certain aspects, compositions are described that include the antisense oligonucleotide conjugates described herein, or pharma- ceutically acceptable salts thereof, for use in therapy. In certain aspects, compositions are described that include the antisense oligonucleotide conjugates described herein, or pharma-ceutically acceptable salts thereof, for use in treating Duchenne muscular dystrophy.

[0130] V. kit The present disclosure also provides a kit for treating patients with genetic disease, the kit comprises a composition comprising at least an antisense oligonucleotide conjugate, or its pharma- ceutically acceptable salt, 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 application of the above method has a wide range of applications to identify antisense molecules suitable for use in treating many other diseases.In one aspect, the kit comprises an antisense oligonucleotide conjugate according to formula (I), formula (II), or formula (III), or its pharma- ceutically acceptable salt. EXAMPLES

[0131] Although the above disclosure has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of this disclosure, certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and are not limiting. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce substantially similar results.

[0132] Example 1 (100 mg / vial composition) Preparation of PPMO#1 hexahydrochloride formulation To 1 L of water for injection, 5.67 g of sodium citrate dihydrate, and 0.24 g of citric acid monohydrate were added. To this citrate buffer, 40 g of D-mannitol, 38 g of sucrose, and 0.02 g of polysorbate 80 were added. 51.0 g of PPMO#1 hydrochloride (6 HCl salt, equivalent to 50.0 g of PPMO#1 free base) was added and mixed well. Upon complete dissolution, the pH of the solution was confirmed to be 6.0-7.0, the osmolality to be 300-500 mOsm / kg, and the concentration of PPMO#1 was measured using an extinction coefficient of 23.1 at 260 nm in 1% ammonium hydroxide. The final solution was filtered through a 0.2 μm bioburden reducing filter. 2 mL of this solution was filled into vials, lyophilized and stoppered and sealed to produce the final formulation vials.

[0133] Reconstitution of PPMO#1 hexahydrochloride formulation For each PPMO#1 Hexahydrochloride formulation vial, 2 mL of Sterile Water for Injection is added through the stopper, without the seal / stopper, using a needle / syringe. The contents of each vial are mixed by gentle swirling without shaking. Visually inspect each vial to ensure the contents are completely dissolved. Gently inverting 5-10 times is recommended to ensure homogeneity. Upon reconstitution, PPMO#1 Hexahydrochloride will be a clear, colorless solution that may have some opalescence. The reconstituted formulation is then taken from the individual vials and diluted with 0.9% Sodium Chloride Injection (saline) to a concentration of approximately 20 mg / mL for infusion.

[0134] analysis Samples were analyzed for appearance, moisture content, reconstitution time, and subvisible particle analysis. The appearance of the lyophilized cake and reconstituted solution were visually confirmed for clarity and the presence of any visible particles. The moisture content in the lyophilized cake was measured by coulometric Karl Fischer titration and found to be less than 1% based on duplicate measurements. Reconstitution time was determined by dissolving the contents of the vial in 2 mL of Water for Injection. Dissolution was achieved by gently swirling (not shaking) and inverting the vial after adding the Water for Injection and was found to be within 5 minutes. Light Obscuration Method USP <788> Subvisible particle analysis was performed using Method 1. A low volume (1 mL) method was used for this analysis, and all analyses met the USP criteria of <6000 particles / container for >10 μm and <600 particles / container for >25 μm.

[0135] Example 2 (400 mg / vial composition) Preparation of PPMO#1 hexahydrochloride formulation To 1 L of water for injection, 5.67 g of sodium citrate dihydrate, and 0.24 g of citric acid monohydrate are added. To this citrate buffer, 40 g of D-mannitol, 38 g of sucrose, and 0.02 g of polysorbate 80 are added. 102.0 g of PPMO#1 hexahydrochloride (6HCl salt, equivalent to 100.0 g of PPMO#1 free base) is added and mixed well. Upon complete dissolution, the pH of the solution is confirmed to be 6.0-7.0, the osmolality to be 300-500 mOsm / kg, and the concentration of PPMO#1 is measured using an extinction coefficient of 23.1 at 260 nm in 1% ammonium hydroxide. The final solution was filtered through a 0.2 μm bioburden reducing filter. 4 mL of this solution was filled into vials, lyophilized, stoppered, and sealed to produce the final formulation vials.

