Compositions for treating muscular dystrophy

Abstract

To provide a method for treating a human subject having a disease or disorder suitable for antisense oligonucleotide therapy comprising an effective amount of an antisense oligonucleotide or an antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof.SOLUTION: In some embodiments, nucleobases of the antisense oligonucleotide are linked to a morpholino ring structure. In some embodiments, morpholino subunits are connected by a phosphorus-containing intersubunit linkage that joins the morpholino nitrogen of one subunit to the 5'-exocyclic carbon of an adjacent morpholino subunit.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 835,836, filed April 18, 2019. The entire teachings of the above application are incorporated by reference in their entirety.

[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 March 19, 2020, is named 8164_51_WO00_SL.txt, and is 51,048 bytes in size.

[0003] The present invention relates to an improved method of treating diseases or disorders amenable to antisense oligonucleotide therapy, such as muscular dystrophy, in a human patient comprising an effective amount of an antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof. [Background technology]

[0004] Antisense technology has been developed using a variety of chemistries to affect gene expression at various levels (transcription, splicing, stability, translation). Most research has focused on the use of antisense compounds to correct or compensate for abnormal or disease-related genes in a wide range of indications. Antisense molecules can inhibit gene expression with specificity, and for this reason, many research efforts on oligomers as modulators of gene expression have focused on inhibiting target gene expression or the function of cis-acting elements. Antisense oligomers are typically directed against either the sense strand (e.g., mRNA) or, in the case of some viral RNA targets, the minus strand. To achieve the desired effect of downregulating a specific gene, oligomers generally promote the decay of the target mRNA, block mRNA translation, or block the function of a cis-acting RNA element, thereby effectively preventing either de novo synthesis of the target protein or viral RNA replication.

[0005] However, such techniques are not useful when the goal is to upregulate the production of a native protein or to compensate for mutations that induce premature termination of translation, such as nonsense or frameshift mutations. In these cases, the defective gene transcript should not be subject to targeted degradation or steric inhibition, and therefore the chemistry of the antisense oligomer should neither promote decay of the target mRNA nor block translation.

[0006] In various genetic disorders, the effect of mutations on the final expression of genes can be regulated through the process of targeted exon skipping during the splicing process. The splicing process is directed by a complex, multi-component mechanism that brings adjacent exon-intron junctions in pre-mRNA into close proximity and cleaves phosphodiester bonds at the ends of introns, with subsequent reconstitution of phosphodiester bonds between exons spliced ​​together. This complex and highly precise process is mediated by sequence motifs in pre-mRNA, which are relatively short, semi-conserved RNA segments, to which various nuclear splicing factors involved in the splicing reaction then bind. By altering the way the splicing machinery reads or recognizes motifs involved in pre-mRNA processing, it is possible to create differently spliced ​​mRNA molecules. It is recognized that the majority of human genes are alternatively spliced ​​during normal gene expression, but the mechanisms involved have not been identified. Bennett et al. (U.S. Patent No. 6,210,892) describe antisense modulation of wild-type cellular mRNA processing using antisense oligomeric analogs that do not induce RNAse H-mediated cleavage of the target RNA, which finds utility in alternatively generating spliced ​​mRNAs lacking specific exons.

[0007] When a normal functional protein is terminated prematurely due to a mutation within it, it has been shown that antisense technology can restore some functional protein production through intervention in the splicing process. It has also been shown that when an exon associated with a disease-causing mutation can be specifically deleted from some genes, a truncated protein product can sometimes be produced that has similar biological properties to the native protein or sufficient biological activity to ameliorate the disease caused by the exon-associated mutation. Kole et al. (U.S. Patent Nos. 5,627,274, 5,916,808, 5,976,879, and 5,665,593) disclose a method for countering aberrant splicing using modified antisense oligomer analogs that do not promote the decay of target pre-mRNA. Bennett et al. (U.S. Patent No. 6,210,892) similarly describe the antisense regulation of wild-type cellular mRNA processing using antisense oligomer analogs that do not induce RNAse H-mediated cleavage of target RNA.

[0008] The process of targeted exon skipping is likely to be particularly useful in long genes where there are many exons and introns, where there is redundancy in the genetic organization of exons, or where proteins can function without one or more specific exons. Efforts to redirect gene processing for the treatment of genetic diseases associated with truncations caused by mutations in various genes have focused on the use of antisense oligomers that (1) fully or partially overlap elements involved in the splicing process, or (2) bind to pre-mRNA at positions sufficiently close to the elements to prevent the binding and function of splicing factors that normally mediate the specific splicing reaction that occurs at that element.

[0009] Duchenne muscular dystrophy (DMD) is caused by defective expression of the protein dystrophin. The gene encoding the protein contains 79 exons spread across more than 2 million nucleotides of DNA. Mutations in any exon, characterized by changing the reading frame of the exon, introducing a stop codon, or removing an entire out-of-frame exon(s) or duplication of one or more exons, can prevent the production of functional dystrophin and result in DMD.

[0010] Mutations, typically deletions of one or more exons, that result in the correct reading frame along the entire dystrophin transcript and thus prevent premature termination of translation of the mRNA into protein have been shown to result in a less severe form of muscular dystrophy, Becker muscular dystrophy (BMD). 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 encodes a protein with a short internal deletion that retains some activity, resulting in the Becker phenotype. For many years, it has been known that the deletion of exon (s) that does not change the reading frame of dystrophin protein causes BMD phenotype, while the exon deletion that causes frameshift causes DMD.Generally, dystrophin mutations, including point mutations and exon deletions that change the reading frame and thus interrupt proper protein translation, lead to DMD.It should also be noted that some BMD and DMD patients have exon deletions that span multiple exons. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 6,210,892 [Patent Document 2] U.S. Patent No. 5,627,274 [Patent Document 3] U.S. Patent No. 5,916,808 Summary of the Invention [Problem to be solved by the invention]

[0012] Although at least one product is approved for the treatment of DMD, patients are in need of improved compositions and methods for treating muscular dystrophies, such as DMD and BMD. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows exon skipping in muscle biopsies over 28 days following a single IV injection of vehicle or 30 mg or 60 mg of PPMO#1 (as the 6HCl salt form) into cynomolgus monkeys. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Summary of the Invention] Provided is a method for treating a human patient with a disease or disorder suitable for antisense oligonucleotide therapy, comprising an effective amount of an antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof. In some embodiments, the nucleobase of the antisense oligonucleotide is linked to a morpholino ring structure. In some embodiments, the morpholino subunits are linked by a phosphorus-containing intersubunit bond that connects the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent morpholino subunit.

[0015] In certain embodiments, the antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to one or more cell membrane-permeable peptides (referred to herein as "CPPs"). In certain aspects, the nucleobases of the antisense oligonucleotide conjugate are linked to morpholino ring structures. In some aspects, the morpholino subunits are joined by phosphorus-containing intersubunit linkages connecting the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent morpholino subunit.

[0016] 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. In some embodiments, the arginine-rich peptide is -(RXR)4-R a (SEQ ID NO: 52), -R-(FFR)3-R a (SEQ ID NO: 53), -BX-(RXR)4-R a (SEQ ID NO: 54), -BXR-(FFR)3-R a (SEQ ID NO: 55), -GLY-R-(FFR)3-R a (SEQ ID NO: 56), -GLY-R5-R a (SEQ ID NO: 59), -R5-R a (SEQ ID NO: 60), -GLY-R6-R a (SEQ ID NO: 57) and -R6-R a (SEQ ID NO: 58), wherein 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.

[0017] A method for treating a human patient with Duchenne muscular dystrophy, the method comprising administering to the human patient a therapeutically effective amount of an antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, comprising a cell membrane-permeable peptide and an oligonucleotide, once every four weeks, wherein the antisense oligonucleotide or antisense oligonucleotide conjugate can bind to a selected target and induce exon skipping in the human dystrophin gene. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate induces skipping of a target region of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of dystrophin pre-mRNA. In certain embodiments, the human patient is administered the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof.

[0018] In some embodiments, the human patient is administered the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, for at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

[0019] In some embodiments, the method comprises administering an antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, comprising a cell membrane-permeable peptide that is an arginine-rich peptide attached to the oligonucleotide. In some embodiments, the antisense oligonucleotide conjugate comprises -GLY-R5-R a (SEQ ID NO: 59), -R5-R a (SEQ ID NO: 60), -GLY-R6-R a (SEQ ID NO: 57), or -R6-R a (SEQ ID NO: 58), wherein R is arginine, and R a is hydrogen or an acyl group.

[0020] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate is provided in a pharmaceutical composition formed by dissolving 0.005 mg / kg to about 300 mg / kg of the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof in an aqueous carrier solution.

[0021] In some embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof, is from about 0.005 mg / kg to about 300 mg / kg. In certain aspects, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof, is at least 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg. In certain embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is about 0.005 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 80 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 20 mg / kg to about 80 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 80 mg / kg, or about 80 mg / kg to about 300 mg / kg. In certain embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is about 0.05 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg.

[0022] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is administered intravenously.

[0023] A method for treating a human patient with Duchenne muscular dystrophy, the method comprising administering to the human patient a pharmaceutical composition comprising a therapeutically effective amount of an antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide conjugate comprises a cell membrane-permeable peptide and an oligonucleotide, or a pharmaceutically acceptable salt thereof, that induces skipping of a target region of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of dystrophin pre-mRNA. In certain embodiments, the human patient is administered the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof.

[0024] In certain embodiments, the antisense oligonucleotide conjugate comprises a cell membrane-permeable peptide that is an arginine-rich peptide. In some aspects, the antisense oligonucleotide conjugate comprises -(RXR)4-R a (SEQ ID NO: 52), -R-(FFR)3-R a (SEQ ID NO: 53), -BX-(RXR)4-R a (SEQ ID NO: 54), -BXR-(FFR)3-R a (SEQ ID NO: 55), -GLY-R-(FFR)3-R a (SEQ ID NO: 56), -GLY-R5-R a , -R5-R a , (SEQ ID NO: 59), -GLY-R6-R a( SEQ ID NO: 57), and -R6-R a (SEQ ID NO: 58), and R ais 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 antisense oligonucleotide conjugate comprises -R-R a (SEQ ID NO: 60) or -R6-R a (SEQ ID NO: 58), and R a is an acyl group. In some embodiments, the antisense oligonucleotide conjugate comprises -R6-R a (SEQ ID NO: 58), and R a is an acyl group. In some embodiments, the arginine-rich peptide is -GLY-R5-R a (SEQ ID NO: 59) or -GLY-R6-R a (SEQ ID NO: 57), and R a is an acyl group. In some embodiments, the arginine-rich peptide is -GLY-R6-R a , (SEQ ID NO: 57), and R a is an acyl group. In some embodiments, the antisense oligonucleotide conjugate comprises -R5-R a (SEQ ID NO: 60) or -R6-R a (SEQ ID NO: 58), and R a is H. In some embodiments, the antisense oligonucleotide conjugate is -R6-R a (SEQ ID NO: 58), and R a is H. In some embodiments, the arginine-rich peptide is -GLY-R5-R a (SEQ ID NO: 59) or -GLY-R6-R a (SEQ ID NO: 57), and R a is H. In some embodiments, the arginine-rich peptide is -GLY-R6-R a (SEQ ID NO: 57), and R a is H.

[0025] In some embodiments, the target region of the antisense oligonucleotide or antisense oligonucleotide conjugate is designated as an annealing site, and the base sequence and annealing site are selected from one of the following: [Table 1-1] [Table 1-2] Each T in SEQ ID NOs: 1 to 51 is thymine or uracil. In a specific embodiment, each T in the base sequence is thymine.

[0026] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate comprises a T' moiety attached to the 5' end of the nucleic acid analog, wherein the T' moiety is selected from the following: [ka] In the formula, R 200 is hydrogen or a cell membrane-permeable peptide, and R 1 is C1-C6 alkyl. In certain embodiments, R 200 is hydrogen.

[0027] In some embodiments, the nucleobase of the antisense oligonucleotide or the antisense oligonucleotide is linked to a morpholino ring structure. In certain aspects, the morpholino subunits are linked by a phosphorus-containing intersubunit linkage that connects the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent morpholino subunit.

[0028] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate is provided in a pharmaceutical composition formed by dissolving 0.005 mg / kg to about 300 mg / kg of the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof in an aqueous carrier solution.

[0029] In some embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof, is from about 0.005 mg / kg to about 300 mg / kg. In certain aspects, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof, is at least 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg. In certain embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is about 0.005 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 80 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 20 mg / kg to about 80 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 80 mg / kg, or about 80 mg / kg to about 300 mg / kg. In certain embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is about 0.05 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg.

[0030] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is administered intravenously.

[0031] 1. A method of treating a human patient with Duchenne muscular dystrophy, the method comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of an antisense oligonucleotide or antisense oligonucleotide conjugate according to formula (I). [ka] Formula (I) or a pharmaceutically acceptable salt thereof to a human patient; each Nu is a nucleobase that together form a targeting sequence, T' in formula (I) is a moiety selected from: [ka] R 100 and R 200 are each independently hydrogen or a cell membrane-permeable peptide; R 1 is C1-C6 alkyl, Each Nu from 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of the following: [Table 4-1] [Table 4-2] Each T in SEQ ID NOs: 1-51 is thymine or uracil, also provided herein. In certain embodiments, 1 to (n+1) and each Nu 5' to 3' of the antisense oligonucleotide or antisense oligonucleotide conjugate corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0032] In some embodiments, T' in the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (I) is [ka] is a moiety selected from R 100 is a cell membrane-penetrating peptide, and R 1 is C 1- In certain embodiments, T' in the antisense oligonucleotide conjugate of formula (I) is [ka] and R 100 is a cell membrane-permeable peptide.

[0033] In some embodiments, the cell membrane-permeable peptide of the antisense oligonucleotide conjugate is an arginine-rich peptide. In certain aspects, the arginine-rich peptide of the antisense oligonucleotide conjugate is -(RXR)4-R a (SEQ ID NO: 52), R-(FFR)3-R a (SEQ ID NO: 53), -BX-(RXR)4-R a (SEQ ID NO: 54), -BXR-(FFR)3-R a (SEQ ID NO: 55), -GLY-R-(FFR)3-R a (SEQ ID NO: 56), -GLY-R5-R a (SEQ ID NO: 59), -R5-R a (SEQ ID NO: 60), -GLY-R6-R a (SEQ ID NO: 57) and -R6-R a (SEQ ID NO: 58), and 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. In certain embodiments, the arginine-rich peptide of the antisense oligonucleotide conjugate is -R-R a (SEQ ID NO: 60) or -R6-R a (SEQ ID NO: 58), and R ais an acyl group. In certain embodiments, the arginine-rich peptide of the antisense oligonucleotide conjugate is -R6-R a (SEQ ID NO: 58), and R a is an acyl group. In certain embodiments, the arginine-rich peptide of the antisense oligonucleotide conjugate is -GLY-R5-R a (SEQ ID NO: 59) or -GLY-R6-R a (SEQ ID NO: 57), and R a is an acyl group. In certain embodiments, the arginine-rich peptide is -GLY-R6-R a (SEQ ID NO: 57), and R a is an acyl group. In some embodiments, the antisense oligonucleotide conjugate comprises -R5-R a (SEQ ID NO: 60) or -R6-R a (SEQ ID NO: 58), and R a is H. In certain embodiments, the antisense oligonucleotide conjugate is -R6-R a (SEQ ID NO: 58), and R a is H. In some embodiments, the arginine-rich peptide is -GLY-R5-R a (SEQ ID NO: 59) or -GLY-R6-R a (SEQ ID NO: 57), and R a is H. In certain embodiments, the arginine-rich peptide is -GLY-R6-R a (SEQ ID NO: 57), and R a is H.

[0034] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate is in free base form. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate is a pharmaceutically acceptable salt. In some embodiments, the antisense oligonucleotide conjugate is a halide salt (e.g., HCl salt). In some aspects, the antisense oligonucleotide conjugate is a monohalide, dihalide, trihalide, tetrahalide, pentahalide, or hexahalide salt. In certain aspects, the antisense oligonucleotide conjugate is an HCl salt. In certain aspects, the HCl salt of the antisense oligonucleotide or antisense oligonucleotide conjugate is 1HCl, 2HCl, 3HCl, 4HCl, 5HCl, or 6HCl salt. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate is provided as a mixture of free base and salt form.

[0035] In some embodiments, the antisense oligonucleotide is eteplirsen, golodirsen, or casimersen. In some embodiments, the antisense oligonucleotide conjugate is PPPMO#1, PPMO#2, or PPMO#3, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide conjugate is PPMO#1·6HCl, PPMO#2·6HCl, or PPMO#3·6HCl.

