Treatment of Duchenne muscular dystrophy using peptide-oligonucleotide conjugates

Abstract

A method of treating a subject with Duchenne muscular dystrophy is disclosed. The method includes administering to the subject (e.g., a subject for whom exon 51 skipping is indicated) 1 mg / kg to 60 mg / kg of a complex of an oligonucleotide and a peptide linked to the oligonucleotide via a covalent bond or a linker. The peptide includes at least one cationic domain including at least four amino acid residues and at least one hydrophobic domain including at least three amino acid residues, where the peptide includes a total of 7 to 40 amino acid residues, and the at least one cationic domain includes a β-alanine residue in combination with an arginine residue and / or a histidine residue. The oligonucleotide includes a total of 12 to 40 consecutive nucleobases, where at least 12 consecutive nucleobases are complementary to a target sequence of the human dystrophin gene.

Description

[Technical field]

[0001] The present invention relates to peptide conjugates of antisense oligonucleotides, compositions containing same, and methods of their use. [Background technology]

[0002] Nucleic acid medicines are genomic medicines that have the potential to transform human healthcare. Research has demonstrated that such therapeutics may be applicable across a broad range of disease areas, including neuromuscular diseases. The application of antisense oligonucleotide-based methods to modulate pre-mRNA splicing in the neuromuscular disease Duchenne muscular dystrophy (DMD) has positioned this monogenic disorder at the forefront of advances in precision medicine.

[0003] However, therapeutic development of these promising antisense therapies has been hampered by poor cell permeability and poor distribution properties - challenges that are further accentuated by the large volume and dispersity of the muscle tissue matrix in DMD.

[0004] DMD affects 1 in 3500 newborn boys. This severe X-linked recessive disorder is caused by mutations in the DMD gene, which encodes the dystrophin protein. The disease is characterized by progressive muscle degeneration and weakness, leading to respiratory failure and cardiac complications, and ultimately to premature death. The majority of mutations underlying DMD are genomic out-of-frame deletions that cause premature truncations of the open reading frame, resulting in the absence of the dystrophin protein.

[0005] Exon skipping therapy uses splice-switching antisense oligonucleotides (SSOs) to target specific regions of the DMD transcript and induce the elimination of individual exons, resulting in the restoration of the abnormal reading frame and, consequently, the production of an internally deleted but partially functional dystrophin protein. Despite the undoubted potential of exon skipping therapy with antisense oligonucleotides for DMD, successful application of this approach is currently limited by relatively inefficient targeting of skeletal muscle and poor targeting of single-stranded oligonucleotides to other affected tissues, such as the heart. In September 2016, the US Food and Drug Administration (FDA) granted accelerated approval of eteplirsen, a modulator of exon 51 splicing. This was the first FDA-approved oligonucleotide to modulate splicing in the US, but the level of dystrophin restoration was disappointing, at approximately 1% of normal levels. Comparison with the allelic disease Becker muscular dystrophy (BMD) and experiments in mdx mice indicate that homogeneous sarcolemmal dystrophin expression, at least −15% of wild-type, is required to protect muscles from exercise-induced damage.

[0006] Therefore, novel antisense oligonucleotide-based therapies are needed to treat devastating genetic disorders such as DMD. Summary of the Invention

[0007] Typically, the invention provides a method of treating a subject with Duchenne muscular dystrophy.

[0008] In one embodiment, the method comprises administering 1 mg / kg to 60 mg / kg of a conjugate of an oligonucleotide and a peptide covalently or via a linker to the oligonucleotide, wherein the peptide comprises at least one cationic domain comprising at least four amino acid residues and at least one hydrophobic domain comprising at least three amino acid residues, wherein the peptide comprises a total of 7 to 40 amino acid residues, and wherein the at least one cationic domain comprises β-alanine residues in combination with arginine and / or histidine residues, and wherein the oligonucleotide comprises a total of 12 to 40 contiguous nucleobases, wherein at least 12 contiguous nucleobases are complementary to a target sequence of the human dystrophin gene.

[0009] In some embodiments, the oligonucleotide comprises the following sequence: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106). In some embodiments, the oligonucleotide is SEQ ID NO: 107 5′-ACCAGAGUAACAGUCUGAGUAGGAGC-3′ 108 5′-CUCAUACCUUCUGCUUGAUGAUC-3′ 109 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ 110 5′-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3′ 111 5′-ACAUCAGGAAGAUGGCAUUUCUAG-3′ 112 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ 113 5′-AUCAUUUUUUCUCAUACCUUCUGCUAG-3′ 114 5′-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3′ 115 5′-CACCCACCAUCACCCUCUGUG-3′ 116 5′-AUCAUCUCGUUGAUAUUCCUCAA-3′ and thymine substituted analogs thereof.

[0010] In some embodiments, each cationic domain has a length between 4 and 12 amino acid residues. In some embodiments, each cationic domain has a length between 4 and 7 amino acid residues. In some embodiments, each cationic domain comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cationic amino acids. In some embodiments, each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% arginine and / or histidine residues.

[0011] In some embodiments, each cationic domain comprises one of the following sequences: RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB[Hyp]B (SEQ ID NO:17), R[Hyp]H[Hyp]HB (SEQ ID NO:18), R[Hyp]RR[Hyp]R (SEQ ID NO:19), or any combination thereof. In some embodiments, each cationic domain comprises one of the following sequences or consists of one of the following sequences: RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB[Hyp]B (SEQ ID NO:17), R[Hyp]H[Hyp]HB (SEQ ID NO:18), R[Hyp]RR[Hyp]R (SEQ ID NO:19), or any combination thereof.

[0012] In some embodiments, the peptide comprises two cationic domains.

[0013] In some embodiments, each hydrophobic domain has a length of 3 to 6 amino acids, preferably each hydrophobic domain has a length of 5 amino acids. In some embodiments, each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids. In some embodiments, each hydrophobic domain comprises phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues, preferably each hydrophobic domain comprises or consists of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues. In some embodiments, the peptide comprises one hydrophobic domain. In some embodiments, each hydrophobic domain comprises one of the following sequences: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26), or any combination thereof. In some embodiments, each hydrophobic domain comprises or consists of one of the following sequences: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26), or any combination thereof.

[0014] In some embodiments, the peptide comprises or consists of two cationic domains and one hydrophobic domain, hi some embodiments, the peptide comprises or consists of one hydrophobic core domain flanked by two cationic arm domains.

[0015] In some embodiments, the peptide is RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), HBHBR (SEQ ID NO:17), HBHBR (SEQ ID NO:18), HBHBR (SEQ ID NO:19), HBHBR (SEQ ID NO:20), HBHBR (SEQ ID NO:21), HBHBR (SEQ ID NO:22), HBHBR (SEQ ID NO:23), HBHBR (SEQ ID NO:24), HBHBR (SEQ ID NO:25), HBHBR (SEQ ID NO:26), HBHBR (SEQ ID NO:27), HBHBR (SEQ ID NO:28), HBHBR (SEQ ID NO:29), HBHBR (SEQ ID NO:30), HBHBR (SEQ ID NO:31), HBHBR (SEQ ID NO:32), HBHBR (SEQ ID NO:33), HBHBR (SEQ ID NO:34), HBHBR (SEQ ID NO:35), HBHBR (SEQ ID NO:36), HBHBR (SEQ ID NO:37), HBHBR (SEQ ID NO:38), HBHBR (SEQ ID NO:39), HBHBR (SEQ ID NO:40 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), and R[Hyp]RR[Hyp]R (SEQ ID NO: 19), respectively, flanked by two cationic arm domains each comprising a sequence selected from YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), and WWPW (SEQ ID NO: 26).

[0016] In some embodiments, the peptide comprises one of the following sequences or consists of one of the following sequences: RBRRBRRFQILYRBRBR (SEQ ID NO:27), RBRRBRRYQFLIRBRBR (SEQ ID NO:31), RBRRBRRILFQYRBRBR (SEQ ID NO:32), RBRRBRFQILYBRBR (SEQ ID NO:35), RBRRBRRFQILYRBHBH (SEQ ID NO:37), RBRRBRRFQILYHBHBR (SEQ ID NO:38), and RBRRBRFQILYRBHBH (SEQ ID NO:44).

[0017] In some embodiments, the peptide comprises or has the amino acid sequence: RBRRBRFQILYBRBR (SEQ ID NO: 35). In some embodiments, the peptide comprises or has the amino acid sequence: RBRRBRRFQILYRBHBH (SEQ ID NO: 37). In some embodiments, the peptide comprises or has the amino acid sequence: RBRRBRFQILYRBHBH (SEQ ID NO: 44).

[0018] In some embodiments, the peptide is attached to the remainder of the complex via its N-terminus. In some embodiments, the C-terminus of the peptide is -NH2. In some embodiments, the peptide is attached to the remainder of the complex via its C-terminus. In some embodiments, the peptide is acylated at its N-terminus.

[0019] In some embodiments, the complex comprises or is of the following structure: [ka]

[0020] In some embodiments, the complex comprises or is of the following structure: [ka]

[0021] In some embodiments, the complex comprises or is of the following structure: [ka]

[0022] In some embodiments, each linker is independently represented by formula (I): T1-(CR 1 R 2 ) n -T2(I) wherein T1 is a divalent group for binding to the peptide and is selected from the group consisting of -NH- and carbonyl; T2 is a divalent group for attaching to an oligonucleotide and is selected from the group consisting of -NH- and carbonyl; n is 1, 2 or 3; Each R 1 is independently -Y 1 -X 1 -Z1 and Where: Y 1 does not exist or is -(CR A1 R A2 ) m -, m is 1, 2, 3 or 4, R A1 and R A2 are each independently hydrogen, OH, or (1-2C)alkyl; X 1 is absent or -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR A3 )-, -N(R A3 )-, -N(R A3 )-C(O)-, -N(R A3 )-C(O)O-, -C(O)-N(R A3 )-, -N(R A3 )C(O)N(R A3 )-, -N(R A3 )C(NR A3 )N(R A3 )-, -SO-, -S-, -SO2-, -S(O)2N(R A3 )- or -N(R A3 )SO2-, and each R A3 is independently selected from hydrogen and methyl; and Z 1 is further an oligonucleotide or is hydrogen, (1-6C) alkyl, (2-6C) alkenyl, (2-6C) alkynyl, aryl, (3-6C) cycloalkyl, (3-6C) cycloalkenyl, or heteroaryl; wherein each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl is selected from the group consisting of (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR A4 R A5 and (1-4C)alkoxy; R A4 and R A5are each independently selected from the group consisting of hydrogen and (1-4C)alkyl, and Each R 2 is independently -Y 2 -X 2 -Z 2 where: Y 2 does not exist or is not a B1 R B2 ] m -, m is an integer selected from 1, 2, 3, or 4, R B1 and R B2 are each independently selected from hydrogen, OH or (1-2C)alkyl; X 2 is absent or -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR B3 )-, -N(R B3 )-, -N(R B3 )-C(O)-, -N(R B3 )-C(O)O-, -C(O)-N(R B3 )-, -N(R B3 )C(O)N(R B3 )-, -N(R B3 )C(NR B3 )N(R B3 )-, -SO-, -S--SO2-, -S(O)2N(R B3 )- or -N(R B3 )SO2-, and R B3 is independently selected from hydrogen or methyl, and Z 2 is selected from hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl, each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl being selected from (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR B4 R B5and (1-4C)alkoxy; R B4 and R B5 are each independently hydrogen or (1-2C)alkyl, provided that when n=1 and T1 and T2 are different from each other, R 1 and R 2 are not both H, n=1, T1 and T2 are different from each other, and R 1 and R 2 When one of the groups is H, R 1 and R 2 The other is not methyl, or n=2 and R 1 and R 2 When each occurrence of is H, T1 and T2 are both -C(O)-, or both -NH-.

[0023] In some embodiments, T2 is -C(O)-.

[0024] In some embodiments, each R 1 is independently -Y 1 -X 1 -Z 1 and Y 1 does not exist or is -(CR A1 R A2 ) m -, m is 1, 2, 3 or 4, R A1 and R A2 are each hydrogen or (1-2C) alkyl; X 1 is absent or -O-, -C(O)-, -C(O)O-, -N(R A3 )-, -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, -N(R A3 )C(O)N(R A3 )-, -N(R A3 )C(NR A3 )N(R A3 )- or -S-, and each R A3 are independently hydrogen or methyl; and Z1 is further an oligonucleotide or is hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl, each of which is selected from the group consisting of (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, and heteroaryl, each of which is selected from the group consisting of (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR A4 R A5 and (1-4C)alkoxy; R M and R A5 are each independently hydrogen or (1-2C)alkyl.

[0025] In some embodiments, each R 1 is independently -Y 1 -X 1 -Z 1 and Y 1 does not exist or is -(CR A1 R A2 ) m -, m is 1, 2, 3, or 4, and R A1 and R" are each independently hydrogen or (1-2C)alkyl; X 1 does not exist or is -O-, -C(O)-, -C(O)O-, -N(R A3 )-, -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, -N(R A3 )C(O)N(R A3 )-, -N(R A3 )C(NR A3 )N(R A3 )- or -S-, and each R A3 is independently hydrogen or methyl; and Z 1is further an oligonucleotide or is hydrogen, (1-6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, each of which (1-6C)alkyl, aryl, (3-6C)cycloalkyl, and heteroaryl are optionally substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituents selected from the group consisting of (1-4C)alkyl, halo, and hydroxy.

[0026] In some embodiments, each R 1 is independently -Y 1 -X 1 -Z 1 and Y 1 does not exist or the formula -(CR A1 R A2 ) m -, m is 1, 2, 3 or 4, R A1 and R A2 are each independently hydrogen or (1-2C)alkyl; X 1 does not exist or is -C(O)-, -C(O)O-, -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, and each R A3 is hydrogen or methyl, and Z 1 is further an oligonucleotide or is hydrogen, (1-6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, each of which (1-6C)alkyl, aryl, (3-6C)cycloalkyl, and heteroaryl are optionally substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituents selected from the group consisting of (1-4C)alkyl, halo, and hydroxy.

[0027] In some embodiments, each R 1 is independently -Y 1 -X 1 -Z 1 and Y 1 is absent, -(CH2)-, or -(CH2CH2)-, X 1 does not exist or is -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, and each R A3 are independently hydrogen or methyl; and Z 1 is hydrogen or (1-2C)alkyl.

[0028] In some embodiments, each R 2 is independently -Y 2 -Z 2 and Y 2 does not exist or is -(CR B1 R B2 ) m -, m is 1, 2, 3 or 4, R B1 and R B2 are each independently hydrogen or (1-2C)alkyl; and Z 2 is hydrogen or (1-6C)alkyl.

[0029] In some embodiments, each R 2 is hydrogen.

[0030] In some embodiments, n is 2 or 3. In some embodiments, n is 1.

[0031] In some embodiments, the linker is an amino acid residue selected from the group consisting of glutamic acid, succinic acid, and gamma-aminobutyric acid residues.

[0032] In some embodiments, the linker comprises or is the following structure: [ka]

[0033] In some embodiments, the linker comprises or is the following structure: [ka]

[0034] In some embodiments, the linker comprises or is the following structure: [ka]

[0035] In some embodiments, the linker comprises or is the following structure: [ka]

[0036] In some embodiments, the linker comprises or is the following structure: [ka]

[0037] In some embodiments, the complex comprises or is of the following structure: [ka]

[0038] In some embodiments, the complex comprises or is of the following structure: [ka]

[0039] In some embodiments, the complex comprises or is of the following structure: [ka]

[0040] In some embodiments, the complex comprises or is of the following structure: [ka]

[0041] In some embodiments, the complex comprises or is of the following structure: [ka]

[0042] In some embodiments, the oligonucleotide is attached at its 3' end to the linker or to the peptide.

[0043] In some embodiments, the conjugate has a 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRBRFQILYBRBR: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCA In some embodiments, the complex comprises or consists of 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO: 113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO: 114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO: 116), or a thymine substituted analog thereof, wherein a free -COOH, if present in the glutamic acid residue, is replaced with -CONH2. In some embodiments, the complex has the structure: [ka]

[0044] In some embodiments, the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR via a glutamic acid residue, where any free -COOH present in the glutamic acid residue is replaced with -CONH2. In some embodiments, the conjugate comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109) having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR via a glutamic acid residue, or a thymine-substituted analog thereof, where any free -COOH present in the glutamic acid residue is replaced with -CONH2. In some embodiments, the conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), having its 3' terminus covalently attached via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR, or a thymine-substituted analog thereof, where a free -COOH, if present in the glutamic acid residue, is replaced with -CONH2. In some embodiments of this paragraph, the conjugate has the following structure: [ka]

[0045] In some embodiments, the conjugate has a 3' end covalently linked via a glutamic acid residue to the N-terminus of the peptide RBRRBRFQILYBRBR-NH2: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGC In some embodiments, the complex comprises or consists of AUUUCUAG-3' (SEQ ID NO: 111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO: 113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO: 114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO: 116), or a thymine substituted analog thereof, wherein if a free -COOH is present in the glutamic acid residue, it is replaced with -CONH2. In some embodiments, the complex has the structure: [ka]

[0046] In some embodiments, the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' terminus covalently attached to the N-terminus of the peptide RBRRBRFQILYBRBR-NH2 via a glutamic acid residue, where any free -COOH present in the glutamic acid residue is replaced with -CONH2. In some embodiments, the conjugate comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109) having its 3' terminus covalently attached to the N-terminus of the peptide RBRRBRFQILYBRBR-NH2 via a glutamic acid residue, or a thymine-substituted analog thereof, where any free -COOH present in the glutamic acid residue is replaced with -CONH2. In some embodiments, the conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), having its 3' terminus covalently linked via a glutamic acid residue to the N-terminus of the peptide RBRRBRFQILYBRBR-NH2, or a thymine-substituted analog thereof, where a free -COOH, if present in the glutamic acid residue, is replaced with -CONH2. In some embodiments of this paragraph, the conjugate has the following structure: [ka]

[0047] In some embodiments, the conjugate has a 3' end covalently linked via a gamma-aminobutyric acid residue to the C-terminus of the peptide Ac-RBRBRFQILYRBHBH, such as 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), ), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof.