[0136] Reconstitution of PPMO#1 hexahydrochloride formulation For each PPMO#1 hexahydrochloride formulation vial, 4 mL of sterile water for injection was added through the stopper, without a seal / stopper, using a needle / syringe. The contents of each vial were mixed by gentle swirling without shaking. Each vial was visually inspected to ensure the contents of each vial were completely dissolved. Gently inverting 5-10 times was recommended to ensure homogeneity. Upon reconstitution, PPMO#1 hexahydrochloride was a clear, colorless solution that may have some opalescence. The reconstituted formulation was then taken from the individual vials and diluted with 0.9% Sodium Chloride Injection (saline) for infusion.

[0137] analysis Samples were analyzed for appearance, moisture content, reconstitution time, and subvisible particle analysis. The appearance of the lyophilized cake and reconstituted solution were visually confirmed for clarity and the presence of any visible particles. The moisture content in the lyophilized cake was measured by coulometric Karl Fischer titration and found to be less than 1% based on duplicate measurements. Reconstitution time was determined by dissolving the contents of the vial in 4 mL of water for injection. Dissolution was achieved by gently swirling (not shaking) and inverting the vial after adding the water for injection and was found to be within 15 minutes. Light Obscuration Method USP <788> Subvisible particle analysis was performed using Method 1. A low volume (1 mL) method was used for this analysis, and all analyses met the USP criteria of <6000 particles / container for >10 μm and <600 particles / container for >25 μm.

[0138] Example 3 (100 mg / vial, polysorbate 80 variation) Preparation of PPMO#1 hexahydrochloride formulations with varying amounts of polysorbate 80 To 1 L of water for injection, 5.67 g of sodium citrate dihydrate and 0.24 g of citric acid monohydrate were added. To this citrate buffer, 40 g of D-mannitol (about 4 wt%) and 38 g of sucrose (about 3.8 wt%) were added. To this solution, four different levels of polysorbate 80 were added: 0 g (no polysorbate 80), 0.002 g (about 0.0002 wt%), 0.02 g (about 0.002 wt%), and 0.2 g (about 0.02 wt%) of polysorbate 80 to prepare four different solutions. 51.0 g of PPMO#1 hexahydrochloride (6HCl salt, equivalent to 50.0 g of PPMO#1 free base) was added to each solution and mixed well. The final components of each solution were: The results are shown in the table. Upon complete dissolution, the pH of the solution was determined to be 6.0-7.0, the osmolality to be 400-500 mOsm / kg, and the concentration of PPMO#1 was measured using an extinction coefficient of 23.1 at 260 nm in 1% ammonium hydroxide. The final solution was filtered through a 0.2 μm bioburden reducing filter. 2 mL of this solution was filled into vials, lyophilized, stoppered, and sealed to generate the final formulation vials.

[0139] [Table 8]

[0140] Reconstitution of PPMO#1 hexahydrochloride formulation For each PPMO#1 Hexahydrochloride formulation vial from Example 3, 4 mL of sterile water for injection was added through the stopper, without a seal / stopper, using a needle / syringe. The contents of each vial were mixed by gentle swirling without shaking. Each vial was visually inspected to ensure the contents of each vial were completely dissolved. Gently inverting 5-10 times is recommended to ensure homogeneity. Upon reconstitution, PPMO#1 Hexahydrochloride was a clear, colorless solution that may have some opalescence. The reconstituted formulation was then taken from each individual vial and diluted with 0.9% Sodium Chloride Injection (saline) to a concentration of approximately 20 mg / mL for infusion.

[0141] analysis Samples from Example 3 were analyzed for appearance, moisture content, reconstitution time, and subvisible particle analysis. The appearance of the lyophilized cake and reconstituted solution were visually confirmed for clarity and the presence of any visible particles. Moisture content in the lyophilized cake was measured by coulometric Karl Fischer titration. Reconstitution time was determined by dissolving the contents of the vial in 2 mL of water for injection. Dissolution was achieved by gently swirling (not shaking) and inverting the vial after adding the water. Light Obscuration Method USP <788> Subvisible particle analysis was performed using Method 1. A low volume (1 mL) method was used for this analysis.

[0142] These samples were subjected to accelerated stability conditions (40°C, 75% RH) for 8 weeks. There was no effect on the appearance and moisture content of the lyophilized cakes, however significant differences in reconstitution times and moderate differences in sub-visible particles were observed over time. The short reconstitution times and lack of formation of sub-visible particles over time can be attributed to the levels of polysorbate present in the solutions. The results from the accelerated stability conditions are summarized below. [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14]