[0036] In some embodiments, the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof is provided in a pharmaceutical composition formed by dissolving 0.005 mg / kg to about 300 mg / kg of the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof in an aqueous carrier solution. In some embodiments, the pharmaceutical composition is formed by dissolving about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#1 in an aqueous carrier solution. In some embodiments, the pharmaceutical composition is formed by dissolving 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, or 100 mg / kg of PPMO#1·6HCl in an aqueous carrier solution. In some embodiments, the pharmaceutical composition is formed by dissolving about 60 mg / kg of a pharmaceutically acceptable salt of PPMO#1 in an aqueous carrier solution. In some embodiments, the pharmaceutical composition is formed by dissolving about 80 mg / kg of a pharmaceutically acceptable salt of PPMO#1 in an aqueous carrier solution. In some embodiments, the pharmaceutical composition is formed by dissolving about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#1 in an aqueous carrier solution. In some embodiments, the pharmaceutical composition is formed by dissolving about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#2 in an aqueous carrier solution. In some embodiments, the pharmaceutical composition is formed by dissolving about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#3 in an aqueous carrier solution.

[0037] In some embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof, is from about 0.005 mg / kg to about 300 mg / kg. In certain aspects, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof, is at least 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg. In certain embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is about 0.005 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 80 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 20 mg / kg to about 80 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 80 mg / kg, or about 80 mg / kg to about 300 mg / kg. In certain embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is about 0.05 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg.In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of PPMO#1 is about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of PPMO#1 is about 30 mg / kg, about 60 mg / kg, about 80 mg / kg, or about 100 mg / kg. In some embodiments, the therapeutically effective amount of the pharmaceutical composition is 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, or 100 mg / kg of PPMO#1·6HCl. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of PPMO#2 is about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of PPMO#3 is about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg.

[0038] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is administered intravenously.

[0039] 1. A method of treating a human patient with Duchenne muscular dystrophy, the method comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of an antisense oligonucleotide conjugate according to formula (II): [ka] (II) or a pharmaceutically acceptable salt thereof, wherein each Nu from 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of the following: [Table 5-1] [Table 5-2] In the formula, 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] In certain embodiments, each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of Formula (II) corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0040] In some embodiments, the antisense oligonucleotide conjugate is in free base form. In some embodiments, the antisense oligonucleotide conjugate is a pharmaceutically acceptable salt. In certain aspects, the antisense oligonucleotide is a halogenated salt. In certain aspects, the antisense oligonucleotide conjugate is a hexahalogenated salt. In certain aspects, the antisense oligonucleotide conjugate is an HCl salt. In certain aspects, the antisense oligonucleotide conjugate is a hexaHCl salt. In some embodiments, the antisense oligonucleotide conjugate is provided as a mixture of free base and salt forms.

[0041] In some embodiments, the antisense oligonucleotide conjugate is provided in a pharmaceutical composition formed by dissolving 0.005 mg / kg to about 300 mg / kg of the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof in an aqueous carrier solution.

[0042] In some embodiments, the therapeutically effective amount of the antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof, is from about 0.005 mg / kg to about 300 mg / kg. In certain aspects, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof, is at least 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg. In certain embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is about 0.005 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 80 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 20 mg / kg to about 80 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 80 mg / kg, or about 80 mg / kg to about 300 mg / kg. In certain embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is about 0.05 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg.In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of PPMO#1 is about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of PPMO#1 is about 30 mg / kg, about 60 mg / kg, about 80 mg / kg, or about 100 mg / kg. In some embodiments, the therapeutically effective amount of the pharmaceutical composition is 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, or 100 mg / kg of PPMO#1·6HCl. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of PPMO#2 is about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of PPMO#3 is about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg.

[0043] In some embodiments, the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof is administered intravenously.

[0044] 1. A method of treating a human patient with Duchenne muscular dystrophy, the method comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of an antisense oligonucleotide conjugate according to formula (IV): [ka] (IV) or a pharmaceutically acceptable salt thereof, wherein each Nu from 1 to (n+1) and from 5' to 3' corresponds to a nucleobase in one of the following: [Table 6-1] [Table 6-2] In the formula, 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] In certain embodiments, each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of formula (IV) corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0045] In some embodiments, the antisense oligonucleotide conjugate is in free base form. In some embodiments, the antisense oligonucleotide is in its pharmaceutically acceptable salt form. In some embodiments, the antisense oligonucleotide conjugate is in the form of a halide salt. In some embodiments, the antisense oligonucleotide conjugate is in the form of a hexahalide salt. In certain aspects, the antisense oligonucleotide conjugate is an HCl salt. In certain embodiments, the HCl salt is a 5HCl salt. In certain embodiments, the HCl salt is a 6HCl salt. In some embodiments, the antisense oligonucleotide conjugate is provided as a mixture of free base and salt forms.

[0046] In some embodiments, the antisense oligonucleotide conjugate is provided in a pharmaceutical composition formed by dissolving 0.005 mg / kg to about 300 mg / kg of the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof in an aqueous carrier solution.

[0047] In some embodiments, the therapeutically effective amount of the antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof, is from about 0.005 mg / kg to about 300 mg / kg. In certain aspects, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or pharmaceutically acceptable salt thereof, is at least 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg. In certain embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is about 0.005 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 80 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 20 mg / kg to about 80 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 80 mg / kg, or about 80 mg / kg to about 300 mg / kg. In certain embodiments, the therapeutically effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is about 0.05 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg.

[0048] In some embodiments, the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof is administered intravenously.

[0049] Provided herein is a method for treating a human patient with Duchenne muscular dystrophy, the method comprising administering to the human patient once every four weeks a therapeutically effective amount of an antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide conjugate comprises a cell membrane-permeable peptide covalently attached to the oligonucleotide, and wherein the antisense oligonucleotide conjugate or the pharmaceutically acceptable salt thereof induces exon skipping in the human dystrophin gene.

[0050] In some embodiments, the antisense oligonucleotide conjugate or pharmaceutically acceptable salt thereof comprises a cell membrane-permeable peptide that is an arginine-rich peptide. In some embodiments, the antisense oligonucleotide conjugate or pharmaceutically acceptable salt thereof comprises a peptide having the structure -GLY-R5-R a (SEQ ID NO: 59), -R5-R a (SEQ ID NO: 60), -GLY-R6-R a (SEQ ID NO: 57), or -R6-R a (SEQ ID NO: 58), wherein R is arginine, and R a is hydrogen or an acyl group.

[0051] In some embodiments, the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof induces skipping of a target region of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of dystrophin pre-mRNA. In some embodiments, the antisense oligonucleotide conjugate is described by Formula (I): [ka] Formula (I) or a pharmaceutically acceptable salt thereof; each Nu is a nucleobase that together form a targeting sequence, T' in formula (I) is a moiety selected from: [ka] R 100 is a cell membrane-penetrating peptide, R 200 is hydrogen, R 1 is C1-C6 alkyl, Each Nu from 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of the following: [Table 7-1] [Table 7-2] T in each of SEQ ID NOs: 1 to 51 is thymine or uracil.

[0052] In some embodiments, each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate or pharmaceutically acceptable salt thereof corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0053] In some embodiments, the antisense oligonucleotide conjugate is PPPMO#1, PPMO#2, or PPMO#3, or a pharmaceutically acceptable salt thereof.

[0054] In some embodiments, the method comprises administering to a human patient a therapeutically effective amount of an antisense oligonucleotide conjugate according to Formula (II): [ka] (II) or a pharmaceutically acceptable salt thereof, wherein each Nu from 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of the following: [Table 9-1] [Table 9-2] In the formula, 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] is.

[0055] In some embodiments, each Nu from 1 to (n+1) and 5' to 3' of the antisense oligonucleotide conjugate of Formula (II) or a pharmaceutically acceptable salt thereof corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0056] In some embodiments, the antisense oligonucleotide conjugate is in free base form.

[0057] In some embodiments, the antisense oligonucleotide conjugate is a pharmaceutically acceptable salt.

[0058] In some embodiments, the antisense oligonucleotide conjugate is a halide salt.

[0059] In some embodiments, the antisense oligonucleotide conjugate is an HCl salt. In some embodiments, the HCl salt of the antisense oligonucleotide conjugate is a 6HCl salt.

[0060] In some embodiments, a therapeutically effective amount of the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof is provided in a pharmaceutical composition formed by dissolving 0.005 mg / kg to about 300 mg / kg of the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, in an aqueous carrier solution. In some embodiments, a therapeutically effective amount of an antisense oligonucleotide conjugate is at least 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg, about 0.005 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 80 mg / kg, about 0.1 mg / kg to about 50 mg / kg , about 0.1 mg / kg to about 25 mg / kg, about 20 mg / kg to about 80 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 80 mg / kg, about 80 mg / kg to about 300 mg / kg, about 0.05 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg.

[0061] In some embodiments, the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof is administered intravenously. [Mode for Carrying Out the Invention]

[0062] Embodiments of the present invention relate to improved methods for treating diseases or disorders amenable to antisense oligonucleotide therapy by administering an effective amount of an antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof. In some embodiments, exon skipping is induced by administering an effective amount of an antisense oligonucleotide that is a phosphorodiamidate morpholino oligonucleotide (PMO) or an antisense oligonucleotide that is a PMO conjugated to a cell membrane-penetrating peptide (PPMO), or a pharmaceutically acceptable salt thereof, which selectively binds to a target sequence.In some embodiments, the invention relates to a method of treating a disease or disorder amenable to antisense oligonucleotide therapy, wherein an effective amount of an antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO) or a pharmaceutically acceptable salt thereof is administered in an amount of at least about 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg, about 0.005 mg / kg to about 200 mg / kg. , about 0.1 mg / kg to about 100.0 mg / kg, about 0.1 mg / kg to about 80 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 20 mg / kg to about 80 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 80 mg / kg, or about 80 mg / kg to about 300 mg / kg, about 0.05 mg / kg, about 0.3 mg / kg, about 1 mg / kg , about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg. In some embodiments, the antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO), or a pharmaceutically acceptable salt thereof, is administered once every 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the antisense oligonucleotide conjugate (e.g., PPMO) or a pharmaceutically acceptable salt thereof, is administered once every 4 weeks.In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of PPMO#1·6HCl dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 60 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 80 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#2 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#3 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, the antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO), or a pharmaceutically acceptable salt thereof, is administered monthly.

[0063] Examples of such diseases or disorders that are suitable for antisense oligonucleotide therapy include muscular dystrophy, such as DMD and BMD, by administering the antisense compound specifically designed to induce exon skipping in human dystrophin gene.Dystrophin plays an important role in muscle function, and various muscle-related diseases are characterized by the mutant form of this gene.Therefore, in certain embodiments, the improved method described herein can be used to induce exon skipping in the mutant form of human dystrophin gene, such as the mutant dystrophin gene found in DMD and BMD.

[0064] 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 causes various forms of muscular dystrophy.To treat this condition, the antisense oligonucleotide or antisense oligonucleotide conjugate 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 " wild type " form of dystrophin, but rather truncated but functional or semi-functional form of dystrophin.

[0065] 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, which are characterized by defective expression of dystrophin protein due to abnormal mRNA splicing. The methods described herein further provide improved treatment options for patients with muscular dystrophies and offer significant and practical advantages over alternative methods of treating related forms of muscular dystrophies. For example, in some embodiments, the methods involve administering antisense compounds to induce exon skipping in the human dystrophin gene for longer periods than previous approaches.

[0066] Thus, in one embodiment, the present invention relates to a method for treating muscular dystrophies, such as DMD and BMD, by inducing exon skipping in a human patient. In some embodiments, exon skipping is induced by administering an effective amount of an antisense oligonucleotide that is a phosphorodiamidate morpholino oligonucleotide (PMO), or an antisense oligonucleotide that is a PMO conjugated to a cell membrane-permeable peptide (PPMO), or a pharmaceutically acceptable salt thereof, which selectively binds to a target sequence in an exon of a dystrophin pre-mRNA. In some embodiments, the present invention relates to a method of treating DMD or BMD, wherein an effective amount of an antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO) or a pharmaceutically acceptable salt thereof is administered in an amount of at least about 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg, about 0.005 mg / kg to about 200 mg / kg, about 0.1 mg / kg kg~about 100mg / kg, about 0.1mg / kg~about 80mg / kg, about 0.1mg / kg~about 50mg / kg, about 0.1mg / kg~about 25mg / kg, about 20mg / kg~about 80mg / kg, about 50mg / kg ~ approx. 100mg / kg, approx. 50mg / kg ~ approx. 80mg / kg, approx. 80mg / kg ~ approx. 300mg / kg, approx. 0.05mg / kg, approx. 0.3mg / kg, approx. 1mg / kg, approx. 2mg / k g, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg.In some embodiments, the antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO), or a pharmaceutically acceptable salt thereof, is administered once every 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the antisense oligonucleotide conjugate (e.g., PPMO) or a pharmaceutically acceptable salt thereof is administered once every 4 weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every 4 weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of PPMO#1·6HCl dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 60 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 80 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#2 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#3 dissolved in an aqueous carrier solution is administered every four weeks.In some embodiments, the antisense oligonucleotide (eg, PMO) or antisense oligonucleotide conjugate (eg, PPMO), or a pharmaceutically acceptable salt thereof, is administered monthly. I. Definition

[0067] In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly defined herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0068] It is understood that the term "a" or "an" entity refers to one or more of that entity, e.g., "a nucleotide sequence" refers to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0069] Furthermore, when used herein, "and / or" is to be understood as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or C; A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0070] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0071] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "TGA(5'-3')" is complementary to the sequence "TCA(5'-3')." Complementarity can be "partial," in which only some of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity is often desirable, some embodiments can include one or more mismatches, preferably 6, 5, 4, 3, 2, or 1 mismatch, relative to the target RNA. Variations at any position within the oligomer are included. In certain embodiments, sequence variations near the ends of the oligomer are generally preferred over variations within the oligomer, and, if present, are typically located within about 6, 5, 4, 3, 2, or 1 nucleotide of the 5' and / or 3' terminus.

[0072] The terms "antisense oligomer," "antisense compound," and "antisense oligonucleotide" are used interchangeably and refer to a sequence of cyclic subunits joined by intersubunit linkages that have base-pairing moieties and hybridize to a target sequence in a nucleic acid (typically RNA) by Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex within the target sequence. The cyclic subunits are based on ribose or another pentose sugar, or, in preferred embodiments, morpholino groups (see discussion of morpholino oligomers below). The oligomer can have exact or approximate sequence complementarity to the target sequence, with sequence variations near the ends of the oligomer generally being preferable to variations within the oligomer.

[0073] Such antisense oligomers can be designed to block or inhibit mRNA translation or inhibit natural pre-mRNA splicing processing and are said to be "directed" or "target" the target sequence to which they hybridize. The target sequence is typically a region containing the AUG start codon of an mRNA, a translation suppressor oligomer, or a splice site or splice suppressor oligomer (SSO) in pre-processed mRNA. Splice site target sequences can include mRNA sequences downstream of the normal splice acceptor junction of the processed mRNA, from 1 to approximately 25 base pairs at its 5' end. Preferred target sequences are any region of the processed mRNA that includes the splice site, is contained entirely within the exon coding sequence, or spans the splice acceptor or donor site. When an oligomer targets a target nucleic acid in the manner described above, it is more generally said to "target" a biological target, such as a protein, virus, or bacterium.

[0074] The terms "antisense oligomer conjugate" and "antisense oligonucleotide conjugate" are used interchangeably and refer to an antisense oligonucleotide bound to a cell membrane-permeable peptide.

[0075] The terms "cell penetrating peptide" and "CPP" are used interchangeably and refer to cationic cell membrane-penetrating peptides, also referred to as transport peptides, carrier peptides, or peptide transduction domains. As demonstrated herein, the peptides have the ability to induce cell membrane penetration within 100% of cells in a given cell culture population, enabling macromolecular translocation within multiple tissues in vivo upon systemic administration. Preferred CPP embodiments are arginine-rich peptides, as further described below.

[0076] The terms "morpholino," "morpholino oligomer," and "PMO" refer to the following general structure: [ka] The term "morpholino" refers to the phosphorodiamidate morpholino oligomer as described in Figure 2 of Summerton, J., et al., Antisense & Nucleic Acid Drug Development, 7: 187-195 (1997). The morpholino described herein includes all stereoisomers and tautomers of the above general structure. The synthesis, structure and binding properties of morpholino oligomers 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, each of which is incorporated herein by reference in its entirety.