[0048] In some embodiments, the complex comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH via a γ-aminobutyric acid residue. In some embodiments, the complex comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH via a γ-aminobutyric acid residue, or a thymine-substituted analog thereof. In some embodiments, the complex comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH via a γ-aminobutyric acid residue, or a thymine-substituted analog thereof.

[0049] In some embodiments, the conjugate has a 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRBRFQILYRBHBH, 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCA UUUCUAG-3' (SEQ ID NO: 111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO: 113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO: 114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO: 116), or a thymine substituted analog thereof, wherein if a free -COOH is present in the glutamic acid residue, it is replaced with -CONH2. In some embodiments, the complex has the structure: [ka]

[0050] In some embodiments, the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' terminus covalently attached via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, where any free -COOH present in the glutamic acid residue is replaced with -CONH2. In some embodiments, the conjugate comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109) having its 3' terminus covalently attached via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine-substituted analog thereof, where any free -COOH present in the glutamic acid residue is replaced with -CONH2. In some embodiments, the conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), having its 3' terminus covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine-substituted analog thereof, where a free -COOH, if present in the glutamic acid residue, is replaced with -CONH2. In some embodiments of this paragraph, the conjugate has the following structure: [ka]

[0051] In some embodiments, the linker is of the following structure: [ka]

[0052] The conjugates have their 3' end covalently linked to the C-terminus of the peptide Ac-RBRBRFQILYBRBR via a β-alanine residue: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACA or thymine substituted analogs thereof.

[0053] In some embodiments, the complex comprises or consists of 5' CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR via a β-alanine residue. In some embodiments, the complex comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109) having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR via a β-alanine residue, or a thymine-substituted analog thereof. In some embodiments, the complex comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115) having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR via a β-alanine residue, or a thymine-substituted analog thereof.

[0054] In some embodiments, the conjugate has a 3' end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRBRFQILYRBHBH, such as 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), ), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof.

[0055] In some embodiments, the complex comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH via a β-alanine residue. In some embodiments, the complex comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109) having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH via a β-alanine residue, or a thymine-substituted analog thereof. In some embodiments, the complex comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115) having its 3' terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH via a β-alanine residue, or a thymine-substituted analog thereof.

[0056] In some embodiments, the oligonucleotide is a morpholino.

[0057] In some embodiments, all morpholino internucleoside linkages are -P(O)(NMe2)O-.

[0058] In some embodiments, the nucleotide comprises the following group at its 5' end: [ka]

[0059] In some embodiments of these and other oligonucleotides described herein, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0060] In some embodiments, the complex is administered intraperitoneally. In some embodiments, the complex is administered by infusion (e.g., intravenous, optionally bolus or continuous infusion, the latter optionally over a period of 10 minutes to 3 hours, e.g., 0.25 to 2 hours, or 0.5 to 1 hour). In some embodiments, the complex is administered by intravenous. In some embodiments, the subject is a candidate for exon 51 skipping.

[0061] In some embodiments, the conjugate is administered to the subject at a frequency of from weekly to monthly. In some embodiments, the frequency is weekly, biweekly, every three weeks, or monthly, and in some embodiments, the frequency is every three months.

[0062] In some embodiments, 40 mg / kg to 60 mg / kg, 30 mg / kg to 50 mg / kg, 30 mg / kg to 40 mg / kg, 40 mg / kg to 50 mg / kg, 50 mg / kg to 60 mg / kg, 35 mg / kg to 45 mg / kg, 45 mg / kg to 55 mg / kg, 35 mg / kg to 55 mg / kg, 30 mg / kg to 45 mg / kg, 35 mg / kg to 50 mg / kg, 40 mg / kg to 55 mg / kg, 45 mg / kg to 60 mg / kg, 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 5 mg / kg to 25 mg / kg, 10 mg / kg to 30 mg / kg, 1 mg / kg to 15 mg / kg, 5 mg / kg to 20 mg / kg, 10 mg / kg to 25 mg / kg, 15 mg / kg to 30 mg / kg, 1 mg / kg to 10 In some embodiments, the conjugate is administered at a dose of 1 mg / kg, 5 mg / kg to 15 mg / kg, 10 mg / kg to 20 mg / kg, 15 mg / kg to 25 mg / kg, 20 mg / kg to 30 mg / kg, 1 mg / kg to 25 mg / kg, 4 mg / kg to 20 mg / kg, 6 mg / kg to 15 mg / kg, or 8 mg / kg to 10 mg / kg.

[0063] In some embodiments, 1 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or 60 mg / kg of the conjugate is administered. In some embodiments, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg of the conjugate is administered.

[0064] In some embodiments, the method is for use in treating the cardiac effects of DMD, including, for example, cardiomyopathy (e.g., dilated, hypertrophic, or restrictive cardiomyopathy), heart failure, and / or cardiac arrhythmias.

[0065] In some embodiments, the methods described above and elsewhere herein are used to treat BMD.

[0066] The present invention also includes the use of the conjugates described herein in the methods described herein. Accordingly, each method of treatment claim herein can be considered to support a claim in the form of a composition as defined therein for use in the indicated method (e.g., treating, preventing, or ameliorating DMD or BMD).

[0067] definition Throughout this specification "X" refers to any form of the amino acid aminohexanoic acid, such as 6-aminohexanoic acid.

[0068] Throughout this specification "B" refers to the amino acid β-alanine.

[0069] Throughout this specification, "[Hyp]" refers to the amino acid hydroxyproline.

[0070] Throughout this specification, "Ac" refers to an acetyl group (CH3-C(O)-).

[0071] Throughout this specification, other capital letters indicate the associated amino acid residue which is genetically encoded according to the generally accepted alphabetic amino acid code.

[0072] The term "alkyl", as used herein, unless otherwise specified, refers to a straight or branched chain hydrocarbon group containing a total of 1 to 20 carbon atoms (e.g., (1-6C)alkyl, (1-4C)alkyl, (1-3C)alkyl, or (1-2C)alkyl). Non-limiting examples of alkyl include methyl, ethyl, 1-methylethyl, propyl, 1-methylbutyl, 1-ethylbutyl, and the like. References to individual alkyl groups such as "propyl" are specific to the straight chain group only, and references to individual branched chain alkyl groups such as "isopropyl" are specific to the branched chain group only.

[0073] The term "alkenyl," as used herein, unless otherwise specified, refers to an aliphatic group having 1, 2, or 3 carbon-carbon double bonds and containing a total of 2 to 20 carbon atoms (e.g., (2-6C)alkenyl, (2-4C)alkenyl, or (2-3C)alkenyl). Non-limiting examples of alkenyl include vinyl, allyl, homoallyl, isoprenyl, and the like. Unless otherwise specified, alkenyl is optionally substituted with 1, 2, 3, 4, or 5 groups selected from the group consisting of carbocyclyl, aryl, heterocyclyl, heteroaryl, oxo, halogen, and hydroxyl.

[0074] The term "alkynyl," as used herein, unless otherwise specified, refers to an aliphatic group containing 1, 2, or 3 carbon-carbon triple bonds and containing a total of 2 to 20 carbon atoms (e.g., (2-6C)alkynyl, (2-4C)alkynyl, or (2-3C)alkynyl). Non-limiting examples of alkynyl include ethynyl, propargyl, homopropargyl, but-2-yn-1-yl, 2-methyl-prop-2-yn-1-yl, and the like. Unless otherwise specified, alkynyl may be optionally substituted with 1, 2, 3, 4, or 5 groups selected from the group consisting of carbocyclyl, aryl, heterocyclyl, heteroaryl, oxo, halogen, and hydroxyl.

[0075] "Arginine-rich" with respect to a cationic domain means that at least 40% of the cationic domain is made up of arginine residues.

[0076] The term "artificial amino acid" as used herein refers to non-biological amino acids (e.g., non-proteinogenic). For example, artificial amino acids may include synthetic amino acids, modified amino acids (e.g., sugar-modified amino acids), unnatural amino acids, man-made amino acids, spacers, and non-peptide bond spacers. Synthetic amino acids may include those chemically synthesized by humans. For the avoidance of doubt, aminohexanoic acid (X) is an artificial amino acid in the context of the present invention. For the avoidance of doubt, β-alanine (B) and hydroxyproline (Hyp) are natural amino acids, not artificial amino acids, in the context of the present invention, since they occur in nature. Artificial amino acids may include, for example, 6-aminohexanoic acid (X), tetrahydroisoquinoline-3-carboxylic acid (TIC), 1-(amino)cyclohexanecarboxylic acid (Cy), 3-azetidinecarboxylic acid (Az), and 11-aminoundecanoic acid.

[0077] The term "aryl" as used herein refers to a carbocyclic ring system containing one, two, or three rings, at least one of which is aromatic. Unsubstituted aryls contain a total of 6 to 14 carbon atoms. The term aryl includes both monovalent and divalent species. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, indanyl, and the like. In certain embodiments, the optionally substituted aryl is an optionally substituted phenyl.

[0078] As used herein, "bridged ring system" means a ring system in which two rings share two or more atoms, see, for example, Advanced Organic Chemistry, Jerry March, 4th Edition, Wiley Interscience, pp. 131-133, 1992. Examples of bridged heterocyclyl ring systems include aza-bicyclo[2.2.1]heptane, 2-oxa-5-azabicyclo[2.2.1]heptane, aza-bicyclo[2.2.2]octane, aza-bicyclo[3.2.1]octane, quinuclidine, and the like.

[0079] As used herein, the term "carbonyl" refers to a group of the following structure: -C(O)-. Non-limiting examples of carbonyl groups include those found in, for example, acetone, ethyl acetate, proteinogenic amino acids, acetamide, and the like.

[0080] As used herein, "cationic" refers to amino acids or amino acid domains that carry an overall positive charge at physiological pH.

[0081] The term “(m-nC)” or “(m-nC) group” used alone or as a prefix, refers to a group having a total of m to n carbon atoms, when unsubstituted.

[0082] The term "complementary" as used herein with respect to a nucleobase sequence refers to a nucleobase sequence having a pattern of consecutive nucleobases that allows an oligonucleotide having the nucleobase sequence to hybridize to another oligonucleotide or nucleic acid under physiological conditions to form a duplex structure. Complementary sequences include Watson-Crick base pairs formed from natural and / or modified nucleobases. Complementary sequences may also include non-Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, hypoxanthine-cytosine) and Hoogsteen base pairs.

[0083] The term "cycloalkyl", as used herein, unless otherwise specified, refers to a saturated carbocyclic ring system containing one or two rings and containing a total of 3 to 10 carbon atoms. Bicyclic cycloalkyls can be arranged as fused ring systems (two bridgehead carbon atoms are directly bonded to each other), bridged ring systems (two bridgehead carbon atoms are bonded to each other via a covalent linker containing at least one carbon atom), and spirocyclic (two rings are fused at the same carbon atom) systems. Non-limiting examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, and the like.

[0084] The term "cycloalkenyl", as used herein, unless otherwise specified, refers to a non-aromatic, unsubstituted, carbocyclic ring system containing one or two rings, one, two or three endocyclic double bonds, and a total of three to ten carbon atoms. Bicyclic cycloalkenyls can be arranged as fused ring systems (two bridgehead carbon atoms are directly bonded to each other), bridged ring systems (two bridgehead carbon atoms are bonded to each other via a covalent linker containing at least one carbon atom), and spiro ring systems (two rings are fused at the same carbon atom). Non-limiting examples of cycloalkenyls include cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 3-cyclohexen-1-yl, cyclooctenyl, and the like.

[0085] "Dystrophin" is a rod-shaped cytoplasmic protein and 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, dystrophin contains an actin-binding domain at approximately 14-240 amino acids and a central rod domain at approximately 253-3040 amino acids. This large central domain is formed by 24 spectrin-like triple-helical elements of approximately 109 amino acids and shares homology with α-actinin and spectrin. The repeats are usually separated by four proline-rich non-repeat segments, also called hinge domains. Repeats 15 and 16 are separated by an 18 amino acid stretch that appears to provide the primary site of proteolytic cleavage of dystrophin. The sequence identity between most repeats ranges from 10-25%. One repeat contains three α-helices, 1, 2 and 3. α-helices 1 and 3 are each formed by seven helical turns, which interact as a coiled coil, presumably through a hydrophobic interface. α-helix 2 has a more complex structure, formed by a sequence of four and three turns separated by glycine or proline residues. Each repeat is encoded by two exons, usually separated by an intron between amino acids 47 and 48 in the first part of α-helix 2. The other intron is at a different position within the repeat, usually interspersed on helix 3. Dystrophin also contains a cysteine-rich domain at about amino acids 3080-3360, a cysteine-rich segment that shows homology to the C-terminal domain of Dictyostelium discoideum α-actinin (i.e., 15 cysteines in 280 amino acids). The carboxy-terminal domain is at about amino acids 3361-3685.

[0086] The amino terminus of dystrophin binds to F-actin, and the carboxy terminus binds to the dystrophin-associated protein complex (DAPC) in muscle cell membranes. DAPC includes dystroglycan, sarcoglycan, α-dystrobrevin, sarcospan, laminin, syntrophin, integrin, and caveolin, and mutations in any of these components cause autosomal muscular dystrophies. Dystrophin deficiency destabilizes DAPC, resulting in reduced levels of its constituent proteins and progressive fiber damage and membrane leakage. In various forms of muscular dystrophies, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), muscle cells produce denatured, functionally defective forms of dystrophin or no dystrophin at all, mainly due to gene sequence mutations that result in incorrect splicing. Predominant expression of defective dystrophin protein or complete lack of dystrophin or dystrophin-like proteins leads to rapid progression of muscle degeneration, as described above. In this regard, a "deficient" dystrophin protein may be characterized by the form of dystrophin produced in a particular subject with DMD or BMD, or the absence of detectable dystrophin, as is known in the art.

[0087] "Exon" refers to a defined portion of a nucleic acid that codes for a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after any portion of a preprocessed (or precursor) RNA has been removed by splicing. A mature RNA molecule may be a functional form of messenger RNA (mRNA) or a non-coding RNA such as rRNA or tRNA. The human dystrophin gene has about 75 exons.

[0088] "Exon skipping" generally refers to a process in which an entire exon or a portion thereof is removed from a given precursor RNA, thereby eliminating its presence in a mature RNA, such as a mature mRNA, that is translated into a protein. Thus, the portion of the protein encoded by the skipped exon is absent in the expressed form of the protein, usually creating an altered, though still functional, form of the protein. In certain embodiments, the skipped exon is an abnormal exon of the human dystrophin gene, which may contain a mutation or other alteration in its sequence that causes abnormal splicing. As described herein, the skipped exon is exon 51 of the human dystrophin gene.

[0089] The term "halo" or "halogeno" as used herein refers to fluoro, chloro, bromo, and iodo.

[0090] "Histidine-rich" with respect to a cationic domain means that at least 40% of the cationic domain is formed from histidine residues.

[0091] The terms "heteroaryl" or "heteroaromatic", as used interchangeably herein, refer to a ring system containing one, two, or three rings, at least one of which is aromatic and contains one to four (e.g., one, two, or three) heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unsubstituted heteroaryl groups contain a total of one to nine carbon atoms. The term heteroaryl includes both monovalent and divalent species. Examples of heteroaryl groups include monocyclic and bicyclic groups containing five to twelve ring members, more commonly five to ten ring members. Heteroaryl groups may be, for example, 5- or 6-membered monocyclic rings, or 9- or 10-membered bicyclic rings, such as bicyclic structures formed from fused five- and six-membered rings or two fused six-membered rings. Each ring may contain up to about four heteroatoms, typically selected from nitrogen, sulfur, and oxygen. Typically, a heteroaryl ring contains up to 3 heteroatoms, more usually up to 2, for example, a single heteroatom. In some embodiments, a heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atom of a heteroaryl ring may be basic, as in the case of imidazole or pyridine, or essentially non-basic, as in the case of indole or pyrrole nitrogen. Generally, the number of basic nitrogen atoms present in a heteroaryl group will be less than 5, including all ring amino group substituents.