[0143] Example 4 (100 mg / vial, sucrose and mannitol variations) Preparation of PPMO#1 hexahydrochloride formulations with varying amounts of sucrose and D-mannitol To 1 L of water for injection, 5.67 g of sodium citrate dihydrate and 0.24 g of citric acid monohydrate were added. Varying amounts of D-mannitol and sucrose were added to this citrate buffer to prepare four different solutions. To each solution, 0.02 g of polysorbate 80 (approximately 0.002 wt%) was added. 51.0 g of PPMO#1 hexahydrochloride (6HCl salt, equivalent to 50.0 g of PPMO#1 free base) was added to each solution and mixed well. The final ingredients of each solution in Example 4 are shown in the table. Upon complete dissolution, the pH of the solution was confirmed as 6.0-7.0 and the osmolality as 400-500 mOsm / kg. The concentration of PPMO#1 was measured using an extinction coefficient of 23.1 at 260 nm in 1% ammonium hydroxide. The final solutions were filtered through a 0.2 μm bioburden reducing filter. 2 mL of this solution was then filled into vials, lyophilized, stoppered and sealed to produce the final formulation vials. [Table 15]

[0144] Reconstitution of PPMO#1 Hexahydrochloride Formulation of Example 4 For each PPMO#1 Hexahydrochloride formulation vial from Example 4, 4 mL of sterile water for injection was added through the stopper, without a seal / stopper, using a needle / syringe. The contents of each vial were mixed by gentle swirling without shaking. Each vial was visually inspected to ensure that the contents of each vial were completely dissolved. Gently inverting 5-10 times is recommended to ensure homogeneity. Upon reconstitution, PPMO#1 Hexahydrochloride was a clear, colorless solution that may have some opalescence. The reconstituted formulation was then taken from each individual vial and diluted with 0.9% Sodium Chloride Injection (saline) to a concentration of approximately 20 mg / mL for infusion.

[0145] analysis Samples of Example 4 were analyzed for appearance, moisture content, reconstitution time, and subvisible particle analysis. The appearance of the lyophilized cake and reconstituted solution were visually confirmed for clarity and the presence of any visible particles. Moisture content in the lyophilized cake was measured by coulometric Karl Fischer titration. Reconstitution time was determined by dissolving the contents of the vial in 2 mL of water for injection. Dissolution was achieved by gently swirling (not shaking) and inverting the vial after adding the water. Light Obscuration Method USP <788> Subvisible particle analysis was performed using Method 1. A low volume (1 mL) method was used for this analysis.

[0146] These samples from Example 4 were subjected to accelerated stability conditions (40° C., 75% RH) for 4 weeks. There was no effect on the appearance of the lyophilized cake, moisture content, or reconstitution time, and sub-visible particles were observed over time. Moisture data is shown in Table 9. [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22]

Claims

1. 1. A composition comprising: a. an antisense oligonucleotide conjugate comprising a cell membrane penetrating peptide covalently bound to a nucleic acid analog, or a pharma- ceutically acceptable salt thereof, wherein the cell membrane penetrating peptide comprises at least two positively charged amino acids, or a pharma- ceutically acceptable salt thereof; b. one or more surfactants; c. one or more sugars; d. one or more buffering agents.

2. The composition of claim 1 , wherein the one or more surfactants are present in an amount of from about 0.001% to about 2% by weight of the composition.

3. The composition of claim 2, wherein the one or more surfactants are present in an amount of from about 0.002% to about 1% by weight of the composition.

4. 4. The composition of claim 2 or 3, wherein the one or more surfactants are selected from the group consisting of polyoxyethylene sorbitan monooleate (Polysorbate 80, Polysorbate 20), polyvinylpyrrolidone (PVP, Povidone, Poloxamer, Poloxamer F188), and mixtures thereof.

5. 5. The composition of any one of claims 1 to 4, wherein the one or more sugars are present in an amount of from about 0.1% to about 11% by weight of the composition.

6. 6. The composition of claim 5, wherein the one or more sugars are present in an amount of about 6% by weight.

7. 7. The composition of claim 5 or 6, wherein the one or more sugars are selected from the group consisting of trehalose, mannitol, sucrose, and mixtures thereof.

8. 8. The composition of claim 7, wherein the sugar is a mixture of mannitol and sucrose.

9. The composition of claim 7 , wherein the sugar is sucrose.

10. 10. The composition of any one of claims 1 to 9, wherein the one or more buffering agents are present in an amount sufficient to provide a pH of from about 6.0 to about 7.0 when the composition is dissolved or dispersed in an aqueous solvent.

11. The composition of claim 10, wherein the one or more buffering agents are present in an amount sufficient to provide a pH of about 6.3 to about 6.

5.

12. 12. The composition of claim 10 or 11, wherein the one or more buffering agents are present in the range of about 10 mM to about 50 mM when the composition is dissolved or dispersed in an aqueous solvent.