[0077] In certain embodiments, the morpholino is conjugated to a "tail" moiety at the 5' or 3' end of the oligomer to enhance its stability and / or solubility. Exemplary tails include: [ka] In the formula, R 200 is hydrogen, or cell membrane-permeable peptide R 1 is C1-C6 alkyl.

[0078] In one embodiment, the exemplary tail moiety, "TEG" or "EG3," refers to the following tail moieties: [ka]

[0079] In one embodiment, the exemplary tail moiety, "GT," refers to the following tail moiety: [ka]

[0080] 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 peptide moieties attached to the antisense oligonucleotides of the present disclosure. In various embodiments, "G" represents a glycine residue attached to "R5 (SEQ ID NO: 60)" by an amide bond, and each "R" represents an arginine residue linked together by an amide bond, thereby "R5" refers to five arginine residues (SEQ ID NO: 60) linked together by amide bonds. 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 and L-arginine residues. In certain embodiments, "-G-R5 (SEQ ID NO: 59)" or "-G-R5-Ac (SEQ ID NO: 59)" is linked to the distal -OH or NH2 of the "tail" portion. In certain embodiments, "-G-R5 (SEQ ID NO: 59)" or "-G-R5-Ac (SEQ ID NO: 59)" is attached to the morpholine ring nitrogen of the 3'-most morpholino subunit of a PMO antisense oligonucleotide of the present disclosure. In some embodiments, "-G-R5 (SEQ ID NO: 59)" or "-G-R5-Ac (SEQ ID NO: 59)" is attached to the 3' end of an antisense oligonucleotide of the present disclosure and is of the formula: [ka] or a pharmaceutically acceptable salt thereof, or [ka]

[0081] As used herein, the terms "-G-R6 (SEQ ID NO: 57)," "-G-R6-Ac (SEQ ID NO: 57)," and "R6G (SEQ ID NO: 57)" are used interchangeably and refer to peptide moieties attached to antisense oligonucleotides of the present disclosure. In various embodiments, "G" represents a glycine residue attached to "R6" (SEQ ID NO: 58) by an amide bond, and each "R" represents an arginine residue attached together by an amide bond, thereby "R6" refers to six arginine residues (SEQ ID NO: 58) attached together by amide bonds. 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- and L-arginine residues. In certain embodiments, "-G-R6 (SEQ ID NO: 57)" or "-G-R6-Ac (SEQ ID NO: 57)" is linked to the distal -OH or -NH of the "tail" portion. In certain embodiments, "-G-R6 (SEQ ID NO: 57)" or "-G-R6-Ac (SEQ ID NO: 57)" is attached to the morpholine ring nitrogen of the 3'-most morpholino subunit of a PMO antisense oligonucleotide of the present disclosure. In some embodiments, "-G-R6 (SEQ ID NO: 57)" or "-G-R6-Ac (SEQ ID NO: 57)" is attached to the 3' end of an antisense oligonucleotide of the present disclosure and is of the formula: [ka]

[0082] The terms "nucleobase" (Nu), "base-pairing moiety," or "base" are used interchangeably and refer to 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 oligomers. Exemplary analogs include hypoxanthine (the base component of inosine), 2,6-diaminopurine, 5-methylcytosine, C5-propynyl-modified pyrimidines, 10-(9-(aminoethoxy)phenoxazinyl) (G-clamp), methyladenine ("Am"), methylguanine ("Gm"), and the like.

[0083] 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 being natural degradation 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.

[0084] 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 oligomers described herein. Examples of extended size nucleobases include those shown below, as well as tautomers thereof: [ka] [ka]

[0085] The term "PPMO" refers to a PMO conjugated to a cell membrane-permeable peptide.

[0086] "Eteplirsen," also known as "AVN-4658," is a PMO having the base sequence 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 1). Eteplirsen is registered under CAS Registry Number 1173755-55-9. Chemical names include: [P-Deoxy-P-(dimethylamino)](2',3'-dideoxy-2',3'-imino-2',3'-seco)(2'a→5')(C-mU-CCAACA-mU-CAAGGAAGA-mU-GGCA-mU-mU-mU-mU-C-mU-AG) (SEQ ID NO: 61), 5'-[P-[4-[[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]carbonyl]-1-piperazinyl]-N,N-dimethylphosphonamidate] and P,2',3'-trideoxy-P-(dimethylamino)-5'-O-{P-[4-(10-hydroxy-2,5,8-trioxadecanoyl)piperazin-1-yl]-N,N-dimethylphosphonamidyl}-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,2', 3'-Trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,2',3'-trideoxy- P-(Dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2' ,3'-Imino-2',3'-secoadenylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoguanylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoguanylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-3'-Secoadenylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoguanylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,2',3'-to P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoguanylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoguanylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino No-2',3'-secoadenylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,2',3'-trideo Examples of such compounds include 2'a→5'-oxy-P-(dimethylamino)-2',3'-imino-2',3'-secocytidylyl-(2'a→5')-P,3'-dideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secothymidylyl-(2'a→5')-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-secoadenylyl-(2'a→5')-2',3'-dideoxy-2',3'-imino-2',3'-secoguanosine.

[0087] Eteplirsen has the following structure (SEQ ID NO: 1 is disclosed below): [ka]

[0088] "PPMO#1" has the following structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, PPMO#1 is in the form of a halide salt. In some embodiments, PPMO#1 is in the form of a hexahalide salt. In some embodiments, PPMO#1 is in the form of an HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a hexaHCl salt.

[0089] "Golodirsen," also known by its code name "SRP-4053," is a PMO having the base sequence 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 7). Golodirsen is registered under CAS Registry Number 1422959-91-8. Its chemical name includes all-P-ambo-[P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-seco](2'a→5')(GTTGCCTCCGGTTCTGAAGGT-GTTC) (SEQ ID NO: 7) 5'-[4-({2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}carbonyl)-N,N-dimethylpiperazine-1-phosphonamidate].

[0090] Golodirsen has the following structure (SEQ ID NO: 7 is disclosed below): [ka]

[0091] "PPMO#2" has the following structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, PPMO#2 is in the form of a halide salt. In some embodiments, PPMO#2 is in the form of a hexahalide salt. In some embodiments, PPMO#2 is in the form of an HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a hexaHCl salt.

[0092] "Casimersen," also known by its code name "SRP-4045," is a PMO having the base sequence 5'- CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 17). Casimersen is registered under CAS Registry Number 1422959-91-8. Chemical names include: The chemical name is all-P-ambo-[P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-seco](2'a→5')(CAATGCCATCCTGGAGTTCCT-G) SEQ ID NO: 17) 5'-[4-({2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}carbonyl)-N,N-dimethylpiperazine-1-phosphonamidate]

[0093] Casimersen has the following chemical structure (SEQ ID NO: 17 is disclosed below): [ka]

[0094] "PPMO#3" has the following structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, PPMO#3 is in the form of a halide salt. In some embodiments, PPMO#3 is in the form of a hexahalide salt. In some embodiments, PPMO#3 is in the form of an HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a hexaHCl salt.

[0095] An "amino acid subunit" or "amino acid residue" is an α-amino acid residue (‐CO-CHR 10 -NH-), or β- or other amino acid residues (e.g., -CO-(CH2) n CHR 10 -NH-), and R 10 is a side chain (which may contain hydrogen), and n is 1 to 6, preferably 1 to 4.

[0096] The term "naturally occurring amino acid" refers to an amino acid that occurs in naturally occurring proteins. The term "unnatural amino acid" refers to an amino acid that does not occur in naturally occurring proteins, examples of which include beta-alanine (β-Ala), 6-aminohexanoic acid (Ahx), and 6-aminopentanoic acid.

[0097] "Exon" refers to a defined section of a nucleic acid that encodes a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after any portion of a pre-processed (or precursor) RNA is removed by splicing. The mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA such as rRNA or tRNA. The human dystrophin gene has approximately 79 exons.

[0098] "Intron" refers to a region of nucleic acid (within a gene) that is not translated into protein. Introns are non-coding sections that are transcribed into precursor mRNA (pre-mRNA) and then spliced ​​out during formation of the mature RNA.

[0099] An "effective amount" or "therapeutically effective amount" refers to the amount of a therapeutic compound, such as an antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, administered to a human patient, either as a single dose or as part of a series, effective to produce a desired therapeutic effect. For antisense oligonucleotides or antisense oligonucleotide conjugates, this effect is generally achieved by inhibiting translation or natural splice processing of a selected target sequence. In some embodiments, an effective amount is at least 0.05 mg / kg of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof. In some embodiments, an effective amount is from about 0.005 mg / kg to about 300 mg / kg. In some embodiments, the therapeutically effective amount is at least 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg. In some embodiments, the therapeutically effective amount is about 0.005 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 80 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 20 mg / kg to about 80 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 80 mg / kg, or about 80 mg / kg to about 300 mg / kg.In some embodiments, the therapeutically effective amount is about 0.05 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg.

[0100] " Exon skipping " generally refers to the process in which an entire exon or a part thereof is removed from a given pre-processed RNA, thereby excluding it from being present in mature RNA, such as the mature mRNA that is translated into protein.Therefore, the part of the protein coded by the skipped exon is not present in the expressed protein form, and typically produces a modified form of protein that is still functional.In certain embodiments, the skipped exon is an abnormal exon from human dystrophin gene, and may contain mutations or other changes in sequence that otherwise cause abnormal splicing.In certain embodiments, the skipped exon is exon 44, 45, 50, 51, 52 or 53 of human dystrophin gene.

[0101] Dystrophin is a rod-shaped cytoplasmic protein that is an integral part of a protein complex that connects the cytoskeleton of muscle fibers through the cell membrane to the surrounding extracellular matrix. Dystrophin contains multiple functional domains. For example, it contains an actin-binding domain at approximately amino acids 14–240 and a central rod domain at approximately amino acids 253–3040. This large central domain is formed by 24 spectrin-like triple-helical elements of approximately 109 amino acids that share homology with α-actinin and spectrin. Repeats are typically interrupted by four proline-rich non-repeat segments, also known as hinge regions. Repeats 15 and 16 are separated by an 18-amino acid stretch that appears to provide the primary site for dystrophin proteolytic cleavage. Sequence identity between most repeats ranges from 10–25%. Each repeat contains three α-helices: 1, 2, and 3. α-helices 1 and 3 are each formed by seven helical turns, which likely interact as a coiled coil via a hydrophobic interface. α-helix 2 has a more complex structure, formed by segments of four and three helical turns separated by glycine or proline residues. Each repeat is encoded by two exons, typically interrupted by an intron between amino acids 47 and 48 in the first part of α-helix 2. Other introns are usually found at different positions within the repeat, scattered across helix 3. Dystrophin also contains a cysteine-rich domain at approximately amino acids 3080–3360, including a cysteine-rich segment (i.e., 15 cysteines out of 280 amino acids), which shows homology to the C-terminal domain of Dictyostelium discoideum α-actinin. The carboxy-terminal domain is located at approximately amino acids 3361–3685.

[0102] The amino terminus of dystrophin binds to F-actin, and the carboxy terminus binds to the dystrophin-associated protein complex (DAPC) in the sarcolemma. DAPC includes dystroglycan, sarcoglycan, integrin, and caveolin, and mutations in any of these components cause autosomal inherited muscular dystrophies. DAPC is destabilized in the absence of dystrophin, resulting in decreased levels of its member proteins and leading to progressive fiber damage and membrane leakage. In various forms of muscular dystrophy, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), muscle cells produce altered and functionally defective forms of dystrophin or no dystrophin at all, primarily due to mutations in the gene sequence that result in incorrect splicing. Predominant expression of defective dystrophin protein or the complete lack of dystrophin or dystrophin-like proteins leads to the rapid progression of muscle degeneration, as described above. In this regard, a "defective" dystrophin protein may be characterized by the form of dystrophin produced in a particular patient with DMD or BMD, or by the absence of detectable dystrophin, as known in the art.

[0103] As used herein, terms such as "function" and "functionality" refer to biological, enzymatic, or therapeutic functions.

[0104] A "functional" dystrophin protein generally refers to a dystrophin protein that has sufficient biological activity to reduce the progressive degradation of muscle tissue, which is otherwise characteristic of muscular dystrophy, compared to the "altered" or "missing" forms of dystrophin present in certain patients with DMD or BMD. In certain embodiments, a functional dystrophin protein may have approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers therebetween) of the in vitro or in vivo biological activity of wild-type dystrophin, as measured according to conventional techniques in the art. As an example, dystrophin-related activity in in vitro muscle cultures can be measured according to myotube size, myofibril organization (or disorganization), contractile activity, and spontaneous clustering of acetylcholine receptors. Animal models are also valuable resources for studying the pathogenesis of diseases and provide a means for testing 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. These and other animal models can be used to measure the functional activity of various dystrophin proteins. This includes truncated forms of dystrophin, such as those produced by specific exon-skipping antisense oligonucleotides or antisense oligonucleotide conjugates.

[0105] The term "restoration" of dystrophin synthesis or production generally refers to the production of dystrophin protein, including truncated dystrophin, in a human patient with muscular dystrophy after treatment with the antisense oligonucleotide or antisense oligonucleotide conjugate described herein or its pharmaceutically acceptable salt.The proportion of dystrophin-positive fibers in a human patient 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 human patient.

[0106] Analysis of the proportion of positive dystrophin fibers can be performed before and / or after treatment, or at time points throughout the course of treatment. In some embodiments, a post-treatment biopsy is taken from the muscle contralateral to the pre-treatment biopsy. Pre- and post-treatment dystrophin expression analysis can be performed using any suitable dystrophin assay. In one embodiment, immunohistochemical detection is performed on tissue sections from muscle biopsies using an antibody that is a marker for dystrophin, such as a monoclonal or polyclonal antibody. For example, the MANDYS106 antibody, a highly sensitive marker for dystrophin, can be used. Any suitable secondary antibody can be used.

[0107] In some embodiments, the percentage of dystrophin-positive fibers is calculated by dividing the number of positive fibers by the total number of fibers counted. Normal muscle samples have 100% dystrophin-positive fibers. Therefore, the percentage of dystrophin-positive fibers can be expressed as a percentage of normal. When counting dystrophin-positive fibers in post-treatment muscle, a baseline can be established using pre-treatment muscle sections from each patient to control for the presence of trace levels of dystrophin in pre-treatment muscle as well as revertant fibers. This can be used as a threshold for counting dystrophin-positive fibers in that patient's post-treatment muscle sections. 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. Furthermore, the percentage of dystrophin-positive fibers can be determined using Western blot analysis using monoclonal or polyclonal anti-dystrophin antibodies. For example, Novacastra's anti-dystrophin antibody NCL-Dys1 can be used. The proportion of dystrophin-positive fibers can also be analyzed by determining the expression of components of the sarcoglycan complex (β, γ) and / or neuronal NOS.

[0108] In some embodiments, treatment with antisense oligonucleotides or antisense oligonucleotide conjugates slows or reduces the progressive respiratory muscle dysfunction and / or failure in DMD patients that would be expected without treatment.In one embodiment, treatment with antisense oligonucleotides or antisense oligonucleotide conjugates can reduce or eliminate the need for ventilatory support that would be expected without treatment.In one embodiment, measurements of respiratory function to track the course of disease 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 weakness.

[0109] In one embodiment, MEP may decline before changes in other pulmonary function tests, including MIP and FVC. In another embodiment, MEP may be an early indicator of respiratory dysfunction. In another embodiment, FVC may be used to measure the total volume of air expelled during forced expiration after maximal inspiration. In DMD patients, FVC increases concurrently 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 approximately 8 to 8.5 percent per year after age 10 to 12. In another embodiment, 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.

[0110] "Isolated" refers to material that is substantially or essentially free from components that normally accompany it in its native state. For example, as used herein, an "isolated polynucleotide" can refer to a polynucleotide that has been purified or removed from the sequence in which it occurs in nature, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment.

[0111] As used herein, "sufficient length" refers to an antisense oligonucleotide or antisense oligonucleotide conjugate that is complementary to at least 8, more typically 8 to 30 consecutive nucleobases in the target dystrophin pre-mRNA. In some embodiments, a sufficiently long antisense oligonucleotide comprises at least 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleobases in the target dystrophin pre-mRNA. In other embodiments, a sufficiently long antisense oligonucleotide comprises at least 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleobases in the target dystrophin pre-mRNA. A sufficiently long antisense oligonucleotide or antisense oligonucleotide conjugate has at least the minimum number of nucleotides capable of specifically hybridizing to any one or more exons 1 to 79 of the dystrophin gene. Preferably, the antisense oligonucleotide or antisense oligonucleotide conjugate has the minimum number of nucleotides capable of specifically hybridizing to one or more exons and inducing exon skipping. In one embodiment, the antisense oligonucleotide or antisense oligonucleotide conjugate has a minimum number of nucleotides capable of specifically hybridizing to and inducing skipping of any one or more of exons 44, 45, 50, 51, 52, or 53 of the human dystrophin gene.