[0092] Examples of heteroaryl include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazenyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, and isoaryl. Examples of such aryl groups include quinolyl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthyridinyl, carbazolyl, phenazinyl, benzisoquinolinyl, pyridopyrazinyl, thieno[2,3-b]furanyl, 2H-furo[3,2-b]-pyranyl, 5H-pyrido[2,3-d]-oxazinyl, 1H-pyrazolo[4,3-d]-oxazolyl, 4H-imidazo[4,5-d]thiazolyl, pyrazino[2,3-d]pyridazinyl, imidazo[2,1-b]thiazolyl, and imidazo[1,2-b][1,2,4]triazinyl. "Heteroaryl" also includes partially aromatic bicyclic or polycyclic ring systems in which at least one ring is aromatic and one or more of the other rings is non-aromatic, saturated or partially saturated, provided that at least one ring contains one or more heteroatoms selected from nitrogen, oxygen or sulfur. Examples of partially aromatic heteroaryl groups include, for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo1,2,3,4-tetrahydroquinolinyl, dihydrobenzthienyl, dihydrobenzfuranyl, 2,3-dihydro-benzo[1,4]dioxinyl, benzo[1,3]dioxoynyl, 2,2-dioxo-1,3-dihydro-2-benzothienyl, 4,5,6,7-tetrahydrobenzofuranyl, indolinyl, 1,2,3,4-tetrahydro-1,8-naphthyridinyl, 1,2,3,4-tetrahydropyrido[2,3-b]pyrazinyl, and 3,4-dihydro-2W-pyrido[3,2-b][1,4]oxazinyl.Examples of 5-membered heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, and tetrazolyl groups. Examples of 6-membered heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and triazinyl. Bicyclic heteroaryl groups include, for example, a benzene ring fused to a 5- or 6-membered ring containing one, two or three ring heteroatoms; a pyridine ring fused to a 5- or 6-membered ring containing one, two or three ring heteroatoms; a pyrimidine ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; a pyrrole ring fused to a 5- or 6-membered ring containing one, two or three ring heteroatoms; a pyrazole ring fused to a 5- or 6-membered ring containing one, two or three ring heteroatoms; a pyrazine ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; an imidazole ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; a 5- or 6-membered ring containing one, two or three ring heteroatoms; an isoxazole ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; a thiazole ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; an isothiazole ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; a thiophene ring fused to a 5- or 6-membered ring containing one, two or three ring heteroatoms; a furan ring fused to a 5- or 6-membered ring containing one, two or three ring heteroatoms; a cyclohexyl ring fused to a 5- or 6-membered heteroaromatic ring containing one, two or three ring heteroatoms; and a cyclopentyl ring fused to a 5- or 6-membered heteroaromatic ring containing one, two or three ring heteroatoms.Specific examples of bicyclic heteroaryl groups containing a 6-membered ring fused to a 5-membered ring include benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzthiazolyl, benzisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, indolinyl, isoindolinyl, purinyl (including, for example, adeninyl, guaninyl, indazolyl, benzodioxolyl and pyrazolopyridinyl groups). Specific examples of bicyclic heteroaryl groups containing two fused 6-membered rings include, but are not limited to, quinolinyl, isoquinolinyl, chromanyl, thiochromanyl, chromenyl, isochromenyl, chromanyl, isochromanyl, benzodioxanyl, quinolidinyl, benzoxazinyl, benzodiazinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, and pteridinyl groups.

[0093] The term "heterocyclyl" as used herein refers to a ring system containing one, two, or three rings, at least one of which is aromatic and contains one to four (e.g., one, two, or three) heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, with the proviso that the ring system does not contain an aromatic ring further containing an intracyclic heteroatom. Unsubstituted heterocyclyl groups contain a total of two to nine carbon atoms. The term heterocyclyl includes both monovalent and divalent species. Examples of heterocyclyl groups include monocyclic and bicyclic groups containing five to twelve ring members, more commonly five to ten ring members. Heterocyclyl groups may be, for example, 5- or 6-membered monocyclic rings, or 9- or 10-membered bicyclic rings, such as bicyclic structures formed from fused five- and six-membered rings or two fused six-membered rings. Each ring may contain up to about four heteroatoms, typically selected from nitrogen, sulfur, and oxygen. Non-limiting examples of heterocyclyl groups include, for example, pyrrolidine, piperazine, piperidine, azepane, 1,4-diazepane, tetrahydrofuran, tetrahydropyran, oxepane, 1,4-dioxepane, tetrahydrothiophene, tetrahydrothiopyran, indoline, benzopyrrolidine, 2,3-dihydrobenzofuran, phthalan, isochroman, and 2,3-dihydrobenzothiophene.

[0094] The term "internucleoside linkage" as used herein refers to a group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. An internucleoside linkage may be an unmodified internucleoside linkage or a modified internucleoside linkage. An "unmodified internucleoside linkage" is a phosphate (-O-P(O)(OH)-O-) internucleoside linkage ("phosphodiester phosphate"). A "modified internucleoside linkage" is an internucleoside linkage other than a phosphodiester phosphate. Two major types of modified internucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, morpholino internucleoside linkages, methylphosphonates, and phosphoramidates. Non-limiting examples of non-phosphorus internucleoside linkages include methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Phosphorothioate linkages are phosphodiester and phosphotriester linkages in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. In some embodiments, the internucleoside linkage is a group of the following structure: [ka] (In the formula, Z is O, S, or Se; Y-XLR 1 and Each X is independently -O-, -S-, -N(-LR 1 )- or L; Each L is independently a covalent bond or a linker (e.g., optionally substituted C 1-60 Aliphatic linker or optionally substituted C 2-60 heteroaliphatic linker), Each R 1 are independently hydrogen, -SSR 2, -O-CO-R 2 , -S-CO-R 2 , optionally substituted C 1-9 heterocyclyl, or a hydrophobic moiety, and Each R 2 C is optionally substituted independently 1-10 Alkyl, optionally substituted C 2-10 Heteroalkyl, optionally substituted C 6-10 Aryl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 1-9 Heterocyclyl or optionally substituted C 1-9 Heterocyclyl C 1-6 It is an alkyl group.

[0095] L is a covalent bond, R 1 When X is hydrogen, Z is oxygen, and all X groups are -O-, the internucleoside group is known as a phosphodiester phosphate. L is a covalent bond, R 1 When R is hydrogen, Z is sulfur, and all X groups are -O-, the internucleoside group is known as a phosphorothioate diester. Z is oxygen, all X groups are -O-, and (1) L is a linker, or (2) R 1 When Z is not hydrogen, the internucleoside group is known as a phosphotriester. Z is sulfur, all X groups are -O-, and (1) L is a linker or (2) R 1 When is not hydrogen, the internucleoside group is known as a phosphorothioate triester. Non-limiting examples of phosphorothioate triester and phosphotriester linkages are described in US 2017 / 0037399, the disclosure of which is incorporated herein by reference.

[0096] "Intron" refers to a nucleic acid region (within a gene) that is not translated into protein. Introns are non-coding portions that are transcribed into a precursor mRNA (pre-mRNA) and then removed by splicing during formation of the mature RNA.

[0097] The term "morpholino," as used herein with respect to types of oligonucleotides, refers to an oligomer of at least 10 morpholino monomeric units interconnected by morpholino internucleoside linkages. A morpholino includes a 5' group and a 3' group. For example, a morpholino may have the following structure: [ka] (In the formula, n is an integer of at least 10 (e.g., 12 to 30) representing the number of morpholino subunits and associated groups L; each B is independently a nucleobase; R 1 is the 5' group (herein R 1 is sometimes referred to as the 5' end), R 2 is the 3' group (herein R 2 is sometimes referred to as the 3' end), and L is (i) a morpholino internucleoside linkage, or (ii) L is R 2 If it is bonded to a , it is a covalent bond.)

[0098] The 5' group of the morpholino can be, for example, a hydroxyl, a hydrophobic moiety, a phosphate, a diphosphate, a triphosphate, a phosphorothioate, a diphosphorothioate, a triphosphorothioate, a phosphorodithioate, a diphosphorodithioate, a triphosphorodithioate, a phosphonate, a phosphoramidate, a bond to a peptide, a bond to a peptide / linker combination, an endosomal escape site, or a neutral organic polymer. In some embodiments, the 5' group is of the following structure: [ka] [ka]

[0099] Preferred 5' groups are hydroxyl and groups of the following structure: [ka] [ka]

[0100] A more preferred 5' group is the following structure: [ka]

[0101] The 3' group of a morpholino can be, for example, a hydrogen, a hydrophobic moiety, a phosphate, a diphosphate, a triphosphate, a phosphorothioate, a diphosphorothioate, a triphosphorothioate, a phosphorodithioate, a diphosphorodithioate, a triphosphorodithioate, a phosphonate, a phosphoramidate, a bond to a peptide, a bond to a peptide / linker combination, an endosomal escape site, or a neutral organic polymer. In a conjugate of an oligonucleotide that is a morpholino and a peptide covalently bonded or linked to the oligonucleotide, the preferred 3' group is a bond to a peptide or a bond to a peptide / linker combination.

[0102] The term "morpholino internucleoside linkage," as used herein, refers to a divalent group of the following structure: [ka] (In the formula, Z is O or S; X 1 is a bond, -CH2-, or -O-, X 2 is a bond, -CH2-O-, or -O-, and Y is -NR2, where each R is independently H or C 1-6 alkyl (e.g., methyl), or both R together with the nitrogen atom to which they are attached are C 2-9 Forming a heterocyclyl (eg, N-piperazinyl). However, X 1 and X 2are not bonds at the same time.)

[0103] The term "morpholino subunit" as used herein refers to the following structure: [ka] (In the formula, B is a nucleobase.)

[0104] The term "nucleobase" as used herein refers to the nitrogen-containing heterocyclic ring found at the 1' position of the ribofuranose / 2'-deoxyribofuranose of a nucleoside. Nucleobases may be unmodified or modified. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6, and O-6 substituted purines, as well as synthetic and natural nucleobases such as 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine, 2-propyl cytos ... These include adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 5-trifluoromethyluracil, 5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids. For example, 5-substituted pyrimidines, 6-azapyrimidines, N2-, N6-, and / or O6-substituted purines. The stability of nucleic acid duplexes can be enhanced using, for example, 5-methylcytosine.Non-limiting examples of nucleobases include 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynylcytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted protoplasts. phosphorus, 5-halo, especially 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp).Modified nucleobases can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deazaadenine, 7-deazaguanine, 2-aminopyridine, or 2-pyridone.Further nucleobases include those disclosed in Merigan et al., US Pat. No. 3, 687, 808, The Concise Encyclopedia Of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288; and Chapters 6 and 15, Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, 163-166 and 442-443.

[0105] The term "nucleoside" as used herein refers to sugar-nucleobase compounds and groups known in the art, as well as modified or unmodified 2'-deoxyribofuranose-nucleobase compounds and groups known in the art. The sugar may be ribofuranose. The sugar may be modified or unmodified. An unmodified ribofuranose-nucleobase is a ribofuranose with an anomeric carbon bond to the unmodified nucleobase. Unmodified ribofuranose-nucleobases are adenosine, cytidine, guanosine, and uridine. Unmodified 2'-deoxyribofuranose-nucleobase compounds are 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, and thymidine. The modified compounds and groups include one or more modifications selected from the group consisting of nucleobase modifications and sugar modifications described herein. The nucleobase modification is the replacement of an unmodified nucleobase with a modified nucleobase. The sugar modification may be, for example, 2'-substituted, locked, carbocyclized, or non-locked. The 2'-substitution may be one in which the 2'-hydroxyl of the ribofuranose is replaced with 2'-fluoro, 2'-methoxy, or 2'-(2-methoxy)ethoxy. Alternatively, the 2'-substitution may be a 2'-(ara)substitution, which corresponds to the following structure: [ka] where B is a nucleobase and R is a 2'-(ara) substitution (e.g., fluoro). The 2'-(ara) substitutions are known in the art and may be identical to other 2'-substituents described herein. In some embodiments, the 2'-(ara) substitution is a 2'-(ara)-F substitution (R is fluoro). The locked modification is the incorporation of a bridge between the 4'-carbon atom and the 2'-carbon atom of the ribofuranose. Nucleosides with locked modifications are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. Bridged nucleic acids are typically used as affinity enhancing nucleosides. "Nucleoside" can also refer to a morpholino subunit.

[0106] The term "nucleotide" as used herein refers to an internucleoside linkage or the following structure: -X 1 -P(X 2 )(R 1 ) 2, a nucleoside bonded to a monovalent group of the formula 1 is O, S, or NH, and X 2 is absent, ═O, or ═S, and each R 1 are independently -OH, -N(R 2 )2, or -O-CH2CH2CN, where each R 2 are independently optionally substituted alkyl, or both R 2 The groups, together with the nitrogen atom to which they are attached, are joined to form an optionally substituted heterocyclyl.

[0107] The term "oligonucleotide" as used herein refers to a structure containing 10 or more consecutive nucleosides covalently linked by internucleoside linkages, a morpholino containing 10 or more morpholino subunits, or a peptide nucleic acid containing 10 or more morpholino subunits. Preferably, the oligonucleotide is a morpholino.

[0108] The term "optionally substituted" refers to a group, structure, or molecule that may be substituted or unsubstituted as described for each group. 1 The term "any CH, CH2, CH3 group or heteroatom (i.e., NH) within the group may be substituted" is used in 1 It means that (any) one of the hydrogen radicals of the group is replaced by the associated defined group.

[0109] As used herein, the term "operably linked" may include the situation where a selected nucleotide sequence and a regulatory nucleotide sequence are covalently linked in such a way that expression of the nucleotide coding sequence is under the control of the regulatory sequence, such that the regulatory sequence is capable of affecting the transcription of the nucleotide coding sequence that forms part or all of the selected nucleotide sequence. If desired, the resulting transcript may be translated into a desired peptide.

[0110] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with individual (e.g., human) tissue and that are free of undue toxicity, irritation, allergic response, or other problem complications, commensurate with a reasonable benefit / risk ratio.

[0111] As used herein, the term "pharmaceutical composition" refers to a composition containing an oligonucleotide as described herein, formulated with a pharma- ceutical acceptable excipient, and manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease of interest.

[0112] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of the conjugates, oligonucleotides, and peptides disclosed herein. A pharmaceutically acceptable salt of any compound described herein may include those that are within the scope of sound medical judgment, suitable for use in contact with human and animal tissues, and free of undue toxicity, irritation, or allergic reaction, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, and separately by reacting the free base with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, and the like. These include lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like.Representative alkali and alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0113] The terms "reduce" or "inhibit" may generally relate to the ability of one or more compounds of the invention to "reduce" a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured by routine techniques in the diagnostic arts. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to one of skill in the art and 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 altered forms of dystrophin expressed in individuals with DMD or BMD. A "reduction" in response may be statistically significant as compared to the response produced by a compound or control composition not including the antisense compound and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction, including all integers in between.

[0114] As used herein, the term "subject" refers to a human or non-human animal (e.g., a mammal) that has been determined by a qualified professional (e.g., a doctor or nurse) to be suffering from or at risk for a disease, disorder, or condition, with or without clinical testing of a sample from the subject as known in the art. Non-limiting examples of diseases, disorders, and conditions include Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

[0115] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring or structure that may replace the furanose ring of a nucleoside. The sugars contained in the nucleosides of the invention may be non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures include simple variations on the naturally occurring furanose ring (e.g., a six-membered ring). Alternative sugars may also include sugar substitutes in which the furanose ring is replaced with another ring system, such as, for example, a morpholino ring system or a hexitol ring system. Non-limiting examples of useful sugar moieties that can be included in the oligonucleotides of the invention include β-D-ribose, β-D-2′-deoxyribose, substituted sugars (e.g., 2′, 5′ and bis substituted sugars), 4′-S-sugars (e.g., 4′-S-ribose, 4′-S-2′-deoxyribose, and 4′-S-2′-substituted ribose), bicyclic sugar moieties (e.g., bicyclic sugars derived from 2′-O-CH2-4′ or 2′-O-(CH2)2-4′ bridged ribose) and sugar surrogates (where the ribose ring is replaced with a morpholino or hexitol ring system).

[0116] As used herein, the terms "treatment" and "treating" refer to the medical management of a subject with the goal of improving, ameliorating, or stabilizing a disease, disorder, or condition (e.g., DMD or BMD). This term includes active treatment (treatment for the improvement of DMD or BMD), palliative treatment (treatment for the relief of symptoms of DMD or BMD), and supportive treatment (treatment to complement other treatments).

[0117] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations thereof mean "including, but not limited to" and are not intended to (and do not) exclude other moieties, additives, components, integers or means. Throughout the description and claims of this specification, the singular encompasses the plural unless the context dictates otherwise. In particular, when the indefinite article is used, the specification is understood to contemplate the plural as well as the singular, unless the context requires otherwise.

[0118] All "complexes" also refer to solvates thereof, including pharma- ceutically acceptable solvates thereof.

[0119] All references to "oligonucleotides" also refer to their salts and / or solvates, including pharma- ceutically acceptable salts and / or solvates thereof.

[0120] All peptides are shown herein in N-terminal to C-terminal (left to right) orientation unless otherwise specified, and all oligonucleotides are shown herein in 5' to 3' (left to right) orientation unless otherwise specified.