13. The composition of any one of claims 10 to 12, wherein the one or more buffering agents are selected from the group consisting of histidine, citrate, maleate, arginine, and mixtures thereof.

14. The composition of claim 13 , wherein the buffer is citrate.

15. 15. The composition of any one of claims 1 to 14, wherein the composition has an osmolality of about 250 mOsm / kg to about 650 mOsm / kg.

16. 16. The composition of claim 15, wherein the composition has an osmolality of about 300 mOsm / kg to about 500 mOsm / kg.

17. 16. The composition of claim 15, wherein the composition has an osmolality of about 400 mOsm / kg to about 600 mOsm / kg.

18. The composition according to any one of claims 1 to 17, wherein the composition is in a lyophilized form.

19. The composition of any one of claims 1 to 18, wherein the composition dissolves within 30 minutes after addition of an aqueous solvent.

20. The composition of any one of claims 1 to 19, wherein the composition dissolves within 15 minutes after addition of an aqueous solvent.

21. The composition of any one of claims 1 to 19, wherein the composition dissolves within 3 minutes after addition of an aqueous solvent.

22. The composition according to any one of claims 19 to 21, wherein the aqueous solvent used to dissolve the composition is sterile water.

23. The composition of any one of claims 1 to 22, wherein the composition comprises from about 50 mg to about 500 mg of the antisense oligonucleotide conjugate, or a pharma- ceutically acceptable salt thereof.

24. The composition of any one of claims 1 to 23, wherein the antisense oligonucleotide conjugate is capable of binding to a selected target and inducing exon skipping in the human dystrophin gene.

25. The composition of any one of claims 1 to 24, wherein the antisense oligonucleotide conjugate comprises a cell membrane penetrating peptide which is an arginine-rich peptide.

26. The arginine-rich peptide is -(RXR) ４ -R ａ (SEQ ID NO:54), R-(FFR) ３ -R ａ (SEQ ID NO: 55), -B-X-(RXR) ４ -R ａ (SEQ ID NO: 56), -B-X-R-(FFR) ３ -R ａ (SEQ ID NO: 57), -GLY-R-(FFR) ３ -R ａ (SEQ ID NO:58), -GLY-R ５ -R ａ (SEQ ID NO:59), -R ５ -R ａ (SEQ ID NO: 60), -GLY-R ６ -R ａ (SEQ ID NO:52) and -R ６ -R ａ (SEQ ID NO:53), ａ 26. The composition of claim 25, wherein: is selected from H, acyl, benzoyl, and stearoyl; R is arginine; X is 6-aminohexanoic acid; B is β-alanine; F is phenylalanine; and GLY (or G) is glycine.

27. The arginine-rich peptide is -GLY-R ５ -R ａ (SEQ ID NO:59), -R ５ -R ａ (SEQ ID NO: 60), -GLY-R ６ -R ａ (SEQ ID NO:52), or -R ６ -R ａ (SEQ ID NO:53), R is arginine, and R ａ The composition of claim 26, wherein is hydrogen or an acyl group.

28. 28. The composition of any one of claims 1 to 27, wherein the antisense oligonucleotide conjugate comprises a sequence complementary to 15 to 35 nucleobases of a target region of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of dystrophin pre-mRNA.

29. The antisense oligonucleotide conjugate has the formula (I): 【Chemical 77】 or a pharma- ceutically acceptable salt thereof, each Nu is a nucleobase which together form a targeting sequence, T, 【Chemical 78】 and 【Chemical Formula 79】 is a moiety selected from R １００ is the cell membrane-penetrating peptide, Each Nu, 1 to n, corresponds 5' to 3' to one of the following nucleobases: 【Table 23-1】 【Table 23-2】 A is 【Chemistry 80】 and C is 【Chemistry 81】 And G is 【Chemistry 82】 and T is 【Chemistry 83】 and U is 【Chemistry 84】 where Gm is a methylated guanine, Am is a methylated adenine, and mC is 【Chemistry 85】 The composition according to any one of claims 1 to 28,

30. The composition of claim 29, wherein each Nu from 1 to n and 5' to 3' of the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:17, or SEQ ID NO:

51.

31. The composition of claim 30, wherein each Nu from 1 to n and 5' to 3' of the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

1.

32. The composition of claim 30, wherein each Nu from 1 to n and 5' to 3' of the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

7.

33. The composition of claim 30, wherein each Nu from 1 to n and 5' to 3' of the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

17.

34. The composition of claim 30, wherein each Nu from 1 to n and 5' to 3' of the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

51.