[0112] "Enhance" or "enhancing," or "increase" or "increasing," or "stimulate" or "stimulating" generally refer to the ability of one or an antisense oligonucleotide or antisense oligonucleotide conjugate to produce or cause a greater physiological response (i.e., a downstream effect) in a cell or subject compared to the response caused by either no antisense oligonucleotide, no antisense oligonucleotide conjugate, or a control compound in the cell or subject. A measurable 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. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points therebetween and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the amount produced by no antisense compound (absence of agent) or a control compound.

[0113] The terms "reduce" or "inhibit" generally refer to the ability of one or more antisense oligonucleotides or antisense oligonucleotide conjugates of the present invention to "reduce" a relevant physiological or cellular response, such as the symptoms of a disease or condition described herein, as measured according to routine techniques in the diagnostic arts. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in the symptoms or pathology of the particular disease or disorder being treated. For example, the relevant physiological or cellular response may include a reduction in the symptoms or pathology of muscular dystrophy, or a reduction in the expression of defective forms of dystrophin, such as the modified forms of dystrophin expressed in individuals with DMD or BMD.

[0114] "Treatment" of an individual (e.g., a mammal, such as a human) or cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be administered 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 disorder. For example, treatment includes any desired effect on the symptoms or pathology of a disease or disorder associated with the dystrophin protein, such as in certain forms of muscular dystrophy, and may include, for example, a minimal change or improvement in one or more measurable markers of the disease or condition being treated. Also included is "prophylactic" treatment, which may be aimed at reducing the rate of progression of the disease or disorder being treated, delaying the onset of the disease or disorder, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0115] In one embodiment, treatment with antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt increases new dystrophin production, and delays or reduces the ambulation disorder that would be expected without treatment.For example, treatment can stabilize, maintain, improve or increase the walking ability (for example, stabilization of walking) of human patients.In some embodiments, treatment maintains or increases the stable walking distance of human patients, as measured by the 6-minute walk test (6MWT) described by, for example, 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 absolute value, percentage change or percent predicted value change.

[0116] The loss of muscle function in DMD patients may occur against the backdrop of normal childhood growth and development. Indeed, children with DMD may demonstrate an increase in walking distance during the 6MWT over the course of approximately one year, despite progressive muscle impairment. In some embodiments, 6MWD from patients with DMD is compared to existing normative data from typically developing control subjects and age- and sex-matched subjects (i.e., patients). In some embodiments, normal growth and development may be accounted for using an age- and height-based equation fitted to normative data. Such an equation can be used to convert 6MWD in patients with DMD into a percent predicted (% predicted) value. In certain 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., age 7 or younger) represent a stabilization of performance rather than an improvement in performance in DMD patients (Henricson et al. PLoS Curr., 2012, version 2, incorporated herein by reference).

[0117] As used herein, a "pediatric patient" is a human patient between the ages of 1 and 21, inclusive. In one embodiment, a pediatric patient is a human patient between the ages of 7 and 21, inclusive.

[0118] "Alkyl" or "alkylene" both refer to saturated straight-chain or branched hydrocarbons. In certain embodiments, alkyl groups are primary, secondary, or tertiary hydrocarbons. In certain embodiments, alkyl groups have 1 to 10 carbon atoms, i.e., C1 to C6. 10and alkyl. In certain embodiments, alkyl groups contain 1 to 6 carbon atoms, i.e., C1-C6 alkyl. This 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 of skill in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, incorporated herein by reference. In certain embodiments, 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.

[0119] "Alkenyl" refers to an unsaturated straight- or branched-chain hydrocarbon radical containing 2 to 18 carbons and containing at least one carbon-to-carbon double bond. Examples include, but are not limited to, ethenyl, propenyl, iso-propenyl, butenyl, iso-butenyl, tert-butenyl, n-pentenyl, and n-hexenyl. The term "lower alkenyl" refers to an alkenyl group, as defined herein, containing 2 to 8 carbons.

[0120] "Alkynyl" refers to an unsaturated straight- or branched-chain hydrocarbon radical containing 2 to 18 carbons with at least one carbon-to-carbon triple bond. Examples include, but are not limited to, ethynyl, propynyl, iso-propynyl, butynyl, iso-butynyl, tert-butynyl, pentynyl, and hexynyl. The term "lower alkynyl" refers to an alkynyl group, as defined herein, containing 2 to 8 carbons.

[0121] "Cycloalkyl" refers to a monocyclic or polycyclic alkyl radical. Examples include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0122] The term "aryl," used alone or as part of a larger moiety, such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to an aromatic ring group having 6 to 14 ring atoms, such as phenyl, 1-naphthyl, 2-naphthyl, 1-anthracyl, and 2-anthracyl. An "aryl" ring may contain one or more substituents. The term "aryl" may be used interchangeably with the term "aryl ring." "Aryl" also includes fused polycyclic aromatic ring systems in which an aromatic ring is fused to one or more rings. Non-limiting examples of useful aryl ring groups include phenyl, hydroxyphenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl, trialkoxyphenyl, alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro, etc., as well as 1-naphthyl, 2-naphthyl, 1-anthracyl, and 2-anthracyl. Also included within the scope of the term "aryl," as used herein, are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as, for example, indanyl, phenanthridinyl, or tetrahydronaphthyl, where the radical or point of attachment is on the aromatic ring.

[0123] The term "acyl" means C(O)R 11 group (where R 11means H, alkyl, or aryl as defined herein. Examples of acyl groups include formyl, acetyl, benzoyl, phenylacetyl, and similar groups.

[0124] "Aralkyl" is a group of the formula -R 12 R 13 refers to the radical of R 12 is an alkylene chain as defined above, and R 13 is one or more aryl radicals as defined above, e.g., benzyl, diphenylmethyl, and the like.

[0125] "Thioalkoxy" refers to a group of the formula -SR 14 refers to the radical of R 14 is an alkyl radical as defined herein. The term "lower thioalkoxy" refers to an alkoxy group, as defined herein, containing 1 to 8 carbons.

[0126] "Alkoxy" means a group of the formula -OR 15 refers to the radical of R 15 is an alkyl radical as defined herein. The term "lower alkoxy" refers to an alkoxy group, as defined herein, containing 1 to 8 carbons. Examples of alkoxy groups include, but are not limited to, methoxy and ethoxy.

[0127] "Alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group.

[0128] "Carbonyl" refers to the C(=O)- radical.

[0129] "Guanidinyl" refers to the H2N(C=NH2)-NH- radical.

[0130] "Amidinyl" refers to the H2N(C=NH2)CH- radical.

[0131] "Amino" refers to the NH2 radical.

[0132] "Alkylamino" refers to a group of the formula -NHR 16 or -NR 16 R 16 Each R refers to a radical. 16 are independently an alkyl radical, as defined herein. The term "lower alkylamino" refers to an alkylamino group, as defined herein, containing 1 to 8 carbons.

[0133] A "heterocycle" refers to a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic, saturated, unsaturated, or aromatic heterocyclic ring containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. The nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be bonded via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Thus, in addition to the heteroaryls listed below, heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiopyranyl, and the like.

[0134] "Heteroaryl" means a 5- to 10-membered aromatic heterocycle having at least one heteroatom selected from nitrogen, oxygen, and sulfur, and containing at least one carbon atom, including both monocyclic and bicyclic ring systems. Representative heteroaryls include pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrirolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl.

[0135] The terms "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkoxy," "optionally substituted thioalkoxy," "optionally substituted alkylamino," "optionally substituted lower alkyl," "optionally substituted lower alkenyl," "optionally substituted lower alkoxy," "optionally substituted lower thioalkoxy," "optionally substituted lower alkylamino," and "optionally substituted heterocyclyl" mean, when substituted, that at least one hydrogen atom is replaced by a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, substituents include: deuterium, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted cycloalkyl, oxo, halogen, -CN, -OR x , N.R. x R y , N.R. x C(=O) R y , N.R. x SO2R y , -NR x C(=O)NR x R y , C(=O) R x , C(=O)OR x , C(=O)NR x R y , -SO m R x and -SO m NR x R y , wherein m is 0, 1, or 2; and R x and R y are the same or different and independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle, or optionally substituted cycloalkyl, and each of the foregoing optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle, and optionally substituted cycloalkyl substituents may be further substituted with oxo, halogen, and —CN.

[0136] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to administration of a compound, drug, or other material other than by direct administration into the central nervous system, thereby entering the system of a human patient and thus being subject to metabolism and other similar processes, e.g., subcutaneous administration.

[0137] As used herein, the phrases "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. The phrase "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the human patient being treated therewith.

[0138] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or any type of formulation auxiliary. Some examples of materials that can serve as pharmaceutically acceptable carriers include, according to the discretion of the formulator, sugars such as lactose, glucose, sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, oat oil, and the like. leaven oil, corn oil, and soybean oil; glycols, such as propylene glycol esters, e.g., ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free distilled water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate; colorants; release agents; coating agents; sweetening agents; flavoring agents; fragrances; preservatives; and antioxidants.

[0139] As used herein, a set of parentheses used within a structural formula indicates that the structural feature between the parentheses is repeated. In some embodiments, the parentheses used may be "[" and "]", and in certain embodiments, the parentheses used to indicate a repeating structural feature may be "(" and ")". In some embodiments, the number of repeating repetitions of the structural feature between the parentheses is the number shown outside the parentheses, e.g., 2, 3, 4, 5, 6, 7, etc. In various embodiments, the number of repeating repetitions of the structural feature between the parentheses is indicated by a variable shown outside the parentheses, such as "Z".

[0140] 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]

[0141] For clarity, the structures of the present disclosure, such as PPMO#1, PPMO#2, and PPMO#3 described above, are continuous from 5' to 3' and include various diagrammatic breaks labeled "Break A," "Break B," and "Break C" for the convenience of depicting the entire structure in a compact form. As will be understood by those skilled in the art, each designation of, for example, "Break A" indicates the continuation of the diagram of the structure at those points. Those skilled in the art will understand that the same is true for each instance of "Break B" and "Break C" in the structures described above. However, none of the diagrammatic breaks are intended to indicate an actual discontinuity in the structures described above, and those skilled in the art would not understand them to mean so.

[0142] To distinguish between different antisense molecules, a nomenclature system for antisense molecules has been proposed and published, which has become particularly suitable when testing several slightly different antisense molecules all directed against the same target region, as shown below. H#A / D(x:y)

[0143] 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 coordinate, and "-" or "+" indicates the intron or exon sequence, respectively. For example, A(-6+18) indicates the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. Since the nearest splice site is the acceptor, these coordinates are preceded by "A." The annealing coordinate at the donor splice site can be written as 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 the 65th to the 85th nucleotide from the start of the exon. II. Methods of the Disclosure

[0144] The present disclosure relates to a method for treating a human patient having a disease or disorder suitable for antisense oligonucleotide therapy by administering an effective amount of an antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO), or a pharmaceutically acceptable salt thereof. The effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, can be administered every 1, 2, 3, or 4 weeks. In some embodiments, the antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO), or a pharmaceutically acceptable salt thereof, is administered every 4 weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every 4 weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of PPMO#1·6HCl dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 60 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 80 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#2 dissolved in an aqueous carrier solution is administered every four weeks.In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#3 dissolved in an aqueous carrier solution is administered every four weeks. In one embodiment, an effective amount of an antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is administered monthly.

[0145] In one embodiment, the method relates to the treatment of a human patient with muscular dystrophy (e.g., DMD) by administering an antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO), or a pharmaceutically acceptable salt thereof, once every four weeks. In some embodiments, a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution at about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg is administered every four weeks. In some embodiments, a PPMO#1·6HCl dissolved in an aqueous carrier solution at about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg is administered every four weeks. In some embodiments, about 60 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 80 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#1 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#2 dissolved in an aqueous carrier solution is administered every four weeks. In some embodiments, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg of a pharmaceutically acceptable salt of PPMO#3 dissolved in an aqueous carrier solution is administered every four weeks. In one embodiment, an effective amount of an antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is administered monthly.

[0146] Some aspects of the present disclosure relate to a method for increasing or restoring muscle cells in a human patient with muscular dystrophy (e.g., DMD), comprising administering to the human patient once every four weeks an effective amount of an antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO), or a pharmaceutically acceptable salt thereof, which is complementary to a nucleotide sequence within a dystrophin transcript and can induce exon skipping in the dystrophin transcript. In one embodiment, the effective amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is administered monthly. Antisense oligonucleotides designed to induce exon skipping

[0147] In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate induces skipping of one or more exons, or portions thereof, in a transcript. In certain embodiments, the one or more exons, or portions thereof, are selected from the group consisting of exon 44, exon 45, exon 50, exon 51, exon 52, exon 53, and any combination thereof. In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate induces skipping of exon 51, exon 45, or exon 53 of a dystrophin transcript.

[0148] In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate induces skipping of exon 51 of the dystrophin transcript. In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate comprises a base sequence complementary to a target region of exon 51 of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from: [Table 10] In each of SEQ ID NOs: 1-6, T is thymine or uracil. In certain embodiments, T in the antisense oligonucleotide or antisense oligonucleotide conjugate is thymine. In certain embodiments, T in the antisense oligonucleotide or antisense oligonucleotide conjugate is uracil.

[0149] In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate induces skipping of exon 53 of the dystrophin transcript. In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate comprises a base sequence complementary to a target region of exon 53 of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from: [Table 11] In each of SEQ ID NOs:7-16, T is thymine or uracil. In certain embodiments, T in the antisense oligonucleotide or antisense oligonucleotide conjugate is thymine. In certain embodiments, T in the antisense oligonucleotide or antisense oligonucleotide conjugate is uracil.

[0150] In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate induces skipping of exon 45 of the dystrophin transcript. In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate comprises a base sequence complementary to a target region of exon 45 of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from: [Table 12] In each of SEQ ID NOs: 17-34, T is thymine or uracil. In certain embodiments, T in the antisense oligonucleotide or antisense oligonucleotide conjugate is thymine. In certain embodiments, T in the antisense oligonucleotide or antisense oligonucleotide conjugate is uracil.

[0151] In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate induces skipping of exon 44 of the dystrophin transcript. In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate comprises a base sequence complementary to a target region of exon 44 of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from: [Table 13] In each of SEQ ID NOs: 35-41, T is thymine or uracil. In certain embodiments, T in the antisense oligonucleotide or antisense oligonucleotide conjugate is thymine. In certain embodiments, T in the antisense oligonucleotide or antisense oligonucleotide conjugate is uracil.

[0152] In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate induces skipping of exon 50 of the dystrophin transcript. In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate comprises a base sequence complementary to a target region of exon 50 of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from: [Table 14] In each of SEQ ID NOs: 42-50, T is thymine or uracil. In certain embodiments, T in the antisense oligonucleotide or antisense oligonucleotide conjugate is thymine. In certain embodiments, T in the antisense oligonucleotide or antisense oligonucleotide conjugate is uracil.

[0153] In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate induces skipping of exon 52 of the dystrophin transcript. In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate comprises a base sequence complementary to a target region of exon 52 of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from: [Table 15] Each T in SEQ ID NO:51 is thymine or uracil. In certain embodiments, the T in the antisense oligonucleotide or antisense oligonucleotide conjugate is thymine. In certain embodiments, the T in the antisense oligonucleotide or antisense oligonucleotide conjugate is uracil.

[0154] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate is a PMO or PPMO, wherein each morpholino ring of the PMO or PPMO is linked to a nucleobase, including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine).