[0121] It is understood that any feature, integer, property, compound, chemical moiety or group described in connection with a particular aspect, embodiment or example of the invention is also applicable to the other aspects, embodiments or examples described herein, except where inconsistent therewith. All of the features disclosed in this specification (including the accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except where at least some of such features and / or steps are mutually exclusive. [Brief description of the drawings]

[0122] [Figure 1] FIG. 1 is a plot showing dose response in major muscle groups for animals (n=2-4) treated with the complex. TA is tibialis anterior. Graphs are plotted as mean ± SD. One animal in the 40 mg / kg injection group showed lower levels in both the TA and heart, below the group mean. [Diagram 2] 2A and 2B are plots showing serum urea and creatinine levels (markers of renal function) pre- and post-dosing (1, 4, and 7 days) in animals (n=2-4). PD=pre-dosing. Graphs are plotted as mean ± SD. [Diagram 3]Figures 3A, 3B, and 3C are plots showing serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels (markers of liver function) pre- and post-dosing (1, 4, and 7 days) in animals (n=2-4). PD=pre-dosing. Graphs are plotted as mean ± SD. [Figure 4A-4C] 4A, 4B, and 4C are plots showing white blood cell counts, neutrophil blood cell counts, and monocyte blood cell counts before and after treatment (1, 4, and 7 days) (n=2-4). PD=pre-treatment. Graphs are plotted as mean ± SD. [Diagram 5] Figure 5 is a plot showing post-administration plasma concentrations of PMO in animals (n=2-4) treated with Complex 1. EOI = end of infusion. [Figure 6] Figure 6 is a plot summarizing the results of tissue biochemical analysis for PMO concentrations in animals (n=2-4) treated with Complex 1. Graphs are plotted as mean ± SD. LLOQ = lower limit of linear quantification (50 ng / g). [Figure 7] Figure 7 is a plot summarizing the results of tissue biochemistry analysis for PMO concentrations in animals (n=2-4) receiving a 40 mg / kg bolus or a 40 mg / kg infusion (30 min) of Complex 1. Graphs are plotted as mean ± SD. LLOQ = lower limit of linear quantification (50 ng / g). [Figure 8] Figure 8 is a plot summarizing the results of tissue biochemistry analysis for PMO concentrations in animals (n=2-4) that received either a 60 mg / kg bolus or a 60 mg / kg infusion (30 min) of Complex 1. Graphs are plotted as mean ± SD. LLOQ = lower limit of linear quantification (50 ng / g). [Figure 9A-B] 9A and B are plots summarizing urinalysis of creatinine and KIM-1 in test animals (n=2-4). Graphs are plotted as mean ± SD. [Figure 10A-10C]10A, 10B, and 10C are plots showing coagulation marker levels (prothrombin time, activated partial thromboplastin time, and fibrinogen levels) before and after (1 and 7 days) administration in animals (n=2-4) administered Complex 1. Graphs are plotted as mean ± SD. [Figure 11] Figure 11 is a plot showing the body weight of the test animals (n = 2 to 4) during the study, plotted as mean ± SD. [Fig. 12A-12I] 12A-12I are plots showing the weights of the indicated tissues from animals dosed with 0 mg / kg, 40 mg / kg, or 60 mg / kg of Complex 1. [Figure 13A-E] Figures 13A-E are plots showing no serum complement activation in CH50, Bb, C3a, or SC5b-9. C5a was not detected during the study. Graphs are plotted as mean ± SD (n = 2-4). [Figure 14] FIG. 14 is a series of plots showing exon 51 skipping by the indicated complexes in biceps, quadriceps, and diaphragm. [Figure 15] FIG. 15 is a series of plots showing exon 51 skipping by the indicated complexes in the left ventricle, right ventricle, and aorta. [Figure 16] FIG. 16 is a plot showing creatine kinase levels in mdx mice one week after treatment with vehicle or Complex 2 (60 mg / kg) and in wild type (WT) mice one week after treatment with vehicle. [Figure 17A] FIG. 17A is a plot showing the exon skipping effect of complex 2 and R6G-PMO in the quadriceps muscle of mdx mice. [Figure 17B] Figure 17B is a plot showing the effect of complex 2 and R6G-PMO on dystrophin restoration in the quadriceps of mdx mice. Results are normalized to WT mice. [Figure 18A] FIG. 18A is a plot showing the exon skipping effects of complex 2 and R6G-PMO in mdx mouse hearts. [Figure 18B] Figure 18B is a plot showing the effect of complex 2 and R6G-PMO on dystrophin restoration in the hearts of mdx mice. Results are normalized to WT mice. [Figure 19A] Figure 19A is a plot showing the effect of exon 51 skipping in the biceps muscle of non-human primates administered Complex 1 Q2W for a total of three doses. Data are presented as mean ± SD. n=3 per group. [Figure 19B] Figure 19B is a plot showing exon 51 skipping efficacy in the quadriceps muscle of non-human primates administered Complex 1 Q2W for a total of three doses. Data are presented as mean ± SD. n=3 per group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0123] Typically, the present invention provides a method of treating a subject with Duchenne muscular dystrophy, comprising administering to the subject (e.g., a subject susceptible to exon 51 skipping) 1 mg / kg to 60 mg / kg of a conjugate of an oligonucleotide and a peptide covalently bonded or linked to the oligonucleotide via a linker.

[0124] In some embodiments, 30 mg / kg to 60 mg / kg (e.g., 40 mg / kg to 60 mg / kg, 30 mg / kg to 50 mg / kg, 30 mg / kg to 40 mg / kg, e.g., 30 mg / kg, 40 mg / kg, 50 mg / kg, or 60 mg / kg) of the conjugate is administered.

[0125] In some embodiments, 40 mg / kg to 50 mg / kg, 50 mg / kg to 60 mg / kg, 35 mg / kg to 45 mg / kg, 45 mg / kg to 55 mg / kg, 35 mg / kg to 55 mg / kg, 30 mg / kg to 45 mg / kg, 35 mg / kg to 50 mg / kg, 40 mg / kg to 55 mg / kg, or 45 mg / kg to 60 mg / kg of the conjugate is administered.

[0126] In some embodiments, 1 mg / kg to 30 mg / kg (e.g., 1 mg / kg to 20 mg / kg, 5 mg / kg to 25 mg / kg, 10 mg / kg to 30 mg / kg, 1 mg / kg to 15 mg / kg, 5 mg / kg to 20 mg / kg, 10 mg / kg to 25 mg / kg, 15 mg / kg to 30 mg / kg, 1 mg / kg to 10 mg / kg, 5 mg / kg to 15 mg / kg, 10 mg / kg to 20 mg / kg, 15 mg / kg to 25 mg / kg, or 20 mg / kg to 30 mg / kg, 1 mg / kg to 25 mg / kg, 4 mg / kg to 20 mg / kg, 6 mg / kg to 15 mg / kg, or 8 mg / kg to 10 mg / kg of the conjugate is administered, e.g., 1 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, In some embodiments, a conjugate is administered at a dose of 1 mg / kg, 25 mg / kg, or 30 mg / kg. In some embodiments, a lower dose (e.g., 1 mg / kg to 30 mg / kg) is administered to reduce drug exposure and increase safety while maintaining sufficient efficacy.

[0127] In some embodiments, the complex is administered by infusion, e.g., intravenous infusion, optionally as a bolus infusion or continuous infusion, the latter optionally over a period of 10 minutes to 3 hours, e.g., 0.25 to 2 hours, or 0.5 to 1 hour.

[0128] The peptide comprises at least one cationic domain comprising at least four amino acid residues and at least one hydrophobic domain comprising at least three amino acid residues, wherein the peptide comprises a total of 7 to 40 amino acid residues and the at least one cationic domain comprises a β-alanine residue in combination with an arginine and / or histidine residue.The oligonucleotide comprises a total of 12 to 40 contiguous nucleobases, wherein at least 12 contiguous nucleobases are complementary to a target sequence of the human dystrophin gene.

[0129] Preferably, the conjugate comprises or consists of 5' CCCAACAATCAAAGAAGAAGGCATTTCTAG-3' (SEQ ID NO: 106) having a 3'-terminus covalently attached to the N-terminus of the peptide RBRRBRFQILYBRBR-NH2 via a glutamic acid residue, where any free -COOH present in the glutamic acid residue is replaced with -CONH2. For example, the 5' group of the conjugate may be a phosphoramidate (e.g., sarcosinamide). Alternatively, the conjugate comprises or consists of 5'-CTCAATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having a 3'-terminus covalently attached to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH via a glutamic acid residue, where any free -COOH present in the glutamic acid residue is replaced with -CONH2. For example, the 5' group of the conjugate may be a phosphoramidate. Alternatively, the conjugate comprises or consists of 5'-CTCACAATCAAAAGAAGAAGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3'-terminus covalently linked to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH via a β-alanine residue. For example, the 5' group of the conjugate may be a phosphoramidate. More preferably, the conjugate comprises or consists of 5'-CCCCAATCAAAAAGGAAAGGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3'-terminus covalently linked to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR via a glutamic acid residue, where any free -COOH present in the glutamic acid residue is replaced with -CONH2. For example, the 5' group of the conjugate may be a phosphoramidate. In some embodiments, the conjugate comprising a glutamic acid residue linker has the following structure: [ka]

[0130] Advantageously, as described in the Examples section, the doses of the complexes described herein can induce skipping of exon 51 of dystrophin even in cardiac tissue of non-human primates without causing persistent liver and kidney toxicity. Advantageously, as described in the Examples section, the doses of the complexes described herein can deliver cargo oligonucleotides to traditionally hard-to-reach tissues. The use of the non-human primate models described herein advantageously facilitates the identification of doses that can be used in other primates, such as humans.

[0131] Preferably, the conjugate is administered to the subject on a weekly to monthly basis (eg, weekly, biweekly, once every three weeks, or monthly), or once every three months.

[0132] Oligonucleotides The oligonucleotides used in the complexes disclosed herein may be complementary to target sites in the dystrophin (DMD) gene. Without wishing to be bound by theory, it is believed that oligonucleotides that hybridize to specific target sites in the human dystrophin gene induce the skipping of exon 51 during splicing of dystrophin pre-mRNA, thereby ameliorating Duchenne muscular dystrophy.

[0133] The oligonucleotide comprises a nucleobase sequence complementary to the human dystrophin gene and can, for example, induce exon 51 skipping. In certain preferred embodiments, the oligonucleotide comprises at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) consecutive nucleobases selected from 5'-CCCAACUCAAGGAAGAUGGCAUUCUAG-3' (SEQ ID NO: 112) or its thymine-substituted analog, 5'-CCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106). In certain preferred embodiments, the oligonucleotide comprises or consists of 5'-CCCACAAAGAAGGCATTTCTAG-3' (SEQ ID NO: 106). Non-limiting examples of oligonucleotides that can be used in the conjugates disclosed herein are listed below. # Sequence number Array 107 5′-ACCAGAGUAACAGUCUGAGUAGGAGC-3′ 108 5′-CUCAUACCUUCUGCUUGAUGAUC-3′ 109 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ 110 5′-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3′ 111 5′-ACAUCAGGAAGAUGGCAUUUCUAG-3′ 112 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ 113 5′-AUCAUUUUUUCUCAUACCUUCUGCUAG-3′ 114 5′-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3′ 115 5′-CACCCACCAUCACCCUCUGUG-3′ 116 5′-AUCAUCUCGUUGAUAUUCCUCAA-3′

[0134] One or more uracils (e.g., all uracils) in the above oligonucleotide sequence are replaced with thymine. For example, the oligonucleotide sequence may be, for example, 5'-CCCAACAUCAAGGAAGAUGGCAUUCUAG-3' (SEQ ID NO: 112). Alternatively, the oligonucleotide sequence may be, for example, 5'-CCCCAACAAAAAGGAAGGCCATTTCTAG-3' (SEQ ID NO: 106).

[0135] In some embodiments of these and other oligonucleotides described herein, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0136] peptide Peptides that may be used in the conjugates described herein include those disclosed in WO 2020030927 and WO 2020115494. Preferably, the peptides included in the conjugates described herein do not contain artificial amino acid residues.

[0137] In some embodiments, the peptide does not contain aminohexanoic acid residues. In some embodiments, the peptide does not contain any form of aminohexanoic acid residues. In some embodiments, the peptide does not contain 6-aminohexanoic acid residues.

[0138] In some embodiments, the peptide contains only, and thus consists of, naturally occurring amino acid residues.

[0139] The peptide is replaced by a natural amino acid. In some embodiments, an artificial amino acid, such as 6-aminohexanoic acid, typically used in cell penetrating peptides, is replaced by an amino acid selected from β-alanine, serine, proline, arginine, and histidine or hydroxyproline.

[0140] In some embodiments, aminohexanoic acid is replaced by β-alanine. In some embodiments, 6-aminohexanoic acid is replaced by β-alanine.

[0141] In some embodiments, aminohexanoic acid is replaced by histidine. In some embodiments, 6-aminohexanoic acid is replaced by histidine.

[0142] In some embodiments, aminohexanoic acid is replaced by hydroxyproline. In some embodiments, 6-aminohexanoic acid is replaced by hydroxyproline.

[0143] In some embodiments, artificial amino acids such as 6-aminohexanoic acid, which are typically used in cell penetrating peptides, may be replaced by any combination of β-alanine, serine, proline, arginine, and histidine or hydroxyproline, e.g., any combination of β-alanine, histidine, and hydroxyproline.

[0144] In some embodiments, peptides having an overall length of 40 amino acid residues or less are provided, which comprise two or more cationic domains, each comprising at least four amino acid residues, and one or more hydrophobic domains, each comprising at least three amino acid residues, wherein at least one cationic domain comprises histidine residues. In some embodiments, at least one cationic domain is histidine-rich.

[0145] In some embodiments, what is meant by histidine-rich is defined herein with respect to the cationic domain.

[0146] Cationic Domain The present invention relates to short cell-penetrating peptides having a specific structure in which there are at least two cationic domains having a certain length.

[0147] In some embodiments, the peptide comprises up to four cationic domains, and up to three cationic domains.

[0148] In some embodiments, the peptide comprises two cationic domains.

[0149] As defined above, the peptide comprises two or more cationic domains, each having a length of at least four amino acid residues.

[0150] In some embodiments, each cationic domain has a length of between 4 and 12 amino acid residues, for example, a length of between 4 and 7 amino acid residues.

[0151] In some embodiments, each cationic domain has a length of 4, 5, 6, or 7 amino acid residues.

[0152] In some embodiments, each cationic domain is of similar length, eg, each respective cationic domain is the same length.

[0153] In some embodiments, each cationic domain contains cationic amino acids, and may contain polar and / or non-polar amino acids.

[0154] The non-polar amino acids may be selected from alanine, β-alanine, proline, glycine, cysteine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, In some embodiments, the non-polar amino acids are uncharged.

[0155] The polar amino acids may be selected from serine, asparagine, hydroxyproline, histidine, arginine, threonine, tyrosine, and glutamine. In some embodiments, the selected polar amino acids do not carry a negative charge.

[0156] The cationic amino acid may be selected from arginine, histidine, and lysine. In some embodiments, the cationic amino acid has a positive charge at physiological pH.

[0157] In some embodiments, each cationic domain does not contain anionic or negatively charged amino acid residues, hi some embodiments, each cationic domain contains arginine, histidine, β-alanine, hydroxyproline, and / or serine residues.

[0158] In some embodiments, each cationic domain comprises or consists of arginine, histidine, β-alanine, hydroxyproline, and / or serine residues.

[0159] In some embodiments, each cationic domain comprises at least 40%, at least 45%, at least 50% cationic amino acids.

[0160] In some embodiments, each cationic domain comprises a majority of cationic amino acids, hi some embodiments, each cationic domain comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% amino acids.

[0161] In some embodiments, each cationic domain has an isoelectric point (pi) of at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 10.5, at least 11.0, at least 11.5, at least 12.0.

[0162] In some embodiments, each cationic domain has an isoelectric point (pi) of at least 10.0.

[0163] In some embodiments, each cationic domain has an isoelectric point (pi) between 10.0 and 13.0.

[0164] In some embodiments, each cationic domain has an isoelectric point (pi) between 10.4 and 12.5.

[0165] In some embodiments, the isoelectric point of the cationic domain is calculated at physiological pH by any suitable means available in the art, in some embodiments by using IPC (www.isoelectric.org), a web-based algorithm developed by Lukasz Kozlowski (Biol. Direct. 2016, 11:55. DOI:10.1186 / s13062-016-0159-9).

[0166] In some embodiments, each cationic domain comprises at least one cationic amino acid, e.g., 1 to 5 cationic amino acids. In some embodiments, each cationic domain comprises at least two cationic amino acids, e.g., 2 to 5 cationic amino acids.

[0167] In some embodiments, each cationic domain is arginine-rich and / or histidine-rich. In some embodiments, a cationic domain may contain both histidine and arginine.

[0168] In some embodiments, each cationic domain comprises a majority of arginine and / or histidine residues.

[0169] In some embodiments, each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% arginine and / or histidine residues. In some embodiments, a cationic domain may comprise at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% arginine residues.

[0170] In some embodiments, the cationic domain may comprise at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% histidine residues.

[0171] In some embodiments, the cationic domain may comprise a total of 1 to 5 histidine and 1 to 5 arginine residues. In some embodiments, the cationic domain may comprise 1 to 5 arginine residues. In some embodiments, the cationic domain may comprise 1 to 5 histidine residues. In some embodiments, the cationic domain may comprise a total of 2 to 5 histidine and 3 to 5 arginine residues. In some embodiments, the cationic domain may comprise 3 to 5 arginine residues. In some embodiments, the cationic domain may comprise 2 to 5 histidine residues.

[0172] In some embodiments, each cationic domain comprises one or more β-alanine residues, hi some embodiments, each cationic domain may comprise a total of 2 to 5 β-alanine residues, for example a total of 2 or 3 β-alanine residues.

[0173] In some embodiments, the cationic domain may include one or more hydroxyproline or serine residues.

[0174] In some embodiments, the cationic domain may comprise one to two hydroxyproline residues.In some embodiments, the cationic domain may comprise one to two serine residues.

[0175] In some embodiments, all of the cationic amino acids in a given cationic domain can be histidines, or, for example, all of the cationic amino acids in a given cationic domain can be arginines.

[0176] In some embodiments, the peptide may include at least one histidine-rich cationic domain.In some embodiments, the peptide may include at least one arginine-rich cationic domain.

[0177] In some embodiments, the peptide may include at least one arginine-rich cationic domain and may include at least one histidine-rich cationic domain.

[0178] In some embodiments, the peptide comprises two arginine-rich cationic domains.

[0179] In some embodiments, the peptide comprises two histidine-rich cationic domains.

[0180] In some embodiments, the peptide comprises two arginine- and histidine-rich cationic domains.

[0181] In some embodiments, the peptide comprises one arginine-rich cationic domain and one histidine-rich cationic domain, hi some embodiments, each cationic domain comprises no more than three consecutive arginine residues, e.g., no more than two consecutive arginine residues.