35. T' of the antisense oligonucleotide conjugate of formula (I) is 【Chemistry 86】 The composition according to any one of claims 28 to 34, wherein

36. The antisense oligonucleotide conjugate has the formula (II): 【Chemistry 87】 or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to (n+1), corresponds 5' to 3' to one of the following nucleobases: 【Table 24-1】 【Table 24-2】 A is 【Chemistry 88】 in Yes, C is 【Chemistry 89】 And G is 【Chemistry 90】 and T is 【Chemistry 91】 and U is 【Chemistry 92】 where Gm is a methylated guanine, Am is a methylated adenine, and mC is 【Chemistry 93】 The composition according to any one of claims 1 to 35,

37. The composition of claim 36, wherein each of 1 to (n+1) Nu of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof corresponds, from 5' to 3', to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, or SEQ ID NO:

51.

38. The composition of claim 37, wherein each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

1.

39. The composition of claim 37, wherein each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

7.

40. The composition of claim 37, wherein each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

17.

41. The composition of claim 37, wherein each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of formula (II) or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

51.

42. The antisense oligonucleotide conjugate has the formula (III): 【Chemistry 94】 or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to (n+1), corresponds 5' to 3' to one of the following nucleobases: 【Table 25-1】 【Table 25-2】 A is 【Chemistry 95】 and C is 【Chemistry 96】 And G is 【Chemistry 97】 and T is 【Chemistry 98】 and U is 【Chemistry 99】 where Gm is a methylated guanine, Am is a methylated adenine, and mC is 【Chemistry 100】 The composition according to any one of claims 1 to 35,

43. The composition of claim 42, wherein each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:17, or SEQ ID NO:

51.

44. The composition of claim 43, wherein each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

1.

45. The composition of claim 43, wherein each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

7.

46. The composition of claim 43, wherein each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

17.

47. The composition of claim 43, wherein each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of formula (III) or a pharma- ceutically acceptable salt thereof corresponds to SEQ ID NO:

51.

48. The composition of any one of claims 1 to 47, wherein the antisense oligonucleotide conjugate is PPMO#1, PPMO#2, PPMO#3, or PPMO#4, or a pharma- ceutically acceptable salt thereof.

49. The composition of any one of claims 1 to 48, wherein the antisense oligonucleotide conjugate is in free base form.

50. The composition of any one of claims 1 to 48, wherein the antisense oligonucleotide conjugate is a pharma- ceutically acceptable salt.

51. 51. The composition of claim 50, wherein the antisense oligonucleotide conjugate is a halogen salt.

52. 52. The composition of claim 50 or 51, wherein the antisense oligonucleotide conjugate is an HCl salt.

53. 53. The composition of claim 52, wherein the HCl salt of the antisense oligonucleotide conjugate is a 1HCl, 2HCl, 3HCl, 4HCl, 5HCl, or 6HCl salt.

54. 54. The composition of claim 53, wherein the antisense oligonucleotide conjugate is the 6HCl salt form of PPMO#1, PPMO#2, PPMO#3, or PPMO#4.

55. 55. The composition of claim 54, wherein the antisense oligonucleotide conjugate is the 6HCl salt form of PPMO#1.

56. 55. The composition of claim 54, wherein the antisense oligonucleotide conjugate is the 6HCl salt form of PPMO#2.

57. 55. The composition of claim 54, wherein the antisense oligonucleotide conjugate is the 6HCl salt form of PPMO#3.

58. 55. The composition of claim 54, wherein the antisense oligonucleotide conjugate is the 6HCl salt form of PPMO#4.

59. A composition according to any one of claims 1 to 58 for use in a method for treating a human patient having Duchenne muscular dystrophy, the method comprising dissolving or dispersing the composition in an aqueous solvent.

60. 60. The composition of claim 59, wherein the amount of aqueous solvent used to dissolve or disperse the composition is sufficient to provide a concentration of the antisense oligonucleotide conjugate of about 20 mg / mL to about 200 mg / mL.

61. 61. The composition of claim 60, wherein the amount of aqueous solvent used to dissolve or disperse the composition is sufficient to provide a concentration of the antisense oligonucleotide conjugate of about 20 mg / mL to about 100 mg / mL.

62. 62. The composition of claim 61, wherein the amount of aqueous solvent used to dissolve or disperse the composition is sufficient to provide a concentration of the antisense oligonucleotide conjugate of about 50 mg / mL.

63. 62. The composition of claim 61, wherein the amount of aqueous solvent used to dissolve or disperse the composition is sufficient to provide a concentration of the antisense oligonucleotide conjugate of about 100 mg / mL.

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