[0155] The antisense oligomer or antisense oligomer conjugate of the present disclosure can use various antisense oligomer chemicals.Examples of oligomer chemicals include, but are not limited to, morpholino oligomers, phosphorothioate-modified oligomers, 2'-O-methyl-modified oligomers, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate oligomers, 2' O-MOE-modified oligomers, 2'-fluoro-modified oligomers, 2'-O,4'C-ethylene-bridged nucleic acids (ENA), tricyclo-DNA, tricyclo-DNA phosphorothioate subunits, 2'-O-[2-(N-methylcarbamoyl)ethyl]-modified oligomers (including any combination of the above).Phosphorothioate and 2'-O-Me-modified chemicals can be combined to produce a 2'-O-Me-phosphorothioate backbone. See, e.g., PCT Publication Nos. WO / 2013 / 112053 and WO / 2009 / 008725, which are incorporated by reference herein in their entireties. Exemplary embodiments of the oligomeric chemicals of the present disclosure are further described herein. A. Peptide Nucleic Acid (PNA)

[0156] Peptide nucleic acids (PNAs) are analogs of DNA, whose backbone is structurally isomorphous with a deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligomers according to Watson-Crick base-pairing rules, mimicking DNA in terms of base pair recognition. PNA backbones are formed by peptide bonds rather than phosphodiester bonds, making them well suited for antisense applications (see structure below). The backbone is uncharged, resulting in PNA / DNA or PNA / RNA duplexes that exhibit exceptional thermal stability. PNAs are not recognized by nucleases or proteases. Non-limiting examples of PNAs are shown below. [ka]

[0157] Despite their radical structural changes relative to their natural structure, PNAs are capable of sequence-specific binding to DNA or RNA in a helical form. PNA characteristics include high binding affinity to complementary DNA or RNA, destabilization caused by single-base mismatches, resistance to nucleases and proteases, salt-independent hybridization with DNA or RNA, and triplex formation with homopurine DNA. PANAGENE™ has developed its proprietary Bts PNA monomer (Bts, benzothiazole-2-sulfonyl group) and proprietary oligomerization process. PNA oligomerization using the Bts PNA monomer consists of repeated cycles of deprotection, coupling, and capping. PNAs can be produced synthetically using any technique known in the art. See, for example, U.S. Patent Nos. 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006, and 7,179,896. Also, for the preparation of PNAs, see U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254:1497-1500, 1991. Each of the above is incorporated by reference in its entirety. B. Locked Nucleic Acid (LNA)

[0158] Antisense oligomers may also contain "locked nucleic acid" subunits (LNAs). "LNAs" are members of a class of modifications called bridged nucleic acids (BNAs). BNAs feature a covalent bond that locks the conformation of the ribose ring to the C30-endo (northern) sugar pucker. In the case of LNAs, the bridge consists of a methylene between the 2'-O and 4'-C positions. LNAs enhance backbone preorganization and base stacking, enhancing hybridization and thermal stability.

[0159] The structure of LNA is described, for example, in Wengel, et al., Chemical Communications (1998) 455; Koshkin et al., Tetrahedron (1998) 54:3607; Jesper Wengel, Accounts of Chem. Research (1999) 32:301; Obika, et al. al., Tetrahedron Letters (1997) 38:8735, Obika, et al., Tetrahedron Letters (1998) 39:5401, and Obika, et al., Bioorganic Medicinal Chemistry (2008) 16:9230, which are incorporated herein by reference in their entirety. Non-limiting examples of LNAs are shown below. [ka]

[0160] The antisense oligomer of the present disclosure may incorporate one or more LNAs. In some cases, the antisense oligomer may be entirely composed of LNAs. Methods for synthesizing individual LNA nucleoside subunits and incorporating them into oligomers are described, for example, in U.S. Patent Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461, each of which is incorporated by reference in its entirety. Typical intersubunit linkers include phosphodiester and phosphorothioate moieties. Alternatively, non-phosphorus-containing linkers can be used. Further embodiments include LNA-containing antisense oligomers, in which each LNA subunit is separated by a DNA subunit. Certain antisense oligomers are composed of alternating LNA and DNA subunits, with the intersubunit linker being phosphorothioate.

[0161] 2'O,4'C-ethylene-bridged nucleic acids (ENAs) are another member of the BNA class. Non-limiting examples are shown below. [ka]

[0162] ENA oligomers and their preparation are described in Obika et al., Tetrahedron Lett (1997) 38(50):8735, which is incorporated herein by reference in its entirety. Antisense oligomers of the present disclosure may incorporate one or more ENA subunits. C. Unlocked Nucleic Acid (UNA)

[0163] Antisense oligomers can also contain unlocked nucleic acid (UNA) subunits.UNA and UNA oligomers are analogs of RNA, in which the C2'-C3' bond of the subunit is broken.LNA is conformationally restricted (compared to DNA and RNA), while UNA is very flexible.UNA is disclosed, for example, in WO2016 / 070166.Non-limiting examples of UNA are shown below. [ka]

[0164] Typical intersubunit linkers include phosphodiester and phosphorothioate moieties. Alternatively, non-phosphorus-containing linkers can be used. D. Phosphorothioates

[0165] "Phosphorothioates" (or S-oligos) are variants of normal DNA in which one of the non-bridging oxygens has been replaced with a sulfur. Non-limiting examples of phosphorothioates are shown below. [ka]

[0166] Sulfurization of internucleotide bonds reduces the action of endonucleases and exonucleases, including 5' to 3' and 3' to 5' DNA POL1 exonuclease, nucleases S1 and P1, RNases, serum nucleases, and snake venom phosphodiesterases. Phosphorothioates are produced by two major routes: the action of a solution of elemental sulfur in carbon disulfide on hydrogen phosphonates, or by sulfurization of phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990, incorporated herein by reference in its entirety). The latter method avoids the insolubility of elemental sulfur in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also result in higher purity phosphorothioates. E. Tricyclo-DNA and Tricyclo-phosphorothioate Subunits

[0167] Tricyclo-DNA (tc-DNA) is a class of constrained DNA analogues in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the backbone and optimize the backbone geometry of the torsion angle γ. Homobasic adenine and thymine-containing tc-DNA form highly stable AT base pairs with complementary RNA. Tricyclo-DNA and its synthesis are described in International Patent Application Publication No. WO2010 / 115993, which is incorporated herein by reference in its entirety. The antisense oligomers of the present disclosure may incorporate one or more tricyclo-DNA subunits. In some cases, the antisense oligomer may be composed entirely of tricyclo-DNA subunits.

[0168] Tricyclo-phosphorothioate subunits are tricyclo-DNA subunits with phosphorothioate intersubunit linkages.Tricyclo-phosphorothioate subunits and their synthesis are described in International Patent Application Publication No. WO2013 / 053928, which is incorporated herein by reference in its entirety.Antisense oligomers of the present disclosure can incorporate one or more tricyclo-DNA subunits.In some cases, antisense oligomers can be completely composed of tricyclo-DNA subunits.Non-limiting examples of tricyclo-DNA / tricyclo-phosphothioate are shown below. [ka] F. 2'O-methyl, 2'O-MOE, and 2'-F oligomers

[0169] A "2'-O-Me oligomer" molecule has a methyl group at the 2'-OH residue of the ribose molecule. 2'-O-Me-RNA behaves the same as (or similar to) DNA, but is protected from nuclease degradation. 2'-O-Me-RNA can also be combined with phosphorothioate oligomers (PTO) for further stabilization. 2'O-Me oligomers (phosphodiester or phosphothioate) can be synthesized according to routine techniques in the art (e.g., see Yoo et al., "2'-O-Me Oligomers," incorporated herein by reference in its entirety). (See, e.g., J. Am. Chem. Soc. Soc. 1999, 12:1016-1017, 2004.) Non-limiting examples of 2'O-Me oligomers are shown below. [ka] 2'O-Me

[0170] 2'-O-Methoxyethyl oligomers (2'-O-MOE) have a methoxyethyl group at the 2'-OH residue of the ribose molecule and are discussed in Martin et al., Helv. Chim. Acta, 78, 486-504, 1995, which is incorporated herein by reference in its entirety. Non-limiting examples of 2'O MOE subunits are shown below. [ka]

[0171] 2'-Fluoro (2'-F) oligomers have a fluorescent radical at the 2' position instead of 2'-OH. Non-limiting examples of 2'-F oligomers are shown below. [ka] 2'-F

[0172] 2'-Fluoro oligomers are further described in WO2004 / 043977, which is incorporated herein by reference in its entirety.

[0173] The 2'O-methyl, 2'O-MOE, and 2'-F oligomers may also contain one or more phosphorothioate (PS) linkages, as illustrated below. [ka]

[0174] Additionally, 2'O-methyl, 2'O-MOE, and 2'-F oligomers may contain PS intersubunit linkages throughout the oligomer, such as, for example, the 2'O-methyl PS oligomer drisapersen shown below. [ka]

[0175] Alternatively, the 2'O-methyl, 2'O-MOE, and / or 2'-F oligomers may contain PS linkages at the termini of the oligomers, as illustrated below. [ka] During the ceremony, R is CH2CH2OCH3 (methoxyethyl or MOE), x, y, and z represent the number of nucleotides contained within the designated 5'-wing, central gap, and 3'-wing regions, respectively.

[0176] The antisense oligomers of the present disclosure may incorporate one or more 2'O-methyl, 2'O-MOE, and 2'-F subunits and may utilize any of the intersubunit linkages described herein. In some cases, the antisense oligomers of the present disclosure may be composed entirely of 2'O-methyl, 2'O-MOE, or 2'-F subunits. One embodiment of the antisense oligomer of the present disclosure consists entirely of 2'O-methyl subunits. G. 2'-O-[2-(N-methylcarbamoyl)ethyl] oligomer (MCE)

[0177] MCE is another example of a 2'-O modified ribonucleoside useful in the antisense oligomers of the present disclosure, where the 2'OH is derivatized to a 2-(N-methylcarbamoyl)ethyl moiety to enhance nuclease resistance. Non-limiting examples of MCE oligomers are shown below. [ka]

[0178] MCE and its synthesis are described in Yamada et al., J. Org. Chem. (2011) 76(9):3042-53, which is incorporated herein by reference in its entirety. Antisense oligomers of the present disclosure may incorporate one or more MCE subunits. H. Stereospecific Oligomers

[0179] Stereospecific oligomers are those in which the stereochemistry of each phosphorus-containing linkage is fixed by synthetic methods such that a substantially stereopure oligomer is produced. Non-limiting examples of stereospecific oligomers are shown below. [ka]

[0180] In the above example, each phosphorus of oligomer has the same stereochemistry. Further examples include the above oligomers. For example, LNA, ENA, tricyclo-DNA, MCE, 2'O-methyl, 2'O-MOE, 2'-F and morpholino-based oligomers can be prepared using stereospecific phosphorus-containing internucleoside linkages, such as phosphorothioate, phosphodiester, phosphoramidate, phosphorodiamidate or other phosphorus-containing internucleoside linkages. Stereospecific oligomers, methods of preparation, chiral controlled synthesis, chiral designs, and chiral auxiliaries for use in preparing such oligomers are described, for example, in WO2017 / 192664, WO2017 / 192679, WO2017 / 062862, WO2017 / 015575, WO2017 / 015555, WO2015 / 107425, WO2015 / 108048, WO 2015 / 108046, WO2015 / 108047, WO2012 / 039448, WO2010 / 064146, WO2011 / 034072, WO2014 / 010250, WO2014 / 012081, WO2013 / 0127858, and WO2011 / 005761, each of which is incorporated herein by reference in its entirety.

[0181] The stereospecific oligomers are R P or S PThe oligomer may have phosphorus-containing internucleoside linkages in any configuration. Chiral phosphorus-containing linkages in which the configuration of the linkage is controlled are referred to as "stereo-pure," while chiral phosphorus-containing linkages in which the configuration of the linkage is not controlled are referred to as "stereoirregular." In certain embodiments, oligomers of the present disclosure include multiple stereo-pure and stereoirregular linkages, such that the resulting oligomer has stereo-pure subunits at pre-specified positions on the oligomer. Examples of the positions of stereo-pure subunits are provided in Figures 7A and 7B of International Patent Application Publication No. WO 2017 / 062862(A2). In one embodiment, all chiral phosphorus-containing linkages in the oligomer are stereo-irregular. In one embodiment, all chiral phosphorus-containing linkages in the oligomer are stereo-pure.

[0182] In one embodiment of an oligomer having n (n is an integer greater than or equal to 1) chiral phosphorus-containing linkages, all n chiral phosphorus-containing linkages in the oligomer are stereoirregular. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1) chiral phosphorus-containing linkages, all n chiral phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1), at least 10% (rounded to the nearest integer) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1), at least 20% (rounded to the nearest integer) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1), at least 30% (rounded to the nearest integer) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1) chiral phosphorus-containing linkages, at least 40% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1) chiral phosphorus-containing linkages, at least 50% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1) chiral phosphorus-containing linkages, at least 60% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1) chiral phosphorus-containing linkages, at least 70% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer greater than or equal to 1), at least 80% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer greater than or equal to 1), at least 90% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure.

[0183] In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), the oligomers have the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), the oligomer contains at least two consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), the oligomer contains at least three consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least four consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least five consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 6 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 7 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 8 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., SP or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 9 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 10 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 11 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 12 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 13 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 14 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 15 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R PIn one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 16 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 17 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 18 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 19 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P contains at least 20 consecutive stereopure phosphorus-containing linkages of I. Morpholino Oligomers

[0184] Exemplary embodiments of the present disclosure include those having the following general structure: [ka] and as depicted in Figure 2 of Summerton, J., et al., Antisense & Nucleic Acid Drug Development, 7: 187-195 (1997). 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 oligomers are described in detail in U.S. Pat. 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.

[0185] In certain embodiments, the morpholino is conjugated to a "tail" moiety at the 5' or 3' end of the oligomer to enhance its stability and / or solubility. Exemplary tails include: [ka] In the formula, R 200 is hydrogen or a cell membrane-permeable peptide, and R 1 is C1-C6 alkyl. In one embodiment, R 200 is hydrogen.

[0186] In various embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate has the formula (I): [ka] (I) or a pharmaceutically acceptable salt thereof; each Nu is a nucleobase that together form a targeting sequence,

[0187] T' is a moiety selected from: [ka] R 100 and R 200 are independently hydrogen or a cell membrane-permeable peptide, and R 1 is C1-C6 alkyl, Each Nu from 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of the following: [Table 16-1] [Table 16-2] 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 and Am is a methylated adenine; m5C is [ka] In certain embodiments, each Nu from 1 to (n+1) and from 5' to 3' corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0188] In various embodiments, R 200 is hydrogen. In various embodiments, T' is [ka] is.

[0189] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (I) is in free base form. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (I) is a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (I) is in a halogenated salt form. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (I) is in a hexahalogenated salt form. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (I) is in its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a hexaHCl salt. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (I) is provided as a mixture of free base and salt forms.

[0190] In various embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate has the formula (II): [ka] (II) or a pharmaceutically acceptable salt thereof, and R 200 is hydrogen or a cell membrane-permeable peptide, wherein each Nu in the 1 to (n+1) positions and 5' to 3' positions corresponds to a nucleobase in one of the following: [Table 17-1] [Table 17-2] 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] In certain embodiments, each Nu from 1 to (n+1) and from 5' to 3' corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0191] In various embodiments, R 200 is hydrogen.

[0192] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (II) is in free base form. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (II) is a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (II) is in a halogenated salt form. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (II) is in a hexahalogenated salt form. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (II) is in its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a hexaHCl salt. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (II) is provided as a mixture of free base and salt forms.

[0193] In various embodiments, the antisense oligonucleotide conjugate has the formula (II): [ka] (III) or a pharmaceutically acceptable salt thereof, and R 200 is hydrogen or a cell membrane-permeable peptide, and each Nu from 1 to (n+1) and from 5' to 3' corresponds to a nucleic acid base in one of the following: [Table 18-1] [Table 18-2] 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] In certain embodiments, each Nu from 1 to (n+1) and from 5' to 3' corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0194] In various embodiments, R 200 is hydrogen.

[0195] In some embodiments, the antisense oligonucleotide conjugate of Formula (III) is in free base form. In some embodiments, the antisense oligonucleotide conjugate of Formula (III) is in its pharmaceutically acceptable salt form. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (III) is in a halide salt form. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (III) is in a hexahalide salt form. In some embodiments, the antisense oligonucleotide conjugate of Formula (III) is in its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a hexaHCl salt. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (III) is provided as a mixture of free base and salt form.

[0196] In various embodiments, the antisense oligonucleotide conjugate is described by formula (IV): [ka] (IV) wherein each Nu from 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of the following: [Table 19-1] [Table 19-2] 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] In certain embodiments, each Nu from 1 to (n+1) and from 5' to 3' corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0197] In various embodiments, the antisense oligonucleotide conjugate has the formula (V): [ka] (V) or a pharmaceutically acceptable salt thereof, wherein each Nu from 1 to (n+1) and from 5' to 3' corresponds to a nucleobase in one of the following: [Table 20-1] [Table 20-2] 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] In certain embodiments, each Nu from 1 to (n+1) and from 5' to 3' corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0198] In various embodiments, R 200 is hydrogen.