[0182] In some embodiments, each cationic domain does not comprise consecutive histidine residues.

[0183] In some embodiments, each cationic domain comprises arginine, histidine, and / or β-alanine residues. In some embodiments, each cationic domain comprises a majority of arginine, histidine, and / or β-alanine residues. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acid residues in each cationic domain are arginine, histidine, and / or β-alanine residues. In some embodiments, each cationic domain consists of arginine, histidine, and / or β-alanine residues.

[0184] In some embodiments, the peptide comprises a first cationic domain comprising arginine and β-alanine residues and a second cationic domain comprising arginine and β-alanine residues.

[0185] In some embodiments, the peptide comprises a first cationic domain comprising arginine and β-alanine residues, and a second cationic domain comprising histidine, β-alanine, and optionally arginine residues.

[0186] In some embodiments, the peptide comprises a first cationic domain comprising arginine and β-alanine residues and a second cationic domain comprising histidine and β-alanine residues.

[0187] In some embodiments, the peptide comprises a first cationic domain consisting of arginine and β-alanine residues and a second cationic domain consisting of arginine and β-alanine residues.

[0188] In some embodiments, the peptide comprises a first cationic domain consisting of arginine and β-alanine residues, and a second cationic domain consisting of arginine, histidine, and β-alanine residues.

[0189] In some embodiments, the peptide comprises at least two cationic domains, e.g., the cationic domains form arms of the peptide. In some embodiments, the cationic domains are located at the N- and C-termini of the peptide. Thus, in some embodiments, the cationic domains may be known as cationic arm domains.

[0190] In some embodiments, the peptide comprises two cationic domains, one located at the N-terminus of the peptide and one located at the C-terminus of the peptide. In some embodiments, it is located at either terminus of the peptide. In some embodiments, no further amino acids or domains are present at the N-terminus and C-terminus of the peptide, except for other groups such as terminal modifications, linkers and / or oligonucleotides. For the avoidance of doubt, such other groups may be present in addition to the "peptide" as described and claimed herein. Thus, in some embodiments, each cationic domain forms a terminus of the peptide. In some embodiments, this does not preclude the presence of further linker groups as described herein.

[0191] In some embodiments, the peptide may contain up to 4 cationic domains, hi some embodiments, the peptide contains 2 cationic domains.

[0192] In some embodiments, the peptide comprises two cationic domains that are both arginine-rich.

[0193] In some embodiments, the peptide comprises one cationic domain that is arginine-rich.

[0194] In some embodiments, the peptide comprises two cationic domains that are both arginine-rich and histidine-rich.

[0195] In some embodiments, the peptide comprises one cationic domain that is arginine-rich and one cationic domain that is histidine-rich.

[0196] In some embodiments, the cationic domain comprises an amino acid unit selected from R, H, B, RR, HH, BB, RH, HR, RB, BR, HB, BH, RBR, RBB, BRR, BBR, BRB, RBH, RHB, HRB, BRH, HRR, RRH, HRH, HBB, BBH, RHR, BHB, HBH, or any combination thereof.

[0197] In some embodiments, the cationic domain may also comprise serine, proline, and / or hydroxyproline residues. In some embodiments, the cationic domain may further comprise an amino acid unit selected from RP, PR, RPR, RRP, PRR, PRP, Hyp, R[Hyp]R, RR[Hyp], [Hyp]RR, [Hyp]R[Hyp], [Hyp][Hyp]R, R[Hyp][Hyp], SB, BS, or any combination thereof, or any combination with the amino acid units listed above.

[0198] In some embodiments, each cationic domain comprises any one of the following sequences: RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB[Hyp]B (SEQ ID NO:17), R[Hyp]H[Hyp]HB (SEQ ID NO:18), R[Hyp]RR[Hyp]R (SEQ ID NO:19), or any combination thereof.

[0199] In some embodiments, each cationic domain comprises or consists of any one of the following sequences: RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB[Hyp]B, R[Hyp]H[Hyp]HB, R[Hyp]RR[Hyp]R (SEQ ID NO:19), or any combination thereof.

[0200] In some embodiments, each cationic domain comprises or consists of one of the following sequences: RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRRBR (SEQ ID NO:4), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), or HBHBR (SEQ ID NO:9).

[0201] In some embodiments, each cationic domain in the peptide can be the same or different, hi some embodiments, each cationic domain in the peptide is different.

[0202] Hydrophobic domain The present invention relates to short cell-penetrating peptides having a specific structure in which there is at least one hydrophobic domain having a certain length.

[0203] As used herein, "hydrophobic" refers to an amino acid or domain that has the ability to repel or not mix with water.

[0204] In some embodiments, the peptide comprises up to 3 hydrophobic domains, or up to 2 hydrophobic domains, hi some embodiments, the peptide comprises 1 hydrophobic domain.

[0205] As defined above, the peptide comprises one or more hydrophobic domains, each having a length of at least three amino acid residues.

[0206] In some embodiments, each hydrophobic domain has a length of 3 to 6 amino acid residues, hi some embodiments, each hydrophobic domain has a length of 5 amino acid residues.

[0207] In some embodiments, each hydrophobic domain may be comprised of apolar, polar, and hydrophobic amino acid residues.

[0208] The hydrophobic amino acid residue may be selected from alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, methionine, tryptophan.

[0209] The non-polar amino acid residue may be selected from proline, glycine, cysteine, alanine, valine, leucine, isoleucine, tryptophan, phenylalanine, and methionine.

[0210] The polar amino acid residue may be selected from serine, asparagine, hydroxyproline, histidine arginine, threonine, tyrosine, glutamine.

[0211] In some embodiments, the hydrophobic domain does not include hydrophilic amino acid residues.

[0212] In some embodiments, each hydrophobic domain comprises a majority of hydrophobic amino acid residues. In some embodiments, each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids. In some embodiments, each hydrophobic domain consists of hydrophobic amino acid residues.

[0213] In some embodiments, each hydrophobic domain has a hydrophobicity of at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.8, at least 1.0, at least 1.1, at least 1.2, or at least 1.3.

[0214] In some embodiments, each hydrophobic domain has a hydrophobicity of at least 0.3, at least 0.35, at least 0.4, or at least 0.45.

[0215] In some embodiments, each hydrophobic domain has a hydrophobicity of at least 1.2, at least 1.25, at least 1.3, or at least 1.35.

[0216] In some embodiments, each hydrophobic domain has a hydrophobicity index between 0.4 and 1.4.

[0217] In some embodiments, each hydrophobic domain has a hydrophobicity index between 0.45 and 0.48.

[0218] In some embodiments, each hydrophobic domain has a hydrophobicity index between 1.27 and 1.39.

[0219] In some embodiments, hydrophobicity is measured as described by White and Wimley: WC Wimley and SH White, "Experimentally determined hydrophobicity scale for proteins at membrane interfaces," Nature Struct Biol 3:842 (1996).

[0220] In some embodiments, each hydrophobic domain comprises at least 3 or at least 4 hydrophobic amino acid residues.

[0221] In some embodiments, each hydrophobic domain comprises a phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residue. In some embodiments, each hydrophobic domain comprises or consists of a phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residue.

[0222] In some embodiments, each hydrophobic domain comprises or consists of a phenylalanine, leucine, isoleucine, tyrosine and / or glutamine residue.

[0223] In some embodiments, each hydrophobic domain comprises or consists of tryptophan and / or proline residues.

[0224] In some embodiments, the peptide comprises one hydrophobic domain. In some embodiments, each hydrophobic domain is located at the center of the peptide. Thus, in some embodiments, the hydrophobic domains may be known as core hydrophobic domains. In some embodiments, each hydrophobic core domain is flanked on both sides by arm domains. In some embodiments, the arm domains may comprise one or more cationic domains and one or more additional hydrophobic domains. In some embodiments, each arm domain comprises a cationic domain.

[0225] In some embodiments, the peptide comprises two arm domains flanking a hydrophobic core domain, where each arm domain comprises a cationic domain.

[0226] In some embodiments, the peptide comprises two cationic arm domains flanking a hydrophobic core domain.

[0227] In some embodiments, each hydrophobic domain comprises one of the following sequences: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26), or any combination thereof.

[0228] In some embodiments, each hydrophobic domain comprises or consists of one of the following sequences: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26), or any combination thereof.

[0229] In some embodiments, each hydrophobic domain comprises or consists of one of the following sequences: FQILY (SEQ ID NO:21), YQFLI (SEQ ID NO:20), or ILFQY (SEQ ID NO:22).

[0230] In some embodiments, each hydrophobic domain comprises or consists of FQILY (SEQ ID NO:21).

[0231] In some embodiments, each hydrophobic domain in the peptide is the same sequence or a different sequence.

[0232] The present invention relates to short cell-penetrating peptides for use in delivering therapeutic cargo molecules in the treatment of disease conditions.

[0233] The peptide has a sequence that is a continuous single molecule, and therefore the domains of the peptide are continuous. In some embodiments, the peptide comprises several domains arranged linearly between the N-terminus and the C-terminus. In some embodiments, the domains are selected from the cationic domain and the hydrophobic domain described above. In some embodiments, the peptide comprises or consists of a cationic domain and a hydrophobic domain, the domains being as defined above.

[0234] Each domain shares common sequence characteristics, as described in the relevant sections above, but the exact sequence of each domain can vary and be altered. Thus, a variety of sequences are possible for each domain. Combinations of each possible domain sequence result in a variety of peptide structures, each of which forms part of the present invention. Features of the peptide structures are described below.

[0235] In some embodiments, a hydrophobic domain separates any two cationic domains, hi some embodiments, each hydrophobic domain is flanked on either side by a cationic domain.

[0236] In some embodiments, a cationic domain is not contiguous with other cationic domains.

[0237] In some embodiments, the peptide comprises one hydrophobic domain flanked by two cationic domains in the following arrangement: [cationic domain]-[hydrophobic domain]-[cationic domain]

[0238] In some embodiments, the hydrophobic domain may be known as a core domain, and each of the cationic domains may be known as an arm domain. In some embodiments, the hydrophobic arm domains flank the cationic core domain on either side.

[0239] In some embodiments, the peptide comprises or consists of two cationic domains and one hydrophobic domain.

[0240] In some embodiments, the peptide consists of a hydrophobic core domain flanked by two cationic arm domains.

[0241] In some embodiments, the peptide is RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), or ), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), and R[Hyp]RR[Hyp]R (SEQ ID NO: 19), respectively, flanked by two cationic arm domains each comprising a sequence selected from YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), and WWPW (SEQ ID NO: 26).

[0242] In some embodiments, the peptide comprises or consists of one hydrophobic core domain comprising a sequence selected from FQILY (SEQ ID NO:21), YQFLI (SEQ ID NO:20), and ILFQY (SEQ ID NO:22), flanked by two cationic arm domains selected from RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRRBR (SEQ ID NO:4), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), and HBHBR (SEQ ID NO:9). In some embodiments, the peptide comprises or consists of one hydrophobic core domain comprising the sequence FQILY (SEQ ID NO:21), flanked by two cationic arm domains selected from RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRRBR (SEQ ID NO:4), BRBR (SEQ ID NO:7), and RBHBH (SEQ ID NO:8).

[0243] In any of these embodiments, further groups such as linkers, terminal modifications and / or oligonucleotides may be present.

[0244] In some embodiments, the peptide is N-terminally modified.

[0245] In some embodiments, the peptide is N-acetylated, N-methylated, N-trifluoroacetylated, N-trifluoromethylsulfonylated, or N-methylsulfonylated, hi some embodiments, the peptide is N-acetylated.

[0246] Optionally, the N-terminus of the peptide may be unmodified.

[0247] In some embodiments, the peptide is N-acetylated.

[0248] In some embodiments, the peptide is C-terminally modified.

[0249] In some embodiments, the peptide comprises a C-terminal modification selected from a carboxy group, a thioacid group, an aminooxy group, a hydrazino group, a thioester group, an azide group, a strained alkyne, a strained alkene, an aldehyde group, a thiol group, or a haloacetyl group.

[0250] Advantageously, the C-terminal modification provides a means for attaching the peptide to the oligonucleotide.

[0251] Thus, the C-terminal modification may comprise a linker or vice versa. In some embodiments, the C-terminal modification may consist of a linker or vice versa. Suitable linkers are described elsewhere herein.

[0252] In some embodiments, the peptide comprises a C-terminal carboxyl group.

[0253] In some embodiments, the C-terminal carboxyl group is provided by a glycine or β-alanine residue.

[0254] In some embodiments, the C-terminal carboxyl group is provided by a β-alanine residue. In some embodiments, the C-terminal β-alanine residue is a linker.

[0255] In some embodiments, each cationic domain may therefore further comprise an N- or C-terminal modification. In some embodiments, the C-terminal cationic domain comprises a C-terminal modification. In some embodiments, the N-terminal cationic domain comprises an N-terminal modification. In some embodiments, the C-terminal cationic domain comprises a linker group. In some embodiments, the C-terminal cationic domain comprises a C-terminal β-alanine. In some embodiments, the N-terminal cationic domain is N-acetylated.

[0256] The peptides of the present invention are defined as having a total length of 40 amino acid residues or less and therefore can be considered oligopeptides.

[0257] In some embodiments, the peptide has a total length of 3 to 30 amino acid residues, for example, 5 to 25 amino acid residues, 10 to 25 amino acid residues, 13 to 23 amino acid residues, or 15 to 20 amino acid residues.

[0258] In some embodiments, the peptide has an overall length of at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 amino acid residues.

[0259] In some embodiments, the peptide is capable of penetrating cells, and therefore may be considered a cell-penetrating peptide.

[0260] In some embodiments, the peptide is for attaching to an oligonucleotide. In some embodiments, the peptide is for transporting an oligonucleotide to a target cell. In some embodiments, the peptide is for delivering an oligonucleotide to a target cell. Thus, the peptide can be considered a carrier peptide.

[0261] In some embodiments, the peptides are capable of penetrating into cells and tissues, for example into the nucleus of a cell, hi some embodiments, into muscle tissue.

[0262] In some embodiments, the peptide may comprise or consist of a peptide selected from any one of the following sequences: RBRRBRRFQILYRBRBR (SEQ ID NO: 27) RBRRBRRFQILYRBRR (SEQ ID NO:28) RBRRBRFQILYRRBRBR (SEQ ID NO: 29) RBRBRFQILYRBRRBRR (SEQ ID NO: 30) RBRRBRRYQFLIRBRBR (SEQ ID NO: 31) RBRRBRRILFQYRBRBR (SEQ ID NO: 32) RBRRBRFQILYRBRBR (SEQ ID NO: 33) RBRRBFQILYRBRRBR (SEQ ID NO: 34) RBRRBRFQILYBRBR (SEQ ID NO: 35) RBRRBFQILYRBRBR (SEQ ID NO: 36) RBRRBRRFQILYRBHBH (SEQ ID NO: 37) RBRRBRRFQILYHBHBR (SEQ ID NO: 38) RBRRBRRFQILYHBRBH (SEQ ID NO: 39) RBRRBRRYQFLIRBHBH (SEQ ID NO: 40) RBRRBRRILFQYRBHBH (SEQ ID NO: 41) RBRHBHRFQILYRBRBR (SEQ ID NO: 42) RBRBBHRFQILYRBHBH (SEQ ID NO: 43) RBRRBRFQILYRBHBH (SEQ ID NO: 44) RBRRBRFQILYHBHBH (SEQ ID NO: 45) RBRRBHFQILYRBHBH (SEQ ID NO: 46) HBRRBRFQILYRBHBH (SEQ ID NO: 47) RBRRBFQILYRBHBH (SEQ ID NO: 48) RBRRBRFQILYBHBH (SEQ ID NO: 49) RBRRBRYQFLIHBHBH (SEQ ID NO:50) RBRRBRILFQYHBHBH (SEQ ID NO:51) RBRRBRRFQILYHBHBH (SEQ ID NO:52)

[0263] In some embodiments, the peptide may comprise or consist of a peptide selected from any one of the following additional sequences: RBRRBRFQILYBRBS (SEQ ID NO:53) RBRRBRFQILYBRB [Hyp] (SEQ ID NO: 54) RBRRBRFQILYBR[Hyp]R (SEQ ID NO: 55) RRBRRBRFQILYBRBR (SEQ ID NO:56) BRRBRRFQILYBRBR (SEQ ID NO:57) RBRRBRWWWBRBR (SEQ ID NO:58) RBRRBRWWPWWBRBR (SEQ ID NO:59) RBRRBRWPWWBRBR (SEQ ID NO: 60) RBRRBRWWPWBRBR (SEQ ID NO:61) RBRRBRRWWWRBRBR (SEQ ID NO:62) RBRRBRRWWPWWRBRBR (SEQ ID NO: 63) RBRRBRRWPWWRBRBR (SEQ ID NO: 64) RBRRBRRWWPWRBRBR (SEQ ID NO: 65) RBRRBRRFQILYBRBR (SEQ ID NO: 66) RBRRBRRFQILYRBR (SEQ ID NO:67) BRBRBWWPWWRBRRBR (SEQ ID NO: 68) RBRRBRRFQILYBHBH (SEQ ID NO: 69) RBRRBRRFQIYRBHBH (SEQ ID NO: 70) RBRRBRFQILYBRBH (SEQ ID NO:71) RBRRBRFQILYR[Hyp]H[Hyp]H (SEQ ID NO: 72) R[Hyp]RR[Hyp]RFQILYRBHBH (SEQ ID NO: 73) R[Hyp]RR[Hyp]RFQILYR[Hyp]H[Hyp]H (SEQ ID NO: 74) RBRRBRWWWRBHBH (SEQ ID NO: 75) RBRRBRWWPRBHBH (SEQ ID NO:76) RBRRBRPWWRBHBH (SEQ ID NO:77) RBRRBRWWPWWRBHBH (SEQ ID NO: 78) RBRRBRWWPWRBHBH (SEQ ID NO:79) RBRRBRWPWWRBHBH (SEQ ID NO: 80) RBRRBRRWWWRBHBH (SEQ ID NO: 81) RBRRBRRWWPWWRBHBH (SEQ ID NO: 82) RBRRBRRWPWWRBHBH (SEQ ID NO: 83) RBRRBRRWWPWRBHBH (SEQ ID NO: 84) RRBRRBRFQILYRBHBH (SEQ ID NO: 85) BRRBRRFQILYRBHBH (SEQ ID NO: 86) RRBRRBRFQILYBHBH (SEQ ID NO: 87) BRRBRRFQILYBHBH (SEQ ID NO: 88) RBRRBHRFQILYRBHBH (SEQ ID NO: 89) RBRRBRFQILY[Hyp]R[Hyp]R (SEQ ID NO: 101) R[Hyp]RR[Hyp]RFQILYBRBR (SEQ ID NO: 102) R[Hyp]RR[Hyp]RFQILY[Hyp]R[Hyp]R (SEQ ID NO: 103) RBRRBRWWWBRBR (SEQ ID NO: 104) RBRRBRWWPWWBRBR (SEQ ID NO: 105)

[0264] In some embodiments, the peptide may comprise or consist of a peptide selected from one of the following sequences: RBRRBRRFQILYRBRBR (SEQ ID NO: 27) RBRRBRRYQFLIRBRBR (SEQ ID NO: 31) RBRRBRRILFQYRBRBR (SEQ ID NO: 32) RBRRBRFQILYBRBR (SEQ ID NO: 35) RBRRBRRFQILYRBHBH (SEQ ID NO: 37) RBRRBRRFQILYHBHBR (SEQ ID NO: 38) RBRRBRFQILYRBHBH (SEQ ID NO: 44)

[0265] In some embodiments, the peptide comprises or consists of the following sequence: RBRRBRFQILYBRBR (SEQ ID NO:35).

[0266] In some embodiments, the peptide comprises or consists of the sequence: RBRRBRRFQILYRBHBH (SEQ ID NO:37).

[0267] In some embodiments, the peptide comprises or consists of the following sequence: RBRRBRFQILYRBHBH (SEQ ID NO:44).

[0268] Complex In some embodiments, the complex comprises a peptide selected from one of the following sequences: RBRRBRFQILYBRBR (SEQ ID NO: 35), RBRRBRRFQILYRBHBH (SEQ ID NO: 37), and RBRRBRFQILYRBHBH (SEQ ID NO: 44).

[0269] In some embodiments, in either case, the peptide may further comprise an N-terminal modification as described above.

[0270] Suitable linkers include, for example, a C-terminal cysteine ​​residue that allows for the formation of a disulfide, thioether or thiol-maleimide bond, a C-terminal aldehyde to form an oxime, click reaction or formation of a morpholino bond with a basic amino acid on the peptide, or a carboxylic acid moiety on the peptide covalently linked to an amino group to form a carboxamide bond.

[0271] In some embodiments, the linker is 1-5 amino acids in length. In some embodiments, the linker may include any linker known in the art. In some embodiments, the linker is selected from any of the following sequences: G, BC, XC, C, GGC, BBC, BXC, XBC, X, XX, B, BB, BX, and XB. In some embodiments, X is 6-aminohexanoic acid. In some embodiments, the linker is a Glu linker.

[0272] In some embodiments, the linker can be a polymer, such as, for example, PEG.

[0273] In some embodiments, the linker is β-alanine.

[0274] In some embodiments, the peptide is attached to the oligonucleotide via a carboxamide bond.

[0275] The linker of the conjugate may form part of the oligonucleotide that binds the peptide. Alternatively, the oligonucleotide may be linked directly to the C-terminus of the peptide. In some embodiments, such as in other embodiments, a linker is not required.

[0276] Alternatively, the peptide can be chemically linked to the oligonucleotide via, for example, a disulfide, alkenyl, alkynyl, aryl, ether, thioether, triazole, amide, carboxamide, urea, thiourea, semicarbazide, carbazide, hydrazine, oxime, phosphate, phosphoramidate, thiophosphate, borane phosphate, iminophosphate, etc., or a thiolmaleimide bond.

[0277] Optionally, a cysteine ​​may be added to the N-terminus of the peptide to allow disulfide bond formation, or the peptide may be bromoacetylated at the N-terminus to allow thioether attachment.

[0278] In some embodiments, the complex is capable of penetrating into cells and tissues, eg, into the nucleus of a cell, eg, into muscle tissue.

[0279] In some embodiments, the oligonucleotide component of the conjugate is as described above under "Oligonucleotides" and elsewhere herein. In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0280] Linker In addition to the above, the conjugates described herein may include a linker that covalently links the peptides described herein to the oligonucleotides described herein. Linkers useful in the present invention can be found in WO2020 / 115494, the disclosure of which is incorporated herein by reference.

[0281] The linkers may independently be of formula (I): T1-(CR 1 R 2 ) n -T2(I) (Wherein, T1 is a divalent group for binding to the peptide and is selected from the group consisting of -NH- and carbonyl; T2 is a divalent group for attaching to an oligonucleotide and is selected from the group consisting of -NH- and carbonyl; n is 1, 2 or 3; Each R 1 is independently -Y 1 -X 1 -Z 1 and Where: Y 1 does not exist or is -(CR A1 R A2 ) m -, m is 1, 2, 3 or 4, R A1 and R A2 are each independently hydrogen, OH, or (1-2C)alkyl; X 1 is absent or -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR A3 )-, -N(R A3 )-, -N(R A3 )-C(O)-, -N(R A3 )-C(O)O-, -C(O)-N(R A3 )-, -N(R A3 )C(O)N(R A3 )-, -N(R A3 )C(NR A3 )N(R A3 )-, -SO-, -S-, -SO2-, -S(O)2N(R A3 )- or -N(R A3 )SO2-, and each R A3 is independently selected from hydrogen and methyl; and Z 1 is further an oligonucleotide or is hydrogen, (1-6C) alkyl, (2-6C) alkenyl, (2-6C) alkynyl, aryl, (3-6C) cycloalkyl, (3-6C) cycloalkenyl, or heteroaryl; wherein each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl is selected from the group consisting of (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR A4 R A5 and (1-4C)alkoxy; R A4 and R A5 are each independently selected from the group consisting of hydrogen and (1-4C)alkyl, and Each R 2 is independently -Y 2 -X 2 -Z 2 where: Y 2 does not exist or is not a B1 R B2 ] m -, m is an integer selected from 1, 2, 3, or 4, R B1 and R B2 are each independently selected from hydrogen, OH or (1-2C)alkyl; X 2 is absent or -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR B3 )-, -N(R B3 )-, -N(R B3 )-C(O)-, -N(R B3 )-C(O)O-, -C(O)-N(R B3 )-, -N(R B3 )C(O)N(R B3 )-, -N(R B3 )C(NR B3 )N(R B3 )-, -SO-, -S--SO2-, -S(O)2N(R B3 )- or -N(R B3 )SO2-, and R B3 is independently selected from hydrogen or methyl, and Z 2is selected from hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl, each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl being selected from (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR B4 R B5 and (1-4C)alkoxy; R B4 and R B5 are each independently hydrogen or (1-2C)alkyl, provided that when n=1 and T1 and T2 are different from each other, R 1 and R 2 are not both H, n=1, T1 and T2 are different from each other, and R 1 and R 2 When one of is H, R 1 and R 2 The other is not methyl, or n=2 and R 1 and R 2 When each occurrence of is H, T1 and T2 are both -C(O)-, or both -NH-.

[0282] In some embodiments, the linker is of the following structure: [ka] [ka] [ka] [ka] [ka]

[0283] Pharmaceutical Compositions The complex of the present invention, or a pharma- ceutically acceptable salt thereof, can be formulated into a pharmaceutical composition.

[0284] In some embodiments, the pharmaceutical composition comprises a complex of the invention or a pharma- ceutically acceptable salt thereof.

[0285] In some embodiments, the pharmaceutical composition may further comprise a pharma- ceutically acceptable diluent, adjuvant, or carrier.

[0286] Suitable pharma- ceutically acceptable diluents, adjuvants or carriers are well known in the art.

[0287] It is to be understood that the pharmaceutical compositions of the present disclosure may further comprise additional known therapeutic agents, drugs, modifications of compounds into prodrugs, and the like, under medical use, for alleviating, ameliorating, preventing, and treating the diseases, disorders, and conditions described herein.

[0288] In some embodiments, the pharmaceutical composition is for use as a medicament, e.g., for use as a medicament in the same manner as described herein for the conjugate, and all features described herein in relation to medical treatment using the conjugate apply to the pharmaceutical composition.

[0289] Thus, in a further aspect of the present invention there is provided a pharmaceutical composition according to the fourth aspect for use as a medicament.In a further aspect there is provided a method of treating a subject with a disease condition comprising administering an effective amount of a pharmaceutical composition as disclosed herein.

[0290] medical use A complex containing the peptide of the present invention can be used as a medicament for the treatment of diseases using the dosages mentioned above.

[0291] The medicament may be in the form of a pharmaceutical composition as defined above.

[0292] Also provided are methods of treating a patient or subject in need of treatment for a disease condition, comprising administering to the patient or subject a therapeutically effective amount of the conjugate. In some embodiments, the medical treatment requires delivery of the oligonucleotide into a cell, e.g., into the nucleus of a cell.

[0293] The disease to be treated may include any disease in which improved penetration of the cell membrane and / or nuclear membrane by oligonucleotides may improve the therapeutic effect.

[0294] In some embodiments, the conjugate is used to treat a disease of the neuromuscular system.

[0295] In some embodiments, the complex is for use in the treatment of a disease caused by splicing defects. In such embodiments, the oligonucleotide may comprise an oligonucleotide capable of preventing or correcting the splicing defect and / or increasing the production of correctly spliced ​​mRNA molecules.

[0296] In some embodiments, the conjugate is used to treat DMD or BMD.

[0297] In some embodiments, the conjugates are used to treat the cardiac effects of diseases such as DMD, including, for example, cardiomyopathy (e.g., dilated, hypertrophic, or restrictive cardiomyopathy), heart failure, and / or cardiac arrhythmias.

[0298] In some embodiments, such that the oligonucleotide of the complex is operable to increase expression of a dystrophin protein, hi some embodiments, the oligonucleotide of the complex is operable to increase expression of a functional dystrophin protein.

[0299] In some embodiments, the complex increases dystrophin expression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. In some embodiments, the complex increases dystrophin expression by up to 50%. In some embodiments, the complex restores dystrophin expression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. In some embodiments, the complex restores dystrophin expression by up to 50%.

[0300] In some embodiments, the complex restores 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of the function of the dystrophin protein, hi some embodiments, the complex restores up to 50% of the function of the dystrophin protein.

[0301] In some embodiments, the oligonucleotide of the complex can act to do so by causing the skipping of exon 51 during transcription of dystrophin.

[0302] In some embodiments, the oligonucleotides of the conjugate cause skipping of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% of one or more exons of the dystrophin gene. In some embodiments, the oligonucleotides of the conjugate cause skipping of up to 50% of one or more exons of the dystrophin gene.

[0303] In some embodiments, the patient or subject to be treated may be any animal or human. In some embodiments, the patient or subject may be a non-human mammal. In some embodiments, the patient or subject may be male or female. In some embodiments, the subject is male.

[0304] In some embodiments, the patient or subject to be treated as described above may be of any age. In some embodiments, the patient or subject to be treated is 0 to 40 years old, such as 0 to 30 years old, such as 0 to 25 years old, such as 0 to 20 years old.

[0305] In some embodiments, the conjugate is for administration to a subject systemically, e.g., by intramedullary, intrathecal, intracerebroventricular, intravitreal, enteral, parenteral, intravenous, intraarterial, intramuscular, intratumor, subcutaneous, oral, or nasal routes.

[0306] In some embodiments, the conjugate is for intravenous administration to a subject.

[0307] In some embodiments, the conjugate is for administration to a subject via intravenous injection.

[0308] In some embodiments, the complex is administered by intravenous injection. In some embodiments, the intravenous injection is a bolus injection. In some embodiments, the intravenous administration lasts for 10 minutes to 3 hours, for example, 0.25 to 2 hours or 0.5 to 1 hour.

[0309] Advantageously, the dosage of the conjugate of the invention may be lower, for example an order of magnitude or more lower, than the dosage required to see any effect from the oligonucleotide alone.

[0310] In some embodiments, following administration of the conjugates of the invention, one or more markers of toxicity are significantly reduced as compared to prior conjugates using currently available peptide carriers.

[0311] A suitable toxicity marker may be a marker of nephrotoxicity.

[0312] Relevant urinary and serum toxicity markers include KIM-1, NGAL, BUN, creatinine, alkaline phosphatase, alanine transferase, and aspartate aminotransferase.

[0313] In some embodiments, the levels of at least one of KIM-1, NGAL, and BUN are reduced following administration of a conjugate of the present invention compared to prior conjugates using currently available peptide carriers.

[0314] In some embodiments, the levels of each of KIM-1, NGAL, and BUN are reduced following administration of a conjugate of the invention as compared to prior conjugates using currently available peptide carriers.

[0315] In some embodiments, the level of each marker is significantly reduced as compared to prior conjugates using currently available peptide carriers.

[0316] In some embodiments, the levels of each marker are reduced by up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% following administration of the conjugates of the present invention compared to prior conjugates using currently available peptide carriers.

[0317] As provided above, the methods of the invention include administering to a subject (e.g., a subject susceptible to exon 51 skipping) 1 mg / kg to 60 mg / kg of a conjugate of an oligonucleotide and a peptide covalently bonded or linked to the oligonucleotide via a linker. In some embodiments, 30 mg / kg to 60 mg / kg (e.g., 40 mg / kg to 60 mg / kg, 30 mg / kg to 50 mg / kg; 30 mg / kg to 40 mg / kg, e.g., 30 mg / kg, 40 mg / kg, 50 mg / kg, or 60 mg / kg) of the conjugate is administered. In some embodiments, 40 mg / kg to 50 mg / kg, 50 mg / kg to 60 mg / kg, 35 mg / kg to 45 mg / kg, 45 mg / kg to 55 mg / kg, 35 mg / kg to 55 mg / kg, 30 mg / kg to 45 mg / kg, 35 mg / kg to 50 mg / kg, 40 mg / kg to 55 mg / kg, or 45 mg / kg to 60 mg / kg of the conjugate is administered. In some embodiments, 1 mg / kg to 30 mg / kg (e.g., 1 mg / kg to 20 mg / kg, 5 mg / kg to 25 mg / kg, 10 mg / kg to 30 mg / kg, 1 mg / kg to 15 mg / kg, 5 mg / kg to 20 mg / kg, 10 mg / kg to 25 mg / kg, 15 mg / kg to 30 mg / kg, 1 mg / kg to 10 mg / kg, 5 mg / kg to 15 mg / kg, 10 mg / kg to 20 mg / kg, 15 mg / kg to 25 mg / kg, or 20 mg / kg to 30 mg / kg, 1 mg / kg to 25 mg / kg, 4 mg / kg to 20 mg / kg, 6 mg / kg to 15 mg / kg, or 8 mg / kg to 10 mg / kg of the conjugate is administered, e.g., 1 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, In some embodiments, a conjugate is administered at a dose of 1 mg / kg, 25 mg / kg, or 30 mg / kg. In some embodiments, a lower dose (e.g., 1 mg / kg to 30 mg / kg) is administered to reduce drug exposure and increase safety while maintaining sufficient efficacy.

[0318] Preparation of peptides The peptides of the invention can be produced by any standard protein synthesis method, such as chemical synthesis, semi-chemical synthesis, or by using an expression system. The invention therefore also relates to nucleotide sequences comprising or consisting of DNA coding for said peptides, expression systems, such as vectors consisting of such sequences with sequences necessary for expression and control of expression, as well as host cells and host organisms transformed with the expression systems.

[0319] Thus, nucleic acids encoding the peptides according to the invention are also provided.

[0320] In some embodiments, the nucleic acid may be provided in an isolated or purified form.

[0321] Also provided is an expression vector comprising a nucleic acid encoding a peptide according to the invention.

[0322] In some embodiments, the vector is a plasmid.

[0323] In some embodiments, the vector comprises a regulatory sequence, e.g., a promoter, operably linked to a nucleic acid encoding a peptide according to the invention. In some embodiments, the expression vector is capable of expressing the peptide when transfected into a suitable cell, e.g., a mammalian cell, a bacterial cell, or a fungal cell.

[0324] Also provided is a host cell comprising the above-described expression vector of the invention.

[0325] The expression vector may be selected depending on the host cell into which the nucleic acid of the present invention may be inserted. Such transformation of the host cell uses conventional techniques such as those taught in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, USA, 2001. The selection of an appropriate vector is within the skill of one skilled in the art. Suitable vectors include plasmids, bacteriophages, cosmids, viruses, etc.

[0326] The produced peptide can be isolated and purified from the host cells by any suitable method, such as, for example, precipitation or chromatographic separation, such as, for example, affinity chromatography.

[0327] Suitable vectors, hosts and recombinant techniques are well known in the art.

[0328] The following examples are intended to illustrate the present invention, but are not intended to limit the invention in any way. EXAMPLES

[0329] Example 1. Preparation of the Complex The conjugates described herein may be prepared, for example, using the techniques described in WO 2020115494 and WO 2020030927.