[0199] In some embodiments, the antisense oligonucleotide conjugate of Formula (V) is in free base form. In some embodiments, the antisense oligonucleotide conjugate of Formula (V) is a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (V) is in a halogenated salt form. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (V) is in a pentahalogenated salt form. In some embodiments, the antisense oligonucleotide conjugate of Formula (V) is in its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a pentahelical salt. In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of Formula (V) is provided as a mixture of the free base and salt forms.

[0200] In various embodiments, the antisense oligonucleotide conjugate is described by formula (VI): [ka] (VI) wherein each Nu from 1 to (n+1) and 5' to 3' corresponds to a nucleobase in one of the following: [Table 21-1] [Table 21-2] 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] In certain embodiments, each Nu from 1 to (n+1) and 5' to 3' corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17.

[0201] In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is selected from the group consisting of eteplirsen, PPMO#1, or a pharmaceutically acceptable salt thereof, golodirsen, PPMO#2, or a pharmaceutically acceptable salt thereof, casimersen, and PPMO#3, or a pharmaceutically acceptable salt thereof. J. Nucleobase Modifications and Substitutions

[0202] In certain embodiments, the antisense oligomers of the present disclosure are composed of RNA nucleobases and DNA nucleobases (often simply referred to in the art as "bases"). RNA bases are commonly known as adenine (A), uracil (U), cytosine (C), and guanine (G). DNA bases are commonly known as adenine (A), thymine (T), cytosine (C), and guanine (G). In various embodiments, the antisense oligomers of the present disclosure are composed of cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I) methylated guanine (Gm) methylated and methylated adenine (Am).

[0203] In certain embodiments, one or more RNA or DNA bases in an oligomer can be modified or replaced with a base other than an RNA or DNA base. Oligomers containing modified or substituted bases include oligomers in which one or more of the purine or pyrimidine bases most commonly found in nucleic acids are replaced with a less common or unnatural base.

[0204] Purine bases contain a pyrimidine ring fused to an imidazole ring, as described by the following general formula: [ka] pudding

[0205] Adenine and guanine are the two most commonly found purine nucleobases in nucleic acids. Other naturally occurring purines include N 6 -methyladenine, N 2 Examples of methylguanine include, but are not limited to, -methylguanine, hypoxanthine, and 7-methylguanine.

[0206] The pyrimidine base contains a six-membered pyrimidine ring, as described by the general formula: [ka] Pyrimidine Core

[0207] Cytosine, uracil, and thymine are the most commonly occurring pyrimidine bases in nucleic acid.Other naturally occurring pyrimidines include, but are not limited to, 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil.In one embodiment, the oligomer described herein contains thymine base instead of uracil.

[0208] Other suitable bases include 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidines (e.g., 2-thiouracil, 2-thiothymine), G-clamps and derivatives thereof, 5-substituted pyrimidines (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, super G, super A, and N4-ethylcytosine, or derivatives thereof; 2 -Cyclopentylguanine (cPent-G), N 2 -cyclopentyl-2-aminopurine (cPent-AP), and N 2 The bases include, but are not limited to, -propyl-2-aminopurine (Pr-AP), pseudouracil, or their derivatives; and degenerate or universal bases such as 2,6-difluorotoluene, or non-existent bases such as abasic sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives (azaribose) in which the ring oxygen is replaced with nitrogen). Examples of Super A, Super G, and Super T derivatives can be found in U.S. Patent No. 6,683,173 (Epoch Biosciences), which is incorporated herein by reference in its entirety. cPent-G, cPent-AP, and Pr-AP have been shown to reduce immunostimulatory activity when incorporated into siRNA (Peacock H. et al. J. Am. Chem. Soc. 2011, 133, 9200). Pseudouracil is a naturally occurring isomerized version of uracil, with a C-glycoside rather than the usual N-glycoside in uridine. Pseudouridine-containing synthetic mRNAs may have an improved safety profile compared to uridine-containing mPvNAs (WO2009 / 127230, incorporated herein by reference in its entirety).

[0209] Certain nucleobases are particularly useful for increasing the binding affinity of the antisense oligomers of the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine). 5-methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6 to 1.2°C, and are currently a more preferred base substitution, particularly when combined with 2'-O-methoxyethyl sugar modifications. Additional exemplary modified nucleobases include those in which at least one hydrogen atom of the nucleobase is replaced with fluorine. Cell membrane-penetrating peptides

[0210] In certain embodiments, the antisense oligonucleotide is conjugated to one or more cell membrane-permeable peptides (referred to herein as "CPPs"). In certain embodiments, one or more CPPs are conjugated to the termini of the antisense oligonucleotide. In certain embodiments, at least one CPP is conjugated to the 5' end of the antisense oligonucleotide. In certain embodiments, at least one CPP is conjugated to the 3' end of the antisense oligonucleotide. In certain embodiments, a first CPP is conjugated to the 5' end of the antisense oligonucleotide and a second CPP is conjugated to the 3' end of the antisense oligonucleotide. In certain embodiments, one CPP is conjugated to the 3' end of the antisense oligonucleotide.

[0211] 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 preferably linked at its carboxy terminus to the 3' and / or 5' end of the antisense oligonucleotide via a linker, which may also be one or more amino acids and preferably has an R selected from H, acyl, acetyl, benzoyl, or stearoyl. a Substituent R containing a In some embodiments, R a is acetyl.

[0212] As seen in the table below, non-limiting examples of CPPs for use herein include -(RXR)-R a (SEQ ID NO: 52), R-(FFR)3-R a (SEQ ID NO: 53), -BX-(RXR)4-R a (SEQ ID NO: 54), -BXR-(FFR)3-R a (SEQ ID NO: 55, -GLY-R-(FFR)3-R a (SEQ ID NO: 56), -GLY-R6-R a (SEQ ID NO: 57), and -R6-R a (SEQ ID NO: 58), wherein 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 "R6 (SEQ ID NO: 58)" is meant to refer to a peptide of six arginine residues (SEQ ID NO: 58) linked together via amide bonds (a single substituent, e.g., R 6 (not SEQ ID NO: 58). In some embodiments, R a is acetyl. Exemplary CPPs are shown in Table 1 (SEQ ID NOs: 52-58). Table 8 [Table 8]

[0213] CPPs, their synthesis, and methods of conjugating to oligomers are further described in U.S. Application Publication No. 2012 / 0289457, and International Patent Application Publication Nos. WO2004 / 097017, WO2009 / 005793, and WO2012 / 150960, the disclosures of which are incorporated herein by reference in their entireties.

[0214] In some embodiments, the antisense oligonucleotide comprises a substituent "Z," defined as a combination of a CPP and a linker. The linker bridges the CPP at its carboxy terminus to the 3' and / or 5' terminus of the oligonucleotide. In various embodiments, the antisense oligonucleotide may comprise only one CPP linked to the 3' terminus of the oligomer. In other embodiments, the antisense oligonucleotide may comprise only one CPP linked to the 5' terminus of the oligomer.

[0215] The linker in Z can comprise, for example, 1, 2, 3, 4, or 5 amino acids.

[0216] In certain embodiments, Z is selected from: -C(O)(CH2)5NH-CPP, -C(O)(CH2)2NH-CPP, -C(O)(CH2)2NHC(O)(CH2)5NH-CPP, -C(O)CHNH-CPP, and the following formula: [ka] wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus.

[0217] In various embodiments, the CPP is an arginine-rich peptide, as defined above and shown in Table 1. In certain embodiments, the arginine-rich CPP is -R-R a (i.e., six arginine residues, SEQ ID NO:58), where R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. a is acetyl. In various embodiments, the CPP is selected from (RXR)4 (SEQ ID NO: 52), (RFF)3R (SEQ ID NO: 53), or R6 (SEQ ID NO: 58), and the linker is selected from the group described above. In some embodiments, the CPP is R6 (SEQ ID NO: 58) and the linker is Gly. In some embodiments, the CPP is R6G (SEQ ID NO: 57).

[0218] In certain embodiments, Z is —C(O)CHNH—R—R covalently attached to the antisense oligomer of the disclosure at the 5′ and / or 3′ end of the oligomer. a ("R6" is disclosed as SEQ ID NO:58), and R a is H, acyl, acetyl, benzoyl, or stearoyl and caps the amino terminus of R6 (SEQ ID NO: 58). a is acetyl. In these non-limiting examples, the CPP is -R-R a (SEQ ID NO: 58), and the linker is -C(O)CHNH-, (i.e., GLY). Z = -C(O)CHNH-R 6- R a ("R6" is disclosed as SEQ ID NO:58) is also exemplified by the following structure: [ka] In the formula, R a is selected from H, acyl, acetyl, benzoyl, and stearoyl.

[0219] In various embodiments, the CPP is -R-R a (SEQ ID NO: 58) and is also exemplified by the following formula: [ka] In the formula, R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. In certain embodiments, the CPP is R6 (SEQ ID NO: 58). In some embodiments, R a is acetyl.

[0220] In some embodiments, the CPP is -(RXR)-R a (SEQ ID NO: 52) and is also exemplified by the following formula: [ka]

[0221] In various embodiments, the CPP is -R-(FFR)3-R a (SEQ ID NO: 53) and is also exemplified by the following formula: [ka]

[0222] In various embodiments, Z is selected from: -C(O)(CH2)5NH-CPP, -C(O)(CH2)2NH-CPP, -C(O)(CH2)2NHC(O)(CH2)5NH-CPP, -C(O)CHNH-CPP, and 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: 53), [ka] (-(RXR)4-R a ) (SEQ ID NO: 52), [ka] or (-R6-R a ) (SEQ ID NO: 58). In some embodiments, R a is acetyl. Pharmaceutically acceptable salts of antisense oligonucleotides and antisense oligonucleotide conjugates

[0223] Certain embodiments of the antisense oligonucleotide conjugates described herein may contain a basic functional group, such as amino or alkylamino, and thus may form pharmaceutically acceptable salts with pharmaceutically acceptable acids.

[0224] In this respect, the term " pharmaceutically acceptable salts " refers to the relatively non-toxic inorganic and organic acid addition salts of the antisense oligonucleotide or antisense oligonucleotide conjugate of the present disclosure.These salts can be prepared in situ during the administration vehicle or dosage form manufacturing process, or can be prepared by separately reacting the purified antisense oligonucleotide conjugate of the present disclosure in its free base form with suitable organic or inorganic acid, and then isolating the salt thus formed during 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 and laurylsulfonate etc. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66:1-19).

[0225] Pharmaceutically acceptable salts of antisense oligonucleotide conjugates include, for example, the conventional non-toxic salts or quaternary ammonium salts of antisense oligonucleotides or antisense oligonucleotide conjugates 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 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 acid), sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothionic acid, etc.

[0226] In certain embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of the present disclosure may contain one or more acidic functional groups, and therefore can form pharmaceutically acceptable salts with pharmaceutically acceptable bases.In these cases, the term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic base addition salts of antisense oligonucleotide conjugates.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 oligonucleotide conjugates in their free acid form with a suitable base, such as a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary, or tertiary amine.Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts.Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc. (See, e.g., Berge et al., supra).

[0227] The salt form may be a complex of multiple cations or anions with the antisense oligomer conjugate, for example, the salt form may be a monohalide, dihalide, trihalide, tetrahalide, pentahalide, or hexahalide. IV. Formulations and Treatments

[0228] Formulations or compositions suitable for therapeutic delivery include formulating an effective amount of an antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. While it is possible for the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, of the present disclosure to be administered alone, it is preferable to administer it as a pharmaceutical formulation (composition).

[0229] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate is provided in a pharmaceutical composition formed by dissolving 0.005 mg / kg to about 300 mg / kg of the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof in an aqueous carrier solution. In some aspects, the aqueous carrier solution is sterile water or saline.

[0230] In some embodiments, the human patient is administered the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, for at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

[0231] The composition may be administered alone or in combination with another therapeutic agent. The additional therapeutic agent may be administered before, simultaneously with, or after administration of the composition containing the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof. For example, the composition may be administered in combination with a steroid and / or an antibiotic. The steroid may be a glucocorticoid or prednisone. Glucocorticoids, such as cortisol, are anti-inflammatory by regulating carbohydrate, fat, and protein metabolism, preventing phospholipid release, reducing eosinophil activity, and many other mechanisms. Mineralocorticoids, such as aldosterone, regulate electrolyte and water levels, primarily by promoting sodium retention in the kidney. Corticosteroids are a class of chemicals that includes steroid hormones naturally produced in the adrenal cortex of vertebrates and laboratory-synthesized analogs of these hormones. Corticosteroids are involved in a wide range of physiological processes, including stress response, immune response, and inflammation control, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. Corticosteroids include betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone.

[0232] Other agents that can be administered include antagonists of the ryanodine receptor, such as dantrolene, which have been shown to enhance antisense-mediated exon skipping in patient cells and mouse models of DMD (G. Kendall et al. Sci Tranl Med 4 164ra160 (2012), incorporated herein by reference).

[0233] Methods for delivery of nucleic acid molecules are described, for example, in Akhtar et al., 1992, Trends Cell Bio., 2:139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar; Sullivan et al., PCT WO 94 / 02595. These and other protocols can be utilized for delivery of virtually any nucleic acid molecule, including the antisense oligonucleotides and antisense oligonucleotide conjugates described herein.

[0234] As described in more detail below, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., as an immersion solution (aqueous or non-aqueous solution or suspension), tablet, e.g., buccal, sublingual, or tablet targeted for systemic absorption, bolus, powder, granule, paste for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension or sustained release formulation; (3) topical application, e.g., as a cream, ointment, or controlled-release patch or spray for application to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam; (5) sublingual administration; (6) intraocular administration; (7) transdermal administration; or (8) nasal administration.

[0235] Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) glycols, such as protease inhibitors. (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free distilled water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffers, (21) polyesters, polycarbonates, and / or polyanhydrides, and (22) other non-toxic compatible materials used in pharmaceutical formulations.

[0236] Additional non-limiting examples of drugs suitable for formulation with the antisense oligonucleotides or antisense oligonucleotide conjugates of the present disclosure include PEG-conjugated nucleic acids, phospholipid-conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) that can enhance drug entry into various tissues; biodegradable polymers, such as poly(DL-lactide-co-glycolide) microspheres for sustained delivery after implantation (Emerich, DF et al., J. Immunol. 1999, 103:111-114, 2001). al., 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutyl cyanoacrylate, which can deliver drugs across the blood-brain barrier and alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).

[0237] Compositions can be prepared that contain surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, branched, and unbranched, or a combination thereof, or long-circulating liposomes or stealth liposomes). The antisense oligonucleotides and antisense oligonucleotide conjugates of the present disclosure can also contain covalently attached PEG molecules of various molecular weights. These formulations provide a method for increasing drug accumulation in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear cell phagocytic system (MPS or RES), thereby allowing for extended blood circulation time and enhanced tissue exposure of encapsulated drugs (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to selectively accumulate in tumors, likely due to extravasation and entrapment in angiogenic target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA compared with conventional cationic liposomes, which are known to accumulate in tissues, particularly MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO96 / 10391; Ansell et al., International PCT Publication No. WO96 / 10390; Holland et al., International PCT Publication No. WO96 / 10392). Long-circulating liposomes also likely protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

[0238] Further embodiments include antisense oligonucleotides or antisense oligonucleotide conjugate compositions prepared for delivery, as described in US Patent Nos. 6,692,911, 7,163,695, and 7,070,807.In this regard, in one embodiment, antisense oligonucleotides or antisense oligonucleotide conjugates or pharmaceutically acceptable salts thereof are provided in a composition comprising a copolymer of lysine and histidine (HK) (described in US Patent Nos. 7,163,695, 7,070,807, and 6,692,911), either alone or in combination with PEG (for example, branched or unbranched PEG, or a mixture of both), in combination with PEG and a targeting moiety, or in combination with a crosslinker.In certain embodiments, antisense oligonucleotides or antisense oligonucleotide conjugates or pharmaceutically acceptable salts thereof are included in a composition comprising gluconate-modified polyhistidine or gluconyl-polyhistidine / transferrin-polylysine. Those skilled in the art will also recognize that amino acids with properties similar to His and Lys can be substituted within the compositions.

[0239] Wetting agents, emulsifying agents, and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0240] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0241] Formulations include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0242] In certain embodiments, the formulation comprises an excipient selected from cyclodextrin, cellulose, liposome, micelle-forming agent, e.g., bile acid, and polymeric carrier, e.g., polyester and polyanhydride, and an antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof. In certain embodiments, the formulation renders the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, of the present disclosure orally bioavailable.

[0243] Methods of preparing these formulations or compositions include the step of bringing into association the antisense oligonucleotide or antisense oligonucleotide conjugate of the present disclosure, or a pharmaceutically acceptable salt thereof, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0244] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing, as an active ingredient, a predetermined amount of the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof. The antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, of the present disclosure may also be administered as a bolus, electuary, or paste.