[0330] 9-Fluorenylmethoxycarbonyl (Fmoc)-protected L-amino acids, benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium (PyBOP), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and Fmoc^-Ala-OH preloaded Wang resin (0.19 or 0.46 mmol / g) were obtained from Merck (Hohenbrunn, Germany). HPLC grade acetonitrile, methanol, and synthesis grade N-methyl-2-pyrrolidone (NMP) were purchased from Fisher Scientific (Loughborough, UK). Peptide synthesis grade N,N-dimethylformamide (DMF) and diethyl ether were obtained from VWR (Leicestershire, UK). Piperidine and trifluoroacetic acid (TFA) were obtained from Alfa Aesar (Heysham, England). PMO was purchased from Gene Tools Inc. (Philomath, USA). Chicken embryo extract and horse serum were obtained from Sera Laboratories International Ltd (West Sussex, UK). Interferon was obtained from Roche Applied Science (Penzberg, Germany). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. MALDI-TOF mass spectrometry was performed using a Voyager DE Pro BioSpectrometry workstation. A 10 mg / mL stock solution of A-cyano-4-hydroxycinnamic acid or sinapic acid in 50% acetonitrile in water was used as matrix. Error bars are shown as ±0.1%.

[0331] Peptides were prepared on a 10 pmol scale using an Intavis Parallel Peptide Synthesizer or on a CEM Liberty Blue chromatograph using Fmoc^-Ala-OH preloaded Wang resin (0.19 or 0.46 mmol / g, Merck Millipore). TMThe peptides were prepared on a 100 pmol scale using a Peptide Synthesizer (Buckingham, UK). Synthesis using an Intavis Parallel Peptide Synthesizer used a double coupling step with a PyBOP / NMM coupling mixture followed by capping with acetic anhydride after each step. Synthesis using a CEM Liberty Blue Peptide Synthesizer performed a single standard coupling for all amino acids except arginine, which was performed with double coupling. Couplings were performed once at 75 °C for 5 min with 60 watts of microwave power, except for the arginine residue, which was coupled twice each. Each deprotection reaction was performed twice at 75 °C, once for 30 s and then for 3 min with 35 watts of microwave power. Once synthesis was complete, the resin was washed with DMF (3 x 50 mL) and the N-terminus of the solid-phase bound peptide was acetylated with acetic anhydride in the presence of DI PEA at room temperature. After N-terminal acetylation, the peptide resin was washed with DMF (3 x 20 mL) and DCM (3 x 20 mL). The peptide was cleaved from the solid support by treatment with a cleavage cocktail consisting of trifluoroacetic acid (TFA):H2O:triisopropylsilane (TIPS) (95%:2.5%:2.5%:3-10 mL) at room temperature for 3 h. After peptide release, excess TFA was removed by sparging with nitrogen. The crude peptide was precipitated by the addition of cold diethyl ether (15-40 mL depending on synthesis scale) and centrifuged at 3200 rpm for 5 min. The crude peptide pellet was washed three times with cold diethyl ether (3 x 15 mL) and purified by RP-HPLC using a Varian 940-LC HPLC System equipped with a 445-LC Scale-up module and a 440-LC fraction collector. Peptides were purified by semi-preparative HPLC on a RP-C18 column (10 x 250 mm, Phenomenex Jupiter) using a linear gradient of CH3CN in 0.1% TFA / H2O at a flow rate of 15 mL / min. Detection was performed at 220 and 260 nm.Fractions containing the desired peptide were combined and lyophilized to give the peptide as a white solid.

[0332] The PMO antisense sequence (5'-CCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106)) for inducing exon 51 skipping of dystrophin can be used, for example, to prepare complex 1. A peptide (Ac-RBRRBRFQILYBRBR) can be conjugated to the 3' end of the PMO via a glutamic acid linker to its C-terminal carboxyl group, where the free -COOH in the glutamic acid residue is replaced with -CONH2, generating the complex [peptide]-CO-CH(CONH2)-CH2CH2CO-[3'-oligonucleotide-5'], which has also been identified as the following structure: [ka] This may be accomplished using PyBOP and HOAt in NMP in the presence of DIPEA in DMSO. In some instances, HBTU may be used instead of PyBOP for activation of the C-terminal carboxyl group of the peptide. In general, to a solution of the peptide in N-methylpyrrolidone (NMP), PyBOP (in NMP), HOAt (in NMP), diisopropylethylamine (DIPEA), and PMO (in DMSO) are added. The resulting mixture may be warmed, for example, to 40° C., and the reaction quenched by the addition of TFA in H2O. The solution may be purified by ion exchange chromatography. For example, the PMO-peptide complex may be purified, for example, using a linear gradient of NaCl(aq.) / CH3CN / phosphate buffer (pH 7.0). Fractions containing the compound of interest may be combined and lyophilized to obtain the resulting peptide-PMO. Removal of excess salt from the peptide-PMO complex may be accomplished by filtration of the fractions collected after ion exchange using a centrifugal filter device. The conjugate may be lyophilized and analyzed by MALDI-TOF. The conjugate may be dissolved in sterile water and filtered through a cellulose acetate membrane before use. The concentration of the peptide-PMO may be determined by the molar absorbance of the conjugate at 265 nm in 0.1 N HCl solution.

[0333] Example 2. Non-human primate studies Complex 1 shown below was diluted to 25 mg / mL in 0.9% sterile saline. [ka] [ka] [ka] and the oligonucleotide is a PMO linked via -P(=O)(NMe2)-O-.

[0334] NHP single-infusion dose-response study The efficacy of exon skipping of complex 1 was tested in non-human primates (NHPs). Specifically, 2-3 year old untreated cynomolgus monkeys received a single intravenous injection of the complex at a dose of 40 mg / kg or 60 mg / kg over 30 minutes (n=2 males and n=2 females per group). A control group (n=1 males and n=1 females) received a control of 0.9% sterile saline. 2-4 year old untreated cynomolgus monkeys received a single intravenous slow bolus injection (1-2 minutes) of 20 mg / kg, 40 mg / kg, or 60 mg / kg of the complex (n=1 males and n=1 females per group).

[0335] Animals were necropsied 1 week after dosing. At routine necropsies, portions of tissues (biceps, gastrocnemius, quadriceps, soleus, tibialis anterior, diaphragm, lung, heart, cerebral cortex, cerebellum, colon, duodenum, esophagus, epidermis, adrenal gland, thyroid, kidney, liver, spleen, pancreas, thymus, testis, and ovary) were collected for exon skipping analysis and tissue biochemistry analysis. Distal samples of liver and kidney were subjected to gross necropsy. During the study period, blood samples were analyzed for clinical chemistry, hematology, complement, and coagulation on days 1, 4, and 7 after dosing and compared to pre-dosing. Additionally, urine was collected on days 1, 4, and 7 after dosing and analyzed for KIM-1 and compared to pre-dosing. Body and organ weight profiles were also evaluated.

[0336] [Table 1]

[0337] Safety assessment Clinical chemistry, hematology, complement, and coagulation were evaluated on days 1, 4, and 7 post-dose and compared to pre-dose. Body and organ weight profiles were also evaluated.

[0338] The clinical chemistry markers used in this study are shown in Table 2. [Table 2]

[0339] The hematological markers used in this study are summarized in Table 3. [Table 3]

[0340] The complement markers used in this study are summarized in Table 4. [Table 4]

[0341] The coagulation markers used in this study are summarized in Table 5. [Table 5]

[0342] In addition, urine samples taken pre- and post-dose on days 1, 4, and 7 were analyzed for KIM-1 and creatinine.

[0343] RT-PCR analysis The level of exon 51 skipping was determined by RT-PCR. Skeletal, cardiac, and smooth muscle tissues were homogenized using a bead-based homogenization method. RNA was extracted using a Maxwell RSC 48 instrument (Promega) and the simplyRNA Tissue Kit (Promega) according to the manufacturer's recommendations. RNA concentration and purity were measured using a ClarioStar (BMG LabTech). Quantified RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, 4368813) under the thermal cycling conditions described in Table 6. [Table 6]

[0344] Nested-PCR was performed as two consecutive PCR reactions. The first PCR was performed using reverse transcribed cDNA template. The second PCR was performed using the product obtained from the first PCR. All primers used in the PCR reactions are shown in Table 7, and thermal cycling conditions are outlined in Table 8. The final PCR products were analyzed by agarose (2%) gel electrophoresis. Gels were prepared using Midori Green Advance Stain (Nippon Genetics). HyperLadder 50 bp (Bioline, BIO-33039) and PCR products were loaded onto an agarose gel and run until an adequate degree of band separation was obtained. Images of the separated gels were then captured using a G:BOX (Syngene) gel imaging system. The non-skipped / native and skipped / Δex51 bands obtained from the nested-PCR gel were subjected to densitometry using ImageJ software (Fiji). Densitometric measurements from band quantification were used to determine the skipping of nhpDMD exon 51 according to the following formula:

[0345] Formula for nhpDMD exon 51 skipping: ([peak area of ​​skipped fragment] / [peak area of ​​skipped fragment + peak area of ​​non-skipped fragment]) x 100

[0346] [Table 7]

[0347] [Table 8]

[0348] The efficiency of exon 51 skipping in non-human primates receiving a bolus or infusion of Complex 1 is summarized in Table 9.

[0349] [Table 9]

[0350] The results are shown in Figures 1 to 13E.

[0351] Figure 1 shows the dose response in the major muscle groups of animals (n = 2-4).

[0352] Figures 2A and 2B show that the post-dosing increases in urea and creatinine (kidney function markers) are transient. The increases seen with infusion dosing are lower than the levels seen with bolus dosing. All increases return to pre-dosing and / or control levels by day 7 post-dosing.

[0353] Figures 3A, 3B, and 3C show that the ALP and ALT profiles are favorable with the infusion. The AST elevations seen with the infusion are likely not lab related and are within the historical control min / max range. Lower values ​​are observed than those seen with the bolus, returning to pre-infusion values ​​by day 4 after infusion.

[0354] Figures 4A, 4B, and 4C show that the slight variations present in the primary hematological parameters following administration of Complex 1 do not reach biologically significant levels. The primary hematological parameters of the infusion administration have the same or lower profile than those seen with the bolus administration.

[0355] Figure 5 shows that the infusion dose resulted in slightly lower plasma concentrations than the bolus dose, but the complex remained in plasma for a longer period (48 hours after administration). The infusion time was 30 minutes.

[0356] Figure 6 shows the dose response with broad-area delivery of Complex 1 detected in traditionally hard-to-reach tissues such as the heart and central nervous system. PMO was detected 7 days after administration. Figures 7 and 8 show that infusion administration results in equivalent or greater levels of PMO concentration and presence in tissues compared to bolus administration.

[0357] For urinary creatinine testing, animal-to-animal variability makes interpretation of the data more complicated, but Figure 9A shows that the transient modulation seen with the 60 mg / kg infusion returned to vehicle control levels by day 4 post-dose. Additionally, urinary testing for KIM-1 (a urinary biomarker of kidney injury) showed no detectable levels of KIM-1 at any time point for any animal in the study (urine was collected pre-dose, 24 hours post-dose, and 4 and 7 days post-dose and analyzed for KIM-1 levels; Figure 9B).

[0358] Figures 10A, 10B, and 10C show that coagulation markers are unaffected by either bolus or infusion administration.

[0359] Figure 11 shows that injection of Complex 1 did not affect body weight profile. No treatment-related changes in organ weight were observed in animals administered Complex 1 (Figures 12A-12I). The observed random organ weight differences were considered to be related to random and / or sexual maturation differences and were unrelated to treatment.

[0360] 13A-13E show serum complement activation in CH50, Bb, C3a or SC5b-9. C5a was not detected during the study.

[0361] The summary of the conclusions is as follows: No deaths, SAEs or AEs were observed when Complex 1 was injected at 40 mg / kg or 60 mg / kg. No macroscopic histopathology was observed following injection of Complex 1 at any dose. The majority of microscopic findings were non-malignant (benign) and non-degenerative and considered to be a known effect of oligosaccharides. The main microscopic findings in the kidneys at 40 mg / kg or 60 mg / kg were minimal to moderate tubular basophilia, minimal to mild tubular basophilic granules, and at 60 mg / kg minimal to mild tubular dilation and tiny multifocal hyaline casts. -Minor, transient and reversible elevations of several hematological markers that were considered benign were observed at all doses. All doses are capable of delivery to tissues critical to addressing DMD pathology. -Minor, transient and reversible elevations in several clinical chemistry markers were observed at all doses. Complex 1 had no effect on organs or body weight.

[0362] Example 3. Additional NHP Studies In additional studies, naive cynomolgus monkeys were administered the above conjugate (conjugate 1) or another conjugate containing the same oligonucleotide sequence as conjugate 1 but linked to a peptide consisting of the sequence (Arg)6Gly. The conjugates were administered intravenously over 30 minutes at 5, 10, 30, or 60 mg / kg (n=3). Saline was used as a control and administered three times Q2W. Biopsies were performed 7 days after each administration and pathological tissue was collected 7 days after the final administration. The levels (%) of exon 51 skipping in biceps, quadriceps, and diaphragm 7 days after the final administration are shown in Figure 14, and the levels (%) of exon 51 skipping in cardiac tissues (left ventricle, right ventricle, and aorta) 7 days after the final administration are shown in Figure 15. As shown in these figures, conjugate 1 showed dramatically improved efficacy in exon 51 skipping over the (Arg)6Gly conjugate in the tissues shown in the figures. In addition, it is noteworthy that Complex 1 showed substantial efficacy in certain tissues at doses as low as 10 mg / kg. Analysis of biopsy samples taken 7 days after the first and second doses, and of a final sample taken 7 days after the final dose, showed that exon skipping was cumulative with repeated doses of Complex 1. The results are shown in Figures 19A and 19B.

[0363] method RNA was extracted, quantified, and normalized to 50 ng / μL in nuclease-free water. 500 ng of normalized RNA was then reverse transcribed. Primers were prepared as 10 uM aliquots from 100 μM stocks. A PCR master mix containing 2X DreamTaq Master Mix, primers, and nuclease-free water was vortexed, thawed, and centrifuged briefly.

[0364] [Table 10]

[0365] [Table 11]

[0366] 22 μl of reaction master mix was added to each thin-walled PCR reaction tube. 3 μl of template cDNA from each sample was then added to each tube and mixed thoroughly. Samples were gently vortexed and the PCR tubes were briefly spun down, taking care to eliminate air bubbles, before proceeding. Tubes were placed in the Proflex and PCR was performed using the following thermal cycling conditions:

[0367] [Table 12]

[0368] After PCR, samples were placed on ice or frozen at -20°C. After preparation for processing, samples were fractionated on a 2% agarose gel and imaged. Bands were analyzed as follows:

[0369] [Table 13]

[0370] Bands were quantified by densitometry using ImageJ software. The 'plot lanes' command was used and the line tool was used to mark the base of the curve. The wand tool was used to select the area under the curve and the area was calculated. The percentage of skipping of nhpDMDex51 was calculated as follows: JPEG2024511955000056.jpg1990

[0371] Example 4. Single dose study in mdx mice Complex 2 was administered intravenously once to mdx mice, and serum creatine kinase levels were measured 7 days after injection. Vehicle (0.9% saline) was used as a control in this study. Wild-type (WT) mice were used as control animals in this study. R6G-PMO was used as a comparative complex. R6G-PMO consists of an identical oligonucleotide sequence to Complex 2, but is linked to a peptide consisting of the sequence (Arg)6Gly. The results are shown in Figures 16, 17A, 17B, 18A, and 18B.

[0372] Complex 2 is shown below. [ka] [ka] [ka] and the oligonucleotide is a PMO linked via -P(=O)(NMe2)-O-.

[0373] The results in FIG. 16 suggest that Complex 2 prevents further muscle damage after administration of a tolerable single dose of Complex 2, as shown by measurement of normalized creatine kinase levels.

[0374] The results in Figures 17A and 17B show that 91% dystrophin restoration was achieved in the quadriceps muscle 7 days after a single administration of Complex 2.

[0375] The results in Figures 18A and 18B show that 26% dystrophin restoration was achieved in the heart 7 days after a single administration of Complex 2.

[0376] Other embodiments Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

[0377] Some embodiments are within the scope of the following numbered paragraphs.

[0378] 1. A method of treating a subject with Duchenne muscular dystrophy, the method comprising administering 1 mg / kg to 60 mg / kg of a conjugate of an oligonucleotide and a peptide covalently or via a linker to the oligonucleotide; the peptide comprises at least one cationic domain comprising at least four amino acid residues and at least one hydrophobic domain comprising at least three amino acid residues, wherein the peptide comprises a total of 7 to 40 amino acid residues, and wherein the at least one cationic domain comprises β-alanine residues in combination with arginine and / or histidine residues; and The method, wherein the oligonucleotide comprises a total of 12 to 40 contiguous nucleobases, at least 12 contiguous nucleobases being complementary to a target sequence of the human dystrophin gene.

[0379] 2. The oligonucleotide is 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), or 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO: 113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO: 114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), 2. The method of paragraph 1, comprising a sequence selected from the group consisting of 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), and thymine substituted analogs thereof.

[0380] 3. The method of paragraph 1 or 2, wherein each cationic domain has a length of between 4 and 12 amino acid residues.

[0381] 4. The method of paragraph 3, wherein each cationic domain has a length of between 4 and 7 amino acid residues.

[0382] 5. The method of any one of paragraphs 1 to 4, wherein each cationic domain comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cationic amino acids.

[0383] 6. The method of any one of paragraphs 1 to 4, wherein each cationic domain contains at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% arginine and / or histidine residues.