[0245] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, lozenges, etc.), the active ingredient may be incorporated into one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) disintegrants, such as sorbitol, ... (6) dissolution retarders, such as paraffin; (7) absorption enhancers, such as quaternary ammonium compounds and surfactants, such as poloxamers and sodium lauryl sulfate; (8) wetting agents, such as cetyl alcohol, glycerol monostearate, and nonionic surfactants; (9) absorbents, such as kaolin and bentonite clay; (10) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (11) coloring agents; and (12) release-controlling agents, such as crospovidone or ethylcellulose. For capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Similar types of solid compositions may also be used as fillers in soft and hard-shell gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0246] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface-active agents, or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0247] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules, pills, and granules, can be optionally obtained or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, using, for example, hydroxypropylmethylcellulose in various proportions to provide the desired release profile, other polymer matrices, liposomes, and / or microspheres. They can also be formulated for rapid release, such as by lyophilization. They can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents and can be compositions that release the active ingredient(s) only, or preferentially, in a certain portion of the digestive tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0248] The liquid dosage form for oral administration of the antisense oligonucleotide or antisense oligonucleotide conjugate of the present disclosure or its pharmaceutically acceptable salt includes pharmaceutically acceptable emulsion, microemulsion, solution, suspension, syrup and elixir.In addition to active ingredient, liquid dosage form can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and their mixtures.

[0249] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0250] In addition to the active compound, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0251] Formulations for rectal or vaginal administration may be presented as suppositories, which may be prepared by mixing one or more antisense oligonucleotides or antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, which are solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active compound(s).

[0252] The preparation or dosage form of topical or transdermal administration of antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt provided herein includes powder, spray, ointment, paste, cream, lotion, gel, solution, patch and inhalant.Antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt can be mixed with pharmaceutically acceptable carrier and any preservative, buffer or propellant that may be required under sterile conditions.Ointment, paste, cream and gel can contain excipients such as animal and vegetable fats, oils, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc and zinc oxide, or their mixtures, in addition to antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt.

[0253] Powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances, in addition to the antisense oligonucleotide or antisense oligonucleotide conjugate provided herein, or a pharmaceutically acceptable salt thereof. Sprays can also contain conventional propellants such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons such as butane and propane.

[0254] Transdermal patch has the additional advantage of providing the controlled delivery of the antisense oligonucleotide or antisense oligonucleotide conjugate of the present disclosure into the body.This dosage form can be prepared by dissolving or dispersing the antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt in suitable medium.Absorption enhancers can also be used to increase the flux of drug across the skin.The rate of this flux can be controlled by providing a rate-controlling membrane or dispersing the drug in a polymer matrix or gel, among other methods known in the art.

[0255] Pharmaceutical compositions suitable for parenteral administration may contain antisense oligonucleotides or antisense oligonucleotide conjugates or their pharmaceutically acceptable salts in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes that make the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0256] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action on the subject oligomers can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. Furthermore, prolonged absorption of the injectable dosage form can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0257] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injection in order to prolong the effect of drugs.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility, among other methods known in the art.Then, the absorption rate of drug depends on its dissolution rate, which in turn depends on crystal size and crystalline form.Alternatively, the delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in oil vehicle.

[0258] Injectable depot forms can be prepared by forming microencapsulated matrices of the subject oligomers in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of oligomer to polymer and the properties of the specific polymer used, the release rate of antisense oligonucleotides or antisense oligonucleotide conjugates can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable preparations can also be prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

[0259] When the antisense oligonucleotides or antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, of the present disclosure are administered as pharmaceuticals to humans and animals, they can be administered per se or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably 10 to 30%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0260] As described above, the formulations or preparations can be administered orally, parenterally, systemically, topically, rectally, or intramuscularly. They are typically administered in a form suitable for each administration route. For example, they are administered in the form of tablets or capsules, by injection, inhalation, eye lotion, ointment, suppository, etc., by injection, infusion, or inhalation, by topical administration with lotion or ointment, and by rectal administration with suppositories.

[0261] Regardless of the route of administration selected, antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt, which can be used in suitable hydrated form, and / or pharmaceutical composition can be formulated into pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.The actual dosage level of active ingredient in pharmaceutical composition can vary to obtain the amount of active ingredient that is effective for achieving the desired therapeutic response for specific patient, composition and mode of administration, without being unacceptably toxic to human patients.

[0262] The selected dosage level will depend upon a variety of factors, including the activity of the particular antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, or an ester, salt, or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, the rate and extent of absorption, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, employed, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0263] Any doctor or veterinarian with ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required.For example, doctor or veterinarian can start the dosage of antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt used in pharmaceutical composition at a level lower than the level required to achieve desired therapeutic effect, and gradually increase the dosage until desired effect is achieved.Generally, the suitable daily dose of antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt is the amount of compound that is the lowest effective dose to produce therapeutic effect.This effective amount generally depends on the factors listed above.

[0264] In some embodiments, the dose of the antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO), or pharmaceutical salt thereof, is about 0.005 mg / kg to about 300 mg / kg. In some embodiments, the dose is at least 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg. In some embodiments, the dose is about 0.005 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 80 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 20 mg / kg to about 80 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 80 mg / kg, or about 80 mg / kg to about 300 mg / kg. In some embodiments, the dose is about 0.05 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg.

[0265] In some embodiments, the antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO), or pharmaceutical salt thereof, is generally administered at regular intervals (e.g., weekly, every two weeks, three weeks, four weeks, or monthly). For example, the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, may be administered by intravenous infusion once a week, every two weeks, three weeks, four weeks, or once a month. In one embodiment, the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, may be administered by intravenous infusion once a week, every two weeks, three weeks, four weeks, or once a month. In some embodiments, the antisense oligonucleotide (e.g., PMO) or antisense oligonucleotide conjugate (e.g., PPMO), or a pharmaceutically acceptable salt thereof, is administered every four weeks.

[0266] Subsequent to administration, a dose of antibiotics, steroids, or other therapeutic agents may be administered or may be administered simultaneously. The treatment regimen may be adjusted (dosage, frequency, route, etc.) as indicated based on the results of immunoassays, other biochemical tests, and physiological tests of the human patient under treatment.

[0267] Nucleic acid molecules can be administered to cells by a variety of methods known to those skilled in the art, including, but not limited to, liposomal encapsulation, iontophoresis, or incorporation into other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, as described herein and known in the art. In certain embodiments, microemulsification technology can be utilized to improve the bioavailability of lipophilic (water-insoluble) pharmaceutical agents. Examples include trimetrine (Dordunoo, SK, et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991), and REV 5901 (Sheen, PC, et al., J. Pharm Sci 80(7), 712-714, 1991. Among other advantages, microemulsification provides enhanced bioavailability by preferentially directing absorption into the lymphatic system instead of the circulatory system, thereby bypassing the liver and preventing breakdown of the compound in the hepatobiliary circulation.

[0268] The formulation contains micelles formed from the antisense oligonucleotides or antisense oligonucleotide conjugates provided herein, or pharmaceutically acceptable salts thereof, and at least one amphiphilic carrier, wherein the micelles have an average diameter of less than about 100 nm. More preferred embodiments provide micelles having an average diameter of less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter of less than about 30 nm or even less than about 20 nm.

[0269] While any suitable amphiphilic carrier is contemplated, currently preferred carriers are generally those that are generally recognized as safe (GRAS) and are capable of both solubilizing the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, and microemulsifying it at a later stage when the solution comes into contact with a complex aqueous phase (such as that found in the human gastrointestinal tract). Amphiphilic components that meet these requirements typically have an HLB (hydrophilic-lipophilic balance) value of 2-20, and their structures contain straight-chain aliphatic radicals ranging from C-6 to C-20. Examples include polyethylene glycolated fatty acid glycerides and polyethylene glycol.

[0270] Examples of amphiphilic carriers include saturated and monounsaturated polyethylene glycolated fatty acid glycerides, such as those obtained from various fully or partially hydrogenated vegetable oils. Such oils can advantageously consist of tri-, di-, and mono-fatty acid glycerides and di- and mono-polyethylene glycol esters of the corresponding fatty acids; particularly preferred fatty acid compositions include capric acid 4-10%, capric acid 3-9%, lauric acid 40-50%, myristic acid 14-24%, palmitic acid 4-14%, and stearic acid 5-15%. Another useful class of amphiphilic carriers includes partially esterified sorbitan and / or sorbitol with saturated or monounsaturated fatty acids (SPAN® series) or the corresponding ethoxylated analogs (TWEEN® series).

[0271] Commercially available amphiphilic carriers, including the Gelucire series, Labrafil, Labrasol, or Lauroglycol (all manufactured and distributed by Gattefosse Corporation, Saint-Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono- and di-laurate, lecithin, polysorbate 80, and the like (manufactured and distributed by numerous companies in the United States and around the world), may be particularly useful.

[0272] In certain embodiments, delivery can be achieved by using liposome, nanocapsule, microparticle, microsphere, lipid particle, vesicle, etc. to introduce antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt into suitable host cells.In particular, antisense oligonucleotide or antisense oligonucleotide conjugate, or its pharmaceutically acceptable salt, can be formulated for delivery by being encapsulated in lipid particle, liposome, vesicle, nanosphere, nanoparticle or similar.The formulation and use of such delivery vehicle can be carried out using known and conventional technology.

[0273] Hydrophilic polymers suitable for use with antisense oligonucleotides or antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, are readily water-soluble, can be covalently attached to vesicle-forming lipids, and are tolerated in vivo without toxic effects (i.e., are biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic acid (also called polylactide), polyglycolic acid (also called polyglycolide), polylactic acid-polyglycolic acid copolymers, and polyvinyl alcohol. In certain embodiments, the polymer has a molecular weight of about 100 or 120 daltons up to about 5,000 or 10,000 daltons, or about 300 daltons to about 5,000 daltons. In other embodiments, the polymer is polyethylene glycol having a molecular weight of about 100 to about 5,000 daltons, or about 300 to about 5,000 daltons. In certain embodiments, the polymer is 750 dalton polyethylene glycol (PEG(750)). Polymers may also be defined by the number of monomers therein, with preferred embodiments utilizing polymers of at least about three monomers, such PEG polymers consisting of three monomers (about 150 daltons).

[0274] Other hydrophilic polymers that may be suitable for use with the antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethylcellulose.

[0275] In certain embodiments, the formulation comprises a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

[0276] Cyclodextrins are cyclic oligosaccharides consisting of six, seven, or eight glucose units, designated by the Greek letters α, β, or γ, respectively. The glucose units are linked by α-1,4-glucosidic bonds. As a result of the chair conformation of the sugar units, all secondary hydroxyl groups (at C-2 and C-3) are located on one side of the ring, while all primary hydroxyl groups at C-6 are located on the other. As a result, the exterior is hydrophilic, making cyclodextrins water-soluble. In contrast, the cavities of cyclodextrins are hydrophobic because they are lined by hydrogen atoms at C-3 and C-5, as well as by ether-like oxygens. These matrices allow complexation with a variety of relatively hydrophobic compounds, including steroid compounds such as 17α-estradiol (see, e.g., van Uden et al., Plant Cell Tiss. Org. Cult. 38:1-3-113 (1994)). Complexation occurs through van der Waals interactions and hydrogen bond formation. For a general review of the chemistry of cyclodextrins, see Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822 (1994).

[0277] The physicochemical properties of cyclodextrin derivatives depend largely on the type and degree of substitution. For example, their water solubility ranges from insoluble (e.g., triacetyl-beta-cyclodextrin) to 147% soluble (w / v) (G-2-beta-cyclodextrin). Furthermore, they are soluble in many organic solvents. The properties of cyclodextrins allow for the control of the solubility of various formulation components by increasing or decreasing their solubility.

[0278] Numerous cyclodextrins and methods for their preparation have been described. For example, Parmeter (I) et al. (U.S. Pat. No. 3,453,259) and Gramera et al. (U.S. Pat. No. 3,459,731) described electrically neutral cyclodextrins. Other derivatives include cyclodextrins with cationic character [Parmeter (II), U.S. Pat. No. 3,453,257], insoluble crosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), and cyclodextrins with anionic character [Parmeter (III), U.S. Pat. No. 3,426,011]. Among cyclodextrin derivatives with anionic character, carboxylic acids, phosphorous acids, phosphinic acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfinic acids, and sulfonic acids are attached to the parent cyclodextrin [Parmeter (III), see above]. Additionally, sulfoalkyl ether cyclodextrin derivatives have been described by Stella et al. (US Pat. No. 5,134,127).

[0279] Liposomes consist of at least one lipid bilayer membrane surrounding an aqueous interior compartment. Liposomes can be characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and typically range in diameter from 0.02 to 0.05 μm, while large unilamellar vesicles (LUVs) are typically larger than 0.05 μm. Large oligolamellar vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 μm. Liposomes with several non-concentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are called multivesicular vesicles.

[0280] Liposome-containing formulations can contain antisense oligonucleotides or antisense oligonucleotide conjugates or pharmaceutically acceptable salts thereof, and liposome membranes are formulated to provide liposomes with increased loading capacity.Alternatively, or in addition, antisense oligonucleotides or antisense oligonucleotide conjugates or pharmaceutically acceptable salts thereof can be contained within or adsorbed onto the liposome bilayer of liposomes.Antisense oligonucleotides or antisense oligonucleotide conjugates can be aggregated with lipid surfactants and loaded into the inner space of liposomes.In these cases, liposome membranes are formulated to resist the disruptive effect of active agent-surfactant aggregates.

[0281] According to one embodiment, the lipid bilayer of the liposome contains lipids derivatized with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the interior space enclosed by the liposome and from the exterior of the lipid bilayer into the surrounding environment.

[0282] The active agent contained within the liposome is in solubilized form. Aggregates of surfactant and active agent (such as emulsions or micelles containing the active agent of interest) may be trapped within the interior space of the liposome. The surfactant acts to disperse and solubilize the active agent and may be selected from any suitable aliphatic, alicyclic, or aromatic surfactant, including, but not limited to, biocompatible lysophosphatidylcholines (LPGs) of various chain lengths (e.g., about C14 to about C20). Polymer-derivatized lipids, such as PEG-lipids, may also be used for micelle formation, as they act to inhibit micelle / membrane fusion and the addition of polymers to the surfactant molecule reduces the CMC of the surfactant, aiding in micelle formation. Surfactants with CMCs in the micromolar range are preferred, and higher CMC surfactants may be used to prepare micelles trapped within the liposome.

[0283] Liposomes can be prepared by any of a variety of techniques known in the art. See, for example, U.S. Patent No. 4,235,871, published PCT application WO96 / 14057, New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104; Lasic DD, Liposomes from physics to applications, Elsevier Science Publishers BV, Amsterdam, 1993. For example, liposomes can be prepared by diffusing a lipid derivatized with a hydrophilic polymer into preformed liposomes at a lipid concentration corresponding to the final molar percentage of the desired derivatized lipid in the liposome, e.g., by exposing the preformed liposomes to micelles composed of lipid-grafted polymers. Liposomes containing hydrophilic polymers can also be formed by homogenization, lipid field hydration, or extrusion techniques, as known in the art.

[0284] In another exemplary formulation procedure, active agent is first dispersed by sonication in lysophosphatidylcholine or other low CMC surfactants (including polymer-grafted lipids) that easily solubilize hydrophobic molecules.Then, the resulting micelle suspension of active agent is used to rehydrate the dried lipid sample containing suitable mole percent of polymer-grafted lipids or cholesterol.Then, the lipid and active agent suspension is formed into liposomes using extrusion techniques known in the art, and the resulting liposomes are separated from the unencapsulated solution by standard column separation.

[0285] In one embodiment, liposomes are prepared to have a substantially uniform size within a selected size range. One effective sizing method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes with a selected uniform pore size, the pore size of which corresponds roughly to the largest size of liposomes that will be extruded through the membrane. See, for example, U.S. Patent No. 4,737,323 (April 12, 1988). In certain embodiments, reagents such as DharmaFECT® and Lipofectamine® can be used to introduce polynucleotides or proteins into cells.

[0286] The release characteristics of a formulation depend on the encapsulation material, the concentration of the encapsulated drug, and the presence of release modifiers. For example, release can be engineered to be pH-dependent, using a pH-sensitive coating that releases only at low pH, such as found in the stomach, or at high pH, ​​such as found in the intestine. Enteric coatings can be used to prevent release from occurring until after passage through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach, followed by a later release in the intestine. Release can also be engineered by including salts or pore-forming agents, which can increase water uptake or drug release by diffusion from the capsule. Excipients that modify the solubility of the drug can also be used to control the release rate. Agents that enhance matrix degradation or release from the matrix can also be incorporated. They can be added to the drug, added as a separate phase (i.e., as microparticles), or co-dissolved in the polymer phase, depending on the compound. In most cases, the amount should be between 0.1 and 30 percent (w / w polymer). Types of degradation accelerators include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine, as well as surfactants such as Tween® and Pluronic®. Pore-forming agents (i.e., water-soluble compounds such as inorganic salts and sugars) that add microstructure to the matrix are added as microparticles. The range is typically 1 to 30 percent (w / w polymer).

[0287] Uptake can also be manipulated by changing the residence time of the particles in the intestine. This can be achieved, for example, by coating the particles with a mucoadhesive polymer or by selecting it as the encapsulating material. Examples include most polymers with free carboxyl groups, such as chitosan, cellulose, and especially polyacrylates (as used herein, polyacrylate refers to polymers containing acrylate groups and modified acrylate groups such as cyanoacrylate and methacrylate).

[0288] In addition to the methods provided herein, antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt can be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.Antisense oligonucleotide or antisense oligonucleotide conjugate or its pharmaceutically acceptable salt, and their corresponding formulations can be administered alone or in combination with other therapeutic strategies in the treatment of muscular dystrophy, such as myoblast transplantation, stem cell therapy, administration of aminoglycoside antibiotics, proteasome inhibitors, and upregulation therapy (for example, upregulation of utrophin, the autosomal paralog of dystrophin).

[0289] The routes of administration described are intended as a guide only, as one skilled in the art can readily determine the optimum route of administration and any dosage for any particular animal and condition. Several approaches have been attempted to introduce functional new genetic material into cells, both in vitro and in vivo (Friedmann (1989) Science, 244:1275-1280). These approaches include incorporation of the gene to be expressed into modified retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl.:5074S-5079S), into non-retroviral vectors (e.g., adeno-associated virus vectors) (Rosenfeld, et al. (1992) Cell 68:143-155; Rosenfeld, et al. (1991) Science 252:431-434), or liposome-mediated delivery of transgenes linked to heterologous promoter-enhancer elements (Friedmann (1989) supra; Brigham, et al. (1989) Am. J. Med. Sci. 298:278-281; ​​Nabel, et al. (1990) Science 249:1285-1288; Hazinski, et al. al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209, and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855), ligand-specific, binding to cation transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624), or the use of naked DNA, expression vectors (Nabel et al. (1990), supra; Wolff et al. (1990) Science, 247:1465-1468). Direct injection of transgenes into tissues results in only local expression (Rosenfeld (1992) supra; Rosenfeld et al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra).The group of Brigham et al. (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) has only reported in vivo transfection of mouse lungs after either intravenous or intratracheal administration of DNA-liposome complexes. An example of a review article on human gene therapy procedures is Anderson, Science (1992) 256:808-813. V. Kit

[0290] The present invention also provides kits for treating human patients with genetic diseases, comprising at least one antisense oligonucleotide or antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof (e.g., one or more antisense oligonucleotides capable of targeting a specific sequence, such as one or more of exons 1-79 (e.g., exon 51) of the dystrophin gene), packaged in a suitable container together with instructions for use. The kit may also contain peripheral reagents, such as buffers, stabilizers, and the like. Those skilled in the art will appreciate that the above-described methods have broad application for identifying antisense molecules suitable for use in treating many other diseases. [Example]

[0291] (VII) Examples Example 1: Exon skipping in cynomolgus monkeys over 28 days following a single intravenous administration of PPMO#1 A total of 20 naive male cynomolgus monkeys were enrolled in the study, with four males in Group A and eight males each in Groups B and C. All animals received a one-hour IV infusion on Day 1. Doses were administered via the saphenous or cephalic vein using an intravenous catheter. Animals in Group A received a vehicle control. Groups B and C received PPMO#1 (as the 6HCl salt) at dose levels of 30 and 60 mg / kg, respectively. The PPMO#1 concentrations in Groups B and C were 3 and 6 mg / mL, respectively, and the dose volume for all animals was 10 mL / kg, as shown in Table 2. Table 2 [Table 2] result:

[0292] Biceps samples were homogenized and processed for dd-PCR to determine the level of exon 51 skipping over 28 days. No exon skipping was detected in biopsy samples from the vehicle-treated group. Exon skipping was detected in animals treated with 30 mg / kg and 60 mg / kg. For both the 30 and 60 mg / kg groups, exon skipping began on day 2 and continued until day 28 (end of study). At day 28, exon skipping levels were 13.3% ± 8.6 and 37.2% ± 7.2 for the 30 and 60 mg / kg doses, respectively. Exon skipping data are shown in Figure 1.

[0293] The level of exon skipping was dose-dependent, with no exon skipping measured in vehicle-treated samples and the highest level detected in the 60 mg / kg group. Exon skipping was measured as early as 24 hours after administration and continued at all time points up to the final study time point, day 28. Exon skipping levels at day 28 were 13.3% ± 8.6 and 37.2% ± 7.2 for the 30 and 60 mg / kg doses, respectively. It is possible that exon skipping persisted beyond 28 days after a single dose. The persistence of exon skipping for 28 days in biceps biopsy samples can be used to support monthly (i.e., once every 4 weeks) dosing regimens in the clinic. The data demonstrate that administering doses of 30 mg / kg or 60 mg / kg every 4 weeks can result in moderately high exon skipping with a duration of effect for at least 28 days. The high potency of PPMO#1, along with its long duration of effect, may translate into therapeutically effective protein expression of functionally truncated dystrophin in DMD patients. Example 2: Evaluation of Renal Safety Parameters Based on Clinical Pathology and Renal Histopathological Evaluation

[0294] A total of 28 naive male cynomolgus monkeys were enrolled in the study, with 4 in Group 1 and 12 each in Groups 2 and 3. All animals received a 1-hour IV infusion on days 1, 29, 57, and 85. Animals in Group 1 received a vehicle control. Groups 2 and 3 received PPMO#1 (as the 6HCl salt) at dose levels of 30 and 60 mg / kg, respectively. The PPMO#1 concentrations in Groups 2 and 3 were 3 and 6 mg / mL, respectively, and the dose volume for all animals was 10 mL / kg.

[0295] All animals were monitored throughout the study using clinical observations and body weight measurements. Blood was collected pre-dose and 1, 2, 4, 8, 12, 16, and 24 hours after the start of each infusion to provide plasma for PK analysis, and urine was collected 8 and 24 hours post-dose. Blood was collected pre-dose on Day 1 and on Days 30, 58, and 86 for hematology and clinical testing. Muscle biopsies and necropsy tissue collection were performed according to the schedule in Table 3 below. Table 3: Study design [Table 3]

[0296] Administration of PPMO#1 (as the 6HCl salt) by intravenous infusion four times on days 1, 29, 57, and 85 was clinically well tolerated under the conditions of this study in cynomolgus monkeys at dose levels of 30 and 60 mg / kg / dose. The highest dose in this study (60 mg / kg) produced a C of 266,000 ± 15,500 ng / mL on day 1. max , and 415,000 ± 254,000 h * AUC in ng / mL inf , and a C of 312000 ± 38100 ng / mL on day 85. max , and 479,000 ± 84,900 h * AUC in ng / mL infThe main microscopic observation in the kidney was the accumulation of basophilic granules in the renal ductal epithelium, which was not considered adverse. There was some reduction in red blood cell mass in the 30 and 60 mg / kg groups, but there were no time- or dose-dependent changes in overall hematological or blood chemistry and urinalysis parameters indicative of significant systemic changes. Example 3: Single Ascending Dose Study

[0297] Patients receive a single dose of PPMO#1 (as the 6HCl salt) (either 0.3, 1.0, 2.0, 4.0, or 6.0 mg / kg) administered as an intravenous (IV) infusion.

[0298] Selected selection criteria: I 2. Have a genetic diagnosis of DMD and an out-of-frame deletion mutation in the DMD gene that is suitable for exon 51 skipping therapy. I 4. Have been receiving a stable dose of oral corticosteroids for at least 12 weeks prior to study drug administration, or have been corticosteroid-free for at least 12 weeks prior to study drug administration.

[0299] Selected exclusion criteria: A. Left ventricular ejection fraction (LVEF) less than 40% based on an echocardiogram performed within the last 3 months. B. Have a QT interval corrected by Fridericia's formula (QTcF) ≥ 450 milliseconds in the ECG. C. Have a forced vital capacity (FVC) less than 40% of predicted within 3 months of screening. D. Platelet count is less than 150 x 103 μL.

[0300] Evaluation criteria:

[0301] Safety endpoints: Safety endpoints included AEs, clinical laboratory tests, safety biomarkers of renal function, ECG, ECHO, physical examination, and vital signs.

[0302] Pharmacokinetic endpoints: The following PK parameters will be calculated: maximum drug concentration (C max ), time to reach maximum blood concentration (T max ), from time 0 to the final measured concentration (AUC 0-t ) and the area under the drug concentration-time curve (AUC) extrapolated to infinity (AUC 0-∞ ), apparent terminal elimination rate constant (λz), apparent terminal elimination half-life (t 1 / 2 ), plasma clearance (CL), terminal volume of distribution (V z ), and steady-state volume of distribution (V ss ). Example 4: Multiple Ascending Dose Study

[0303] Patients will be assigned to one of four cohorts: 4.0, 10.0, 16.0, or 20.0 mg / kg of PPMO#1 (as the 6HCl salt). Part A Dosing and Study Evaluations:

[0304] PPMO#1 (as the 6HCl salt) will be administered intravenously every 4 weeks (defined as every 28 days [± 4 days]). Each cohort will complete at least 12 weeks of treatment. Patients will continue to receive the drug at their assigned cohort's dose level every 4 weeks until the maximum tolerated dose (MTD) is identified.

[0305] All patients will undergo additional muscle and skin biopsies (to assess exon skipping and exploratory exon skipping, dystrophin protein production, and tissue PPMO levels) at week 12 of participation in Part A.

[0306] Assessments of pulmonary and physical function and quality of life will be performed every 12 weeks (± 4 days) after the first dose.

[0307] Transition from Part A to Part B of this study will occur once the MTD is determined in Part A. Part B - Dose Expansion

[0308] Patients will progress directly from Part A to Part B once the MTD is determined in Part A. Once the MTD is established, the next dose administered to all patients will be the MTD. Treatment and observation period

[0309] Patients enrolled in Part B of the study will receive additional treatment with PPMO#1 (as the 6HCl salt) administered IV every 4 weeks at the MTD determined in Part A. Patients will continue to receive PPMO#1 every 4 weeks for a minimum of 24 weeks at the MTD determined in Part A. Clinical laboratory tests, other safety assessments, and functional and quality of life assessments performed in Part A will also be conducted in Part B.

[0310] All patients will also undergo additional muscle and skin biopsies (to assess exon skipping and exploratory exon skipping, dystrophin protein production, and tissue PPMO levels) at week 24 of Part B participation. Safety follow-up period (required for all patients)

[0311] All patients will return to the clinic for an End-of-Study / Early Discontinuation Visit up to 4 weeks (± 4 days) after the last dose of PPMO#1. This includes both patients who complete the study and those who discontinue participation early. Vital sign measurements, physical examination findings, laboratory tests, ECG findings, changes in concomitant medications, and AEs will be assessed at this visit. Safety and efficacy data collection will not be interrupted at any time during this study.

[0312] Selected selection criteria: A. Patients with a genetic diagnosis of DMD and an out-of-frame deletion mutation in the DMD gene suitable for exon 51 skipping therapy. B. Have been receiving a stable dose of oral corticosteroids for at least 12 weeks prior to study drug administration, or have not been receiving corticosteroids for at least 12 weeks prior to study drug administration.

[0313] Selected exclusion criteria: A. LVEF < 40.0% based on an echocardiogram performed within the last 12 weeks. B. Having a QT interval corrected by Fridericia's formula ≥ 450 milliseconds in the ECG. C. Have an FVC < 40.0% predicted (according to the American Thoracic Society / European Respiratory Society criteria) within 12 weeks.

[0314] Dose / Route / Regimen (Test Article):

[0315] In Part A (MAD for dose determination), patients will receive escalating doses of PPMO#1 administered by 60-minute (±5-minute) IV infusion every 4 weeks, starting at the dose level of their assigned cohort (4.0, 10.0, 16.0, or 20.0 mg / kg).

[0316] In Part B (dose expansion), patients will receive doses of PPMO#1 every 4 weeks at the MTD determined in Part A, administered by 60-minute (±5-minute) IV infusion. *********************

[0317] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The present invention provides, for example, the following items. (Item 1) 1. A method of treating a human patient with Duchenne muscular dystrophy, said method comprising: administering to the human patient once every four weeks a therapeutically effective amount of an antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide conjugate comprises a cell membrane-permeable peptide covalently attached to an oligonucleotide; The method, wherein the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof induces exon skipping in the human dystrophin gene. (Item 2) Item 3. The method according to Item 1, wherein the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof comprises a cell membrane-permeable peptide that is an arginine-rich peptide. The antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof is -GLY-R5-R a (SEQ ID NO: 59), -R5-R a (SEQ ID NO: 60), -GLY-R6-R a (SEQ ID NO: 57) or -R6-R a (SEQ ID NO: 58), wherein R is arginine, and R a is hydrogen or an acyl group. (Item 4) 4. The method of any one of items 1 to 3, wherein the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof induces skipping of a target region of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of dystrophin pre-mRNA. (Item 5) The antisense oligonucleotide conjugate has the formula (I): [ka] or a pharmaceutically acceptable salt thereof; each Nu is a nucleobase that together form a targeting sequence, T' in formula (I) is a moiety selected from: [ka] R 100 is a cell membrane-penetrating peptide, R 200 is hydrogen, R 1 is C1-C6 alkyl, Each Nu from 1 to (n+1) and 5' to 3' corresponds to the nucleobase in one of the following: [Table 22-1] [Table 22-2] 5. The method according to any one of items 1 to 4, wherein T in each of SEQ ID NOs: 1 to 51 is thymine or uracil. (Item 6) Item 6. The method of item 5, wherein each Nu in the 5' to 3' positions of 1 to (n+1) of the antisense oligonucleotide conjugates or pharmaceutically acceptable salts thereof corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17. (Item 7) 2. The method of claim 1, wherein the antisense oligonucleotide conjugate is PPPMO#1, PPMO#2, or PPMO#3, or a pharmaceutically acceptable salt thereof. (Item 8) The method comprises administering to a patient a therapeutically effective amount of an antisense oligonucleotide conjugate according to formula (II). [ka] or a pharmaceutically acceptable salt thereof, wherein each Nu from 1 to (n+1) and 5' to 3' corresponds to said nucleobase in one of the following: [Table 23-1] [Table 23-2] 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] Item 1. The method according to item 1, (Item 9) Item 9. The method of item 8, wherein 1 to (n+1) of the antisense oligonucleotide conjugate of formula (II) or a pharmaceutically acceptable salt thereof and each Nu from 5' to 3' corresponds to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 17. (Item 10) 10. The method of any one of items 1 to 9, wherein the antisense oligonucleotide conjugate is in free base form. (Item 11) 10. The method of any one of items 1 to 9, wherein the antisense oligonucleotide conjugate is a pharmaceutically acceptable salt. (Item 12) 12. The method of claim 11, wherein the antisense oligonucleotide conjugate is a halide salt. (Item 13) 13. The method of any one of items 11 to 12, wherein the antisense oligonucleotide conjugate is an HCl salt. (Item 14) 14. The method of claim 13, wherein the HCl salt of the antisense oligonucleotide conjugate is a hexaHCl salt. (Item 15) 15. The method of any one of items 1 to 14, wherein the therapeutically effective amount of the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof is provided in a pharmaceutical composition formed by dissolving 0.005 mg / kg to about 300 mg / kg of the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, in an aqueous carrier solution. (Item 16) The therapeutically effective amount of the antisense oligonucleotide conjugate is at least 0.05 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 10 mg / kg, 16 mg / kg, 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 80 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, or 275 mg / kg, about 0.005 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 80 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg Item 16. The method of item 15, wherein the dose is about 25 mg / kg, about 20 mg / kg to about 80 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 80 mg / kg, about 80 mg / kg to about 300 mg / kg, about 0.05 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 60 mg / kg, about 80 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, or about 300 mg / kg. (Item 17) 17. The method of any one of items 1 to 16, wherein the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof is administered intravenously.

Claims

[Claim 1] The invention described in this specification.

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