[0384] 7. The method of any one of paragraphs 1 to 4, wherein each cationic domain comprises one of the following sequences: RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB[Hyp]B (SEQ ID NO:17), R[Hyp]H[Hyp]HB (SEQ ID NO:18), R[Hyp]RR[Hyp]R (SEQ ID NO:19), or any combination thereof.

[0385] 8. The method of any one of paragraphs 1 to 4, wherein each cationic domain comprises or consists of one of the following sequences: RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB[Hyp]B (SEQ ID NO:17), R[Hyp]H[Hyp]HB (SEQ ID NO:18), R[Hyp]RR[Hyp]R (SEQ ID NO:19), or any combination thereof.

[0386] 9. The method of any one of the above paragraphs, wherein the peptide comprises two cationic domains.

[0387] 10. The method of any one of the above paragraphs, wherein each hydrophobic domain has a length of 3 to 6 amino acids, preferably, each hydrophobic domain has a length of 5 amino acids.

[0388] 11. The method of any one of the above paragraphs, wherein each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids.

[0389] 12. The method of any one of the above paragraphs, wherein each hydrophobic domain comprises a phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residue, preferably each hydrophobic domain comprises or consists of a phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residue.

[0390] 13. The method of any one of the above paragraphs, wherein the peptide comprises one hydrophobic domain.

[0391] 14. The method of any one of the above paragraphs, wherein each hydrophobic domain comprises one of the following sequences: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26), or any combination thereof.

[0392] 15. The method of any one of the above paragraphs, wherein each hydrophobic domain comprises or consists of one of the following sequences: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26), or any combination thereof.

[0393] 16. The method of any one of the above paragraphs, wherein the peptide comprises or consists of two cationic domains and one hydrophobic domain.

[0394] 17. The method of any one of the above paragraphs, wherein the peptide comprises or consists of two cationic domains and one hydrophobic domain.

[0395] 18. The above peptides are RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17). 5. The method of any one of paragraphs 1 to 4, comprising, or consisting of, one hydrophobic domain comprising a sequence selected from YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), and WWPW (SEQ ID NO:26), flanked by two cationic arm domains each comprising a sequence selected from YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), and WWPW (SEQ ID NO:26).

[0396] 19. The method of any one of paragraphs 1 to 4, wherein the peptide comprises one of the following sequences or consists of one of the following sequences: RBRRBRRFQILYRBRBR (SEQ ID NO: 27), RBRRBRRYQFLIRBRBR (SEQ ID NO: 31), RBRRBRRILFQYRBRBR (SEQ ID NO: 32), RBRRBRFQILYBRBR (SEQ ID NO: 35), RBRRBRRFQILYRBHBH (SEQ ID NO: 37), RBRRBRRFQILYHBHBR (SEQ ID NO: 38), and RBRRBRFQILYRBHBH (SEQ ID NO: 44).

[0397] 20. The method of any one of paragraphs 1 to 4, wherein the peptide has the following amino acid sequence: RBRRBRFQILYBRBR (sequence number 35).

[0398] 21. The method of any one of paragraphs 1 to 4, wherein the peptide has the following amino acid sequence: RBRRBRRFQILYRBHBH (sequence number 37).

[0399] 22. The method of any one of paragraphs 1 to 4, wherein the peptide has the following amino acid sequence: RBRRBRFQILYRBHBH (sequence number 44).

[0400] 23. The method of any one of paragraphs 1 to 22, wherein the peptide is attached to the remainder of the complex via its N-terminus.

[0401] 24. The method of paragraph 23, wherein the C-terminus of the peptide is -NH2.

[0402] 25. The method of any one of paragraphs 1 to 22, wherein the peptide is attached to the remainder of the complex via its C-terminus.

[0403] 26. The method of paragraph 25, wherein the peptide is acylated at its N-terminus.

[0404] 27. The method of any one of the above paragraphs, wherein the complex comprises or is of the following structure: [ka]

[0405] 28. The method of any one of paragraphs 1 to 26, wherein the complex comprises or has the following structure: [ka]

[0406] 29. The method of any one of paragraphs 1 to 26, wherein the complex comprises or is of the following structure: [ka]

[0407] 30. The method of any one of the above paragraphs, wherein each linker is independently represented by formula (I): T1-(CR 1 R 2 ) n -T2(I) (Wherein, T1 is a divalent group for binding to the peptide and is selected from the group consisting of -NH- and carbonyl; T2 is a divalent group for attaching to an oligonucleotide and is selected from the group consisting of -NH- and carbonyl; n is 1, 2 or 3; Each R 1 is independently -Y 1 -X 1 -Z 1 and Where: Y 1 does not exist or is -(CR A1 R A2 ) m -, m is 1, 2, 3 or 4, R A1 and R A2are each independently hydrogen, OH, or (1-2C)alkyl; X 1 is absent or -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR A3 )-, -N(R A3 )-, -N(R A3 )-C(O)-, -N(R A3 )-C(O)O-, -C(O)-N(R A3 )-, -N(R A3 )C(O)N(R A3 )-, -N(R A3 )C(NR A3 )N(R A3 )-, -SO-, -S-, -SO2-, -S(O)2N(R A3 )- or -N(R A3 )SO2-, and each R A3 is independently selected from hydrogen and methyl; and Z 1 is further an oligonucleotide or is hydrogen, (1-6C) alkyl, (2-6C) alkenyl, (2-6C) alkynyl, aryl, (3-6C) cycloalkyl, (3-6C) cycloalkenyl, or heteroaryl; wherein each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl is selected from the group consisting of (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR A4 R A5 and (1-4C)alkoxy; R A4 and R A5 are each independently selected from the group consisting of hydrogen and (1-4C)alkyl, and Each R 2 is independently -Y 2 -X 2 -Z 2 where: Y 2 does not exist or is not a B1 R B2 ]m -, m is an integer selected from 1, 2, 3, or 4, R B1 and R B2 are each independently selected from hydrogen, OH or (1-2C)alkyl; X 2 is absent or -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR B3 )-, -N(R B3 )-, -N(R B3 )-C(O)-, -N(R B3 )-C(O)O-, -C(O)-N(R B3 )-, -N(R B3 )C(O)N(R B3 )-, -N(R B3 )C(NR B3 )N(R B3 )-, -SO-, -S--SO2-, -S(O)2N(R B3 )- or -N(R B3 )SO2-, and R B3 is independently selected from hydrogen or methyl, and Z 2 is selected from hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl, each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl being selected from (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR B4 R B5 and (1-4C)alkoxy; R B4 and R B5 are each independently hydrogen or (1-2C)alkyl, provided that when n=1 and T1 and T2 are different from each other, R 1 and R 2 are not both H, n=1, T1 and T2 are different from each other, and R 1 and R 2When one of is H, R 1 and R 2 The other is not methyl, or n=2 and R 1 and R 2 When each occurrence of is H, T1 and T2 are both -C(O)-, or both -NH-.

[0408] 31. The method of paragraph 30, wherein T2 is -C(O)-.

[0409] 32. Each R 1 is independently -Y 1 -X 1 -Z 1 and Y 1 does not exist or is -(CR A1 R A2 ) m -, m is 1, 2, 3 or 4, and R A1 and R A2 are each hydrogen or (1-2C) alkyl; X 1 is absent or -O-, -C(O)-, -C(O)O-, -N(R A3 )-, -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, -N(R A3 )C(O)N(R A3 )-, -N(R A3 )C(NR A3 )N(R A3 )- or -S-, and each R A3 are independently hydrogen or methyl; and Z 1 is further an oligonucleotide or is hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl, each of which is selected from the group consisting of (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, and heteroaryl, each of which is selected from the group consisting of (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR A4R A5 and (1-4C)alkoxy; R M and R A5 32. The method of paragraph 30 or 31, wherein each is independently hydrogen or (1-2C)alkyl.

[0410] 33. Each R 1 is independently -Y 1 -X 1 -Z 1 and Y 1 does not exist or is -(CR A1 R A2 ) m -, m is 1, 2, 3, or 4, and R A1 and R" are each independently hydrogen or (1-2C)alkyl; X 1 does not exist or is -O-, -C(O)-, -C(O)O-, -N(R A3 )-, -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, -N(R A3 )C(O)N(R A3 )-, -N(R A3 )C(NR A3 )N(R A3 )- or -S-, and each R A3 is independently hydrogen or methyl; and Z 1 is further an oligonucleotide or is hydrogen, (1-6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, each of which (1-6C)alkyl, aryl, (3-6C)cycloalkyl, and heteroaryl are optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituents selected from the group consisting of (1-4C)alkyl, halo, and hydroxy.

[0411] 34. Each R 1 is independently -Y 1-X 1 -Z 1 and Y 1 does not exist or the formula -(CR A1 R A2 ) m -, m is 1, 2, 3 or 4, R A1 and R A2 are each independently hydrogen or (1-2C)alkyl; X 1 does not exist or is -C(O)-, -C(O)O-, -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, and each R A3 is hydrogen or methyl, and Z 1 is further an oligonucleotide or is hydrogen, (1-6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, each of which (1-6C)alkyl, aryl, (3-6C)cycloalkyl, and heteroaryl are optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituents selected from the group consisting of (1-4C)alkyl, halo, and hydroxy.

[0412] 35. Each R 1 is independently -Y 1 -X 1 -Z 1 and Y 1 is absent, -(CH2)-, or -(CH2CH2)-, X 1 does not exist or is -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, and each R A3 are independently hydrogen or methyl; and Z 1 32. The method of paragraph 30 or 31, wherein is hydrogen or (1-2C)alkyl.

[0413] 36. Each R 2 is independently -Y 2-Z 2 and Y 2 does not exist or is -(CR B1 R B2 ) m -, m is 1, 2, 3 or 4, R B1 and R B2 are each independently hydrogen or (1-2C)alkyl; and Z 2 36. The method of any one of paragraphs 30 to 35, wherein is hydrogen or (1-6C)alkyl.

[0414] 37. The method of any one of paragraphs 30 to 36, wherein each R2 is hydrogen.

[0415] 38. The method of any one of paragraphs 30 to 37, wherein n is 2 or 3.

[0416] 39. The method of any one of paragraphs 30 to 37, wherein n is 1.

[0417] 40. The method of any one of paragraphs 1 to 39, wherein the linker is an amino acid residue selected from the group consisting of glutamic acid, succinic acid, and gamma-aminobutyric acid residues.

[0418] 41. The method of any one of paragraphs 1 to 39, wherein the linker has the following structure: [ka]

[0419] 42. The method of any one of paragraphs 1 to 39, wherein the linker has the following structure: [ka]

[0420] 43. The method of any one of paragraphs 1 to 39, wherein the linker has the following structure: [ka]

[0421] 44. The method of any one of paragraphs 1 to 39, wherein the linker has the following structure: [ka]

[0422] 45. The method of any one of paragraphs 1 to 39, wherein the linker has the following structure: [ka]

[0423] 46. ​​The method of any one of paragraphs 1 to 39, wherein the complex comprises or has the following structure: [ka]

[0424] 47. The method of any one of paragraphs 1 to 39, wherein the complex comprises or has the following structure: [ka]

[0425] 48. The method of any one of paragraphs 1 to 39, wherein the complex comprises or has the following structure: [ka]

[0426] 49. The method of any one of paragraphs 1 to 39, wherein the complex comprises or is of the following structure: [ka]

[0427] 50. The method of any one of paragraphs 1 to 39, wherein the complex comprises or has the following structure: [ka]

[0428] 51. The method of any one of paragraphs 1 to 50, wherein the oligonucleotide is attached at its 3' end to the linker or the peptide.

[0429] 52. The above conjugates include the following peptides: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 111), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 112), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 113), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 114), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 115), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 116), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 117), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 118), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 119), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 120), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' 111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof, wherein if a free -COOH is present in said glutamic acid residue, it is replaced with -CONH2.

[0430] 53. The method of paragraph 52, wherein the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' end covalently linked to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR via a glutamic acid residue, wherein any free -COOH present in the glutamic acid residue is replaced with -CONH2.

[0431] 54. The method of paragraph 52, wherein the conjugate comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR, or a thymine-substituted analog thereof, wherein any free -COOH present in the glutamic acid residue is replaced with -CONH2.

[0432] 55. The method of paragraph 52, wherein the conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR, or a thymine-substituted analog thereof, wherein any free -COOH present in the glutamic acid residue is replaced with -CONH2.

[0433] 56. The above conjugates are 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 111), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 112), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 113), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 114), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 115), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 116), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 117), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 118), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 119), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 120), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO 111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116) or a thymine substituted analog thereof, wherein if a free -COOH is present in the glutamic acid residue it is replaced with -CONH2.

[0434] 57. The method of paragraph 56, wherein the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' end covalently linked via a glutamic acid residue to the N-terminus of the peptide RBRRBRFQILYBRBR-NH2, wherein any free -COOH present in the glutamic acid residue is replaced with -CONH2.

[0435] 58. The method of paragraph 56, wherein the conjugate comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), having its 3' end covalently linked via a glutamic acid residue to the N-terminus of the peptide RBRRBRFQILYBRBR-NH2, or a thymine-substituted analog thereof, wherein any free -COOH present in the glutamic acid residue is replaced with -CONH2.

[0436] 59. The method of paragraph 56, wherein the conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), having its 3' end covalently linked via a glutamic acid residue to the N-terminus of the peptide RBRRBRFQILYBRBR-NH2, or a thymine-substituted analog thereof, wherein any free -COOH present in the glutamic acid residue is replaced with -CONH2.

[0437] 60. The above conjugates have a 3' end covalently linked to the C-terminus of the peptide Ac-RBRBRFQILYRBHBH via a γ-aminobutyric acid residue: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAG 2. The method of paragraph 1, comprising or consisting of GAAGAUGGCAUUUCUAG-3' (SEQ ID NO:111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof.

[0438] 61. The method of paragraph 60, wherein the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' end covalently linked to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH via a γ-aminobutyric acid residue.

[0439] 62. The method of paragraph 60, wherein the conjugate comprises or consists of 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), having its 3′ end covalently linked via a γ-aminobutyric acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine-substituted analog thereof.

[0440] 63. The method of paragraph 60, wherein the conjugate comprises or consists of 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), having its 3′ end covalently linked via a γ-aminobutyric acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine-substituted analog thereof.

[0441] 64. The above conjugates are 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 111), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 112), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 113), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 114), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 115), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 116), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 117), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 118), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 119), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 120), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO 111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof, wherein if a free -COOH is present in the glutamic acid residue, it is replaced with -CONH2.

[0442] 65. The method of paragraph 64, wherein the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, wherein any free -COOH present in the glutamic acid residue is replaced with -CONH2.

[0443] 66. The method of paragraph 64, wherein the conjugate comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine-substituted analog thereof, wherein any free -COOH present in the glutamic acid residue is replaced with -CONH2.

[0444] 67. The method of paragraph 64, wherein the conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine-substituted analog thereof, wherein any free -COOH present in the glutamic acid residue is replaced with -CONH2.

[0445] 68. The method of any one of paragraphs 52 to 59 and 64 to 67, wherein the linker has the following structure: [ka]

[0446] 69. The above conjugates have their 3'-terminus covalently linked to the C-terminus of the peptide Ac-RBRBRFQILYBRBR via a β-alanine residue: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAG 2. The method of paragraph 1, comprising or consisting of AAGAUGGCAUUUCUAG-3' (SEQ ID NO:111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof.

[0447] 70. The method of paragraph 69, wherein the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' end covalently linked to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR via a β-alanine residue.

[0448] 71. The method of paragraph 69, wherein the conjugate comprises or consists of 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), having its 3′ end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR, or a thymine-substituted analog thereof.

[0449] 72. The method of paragraph 69, wherein the conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), having its 3' end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR, or a thymine-substituted analog thereof.

[0450] 73. The above conjugates have their 3'-terminus covalently linked to the C-terminus of the peptide Ac-RBRBRFQILYRBHBH via a β-alanine residue: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGG 2. The method of paragraph 1, comprising or consisting of AAGAUGGCAUUUCUAG-3' (SEQ ID NO:111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof.

[0451] 74. The method of paragraph 73, wherein the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' end covalently linked to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH via a β-alanine residue.

[0452] 75. The method of paragraph 73, wherein the conjugate comprises or consists of 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), having its 3′ end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine-substituted analog thereof.

[0453] 76. The method of paragraph 73, wherein the conjugate comprises or consists of 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), having its 3′ end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine-substituted analog thereof.

[0454] 77. The method of any one of paragraphs 1 to 76, wherein the oligonucleotide is a morpholino.

[0455] 78. The method of paragraph 77, wherein all morpholino internucleoside linkages are -P(O)(NMe2)O-.

[0456] 79. The method of paragraph 78, wherein the nucleotide comprises at its 5' end the following group: [ka]

[0457] 80. The method of any one of paragraphs 1 to 78, wherein the nucleotide comprises the following group at its 5' end: [ka]

[0458] 81. The method of any one of the above paragraphs, wherein the complex is administered intraperitoneally.

[0459] 82. The method of paragraph 81, wherein the complex is administered by injection.

[0460] 83. The method of paragraph 82, wherein the complex is administered intravenously.

[0461] 84. The method of any one of the above paragraphs, wherein the subject is a candidate for exon 51 skipping.

[0462] 85. The method of any one of the above paragraphs, wherein the conjugate is administered to the subject at a frequency ranging from every week to every three months.

[0463] 86. The method of any one of the above paragraphs, wherein the conjugate is administered to the subject on a weekly to monthly basis.

[0464] 87. The method of paragraph 86, wherein the frequency is weekly, biweekly, or monthly.

[0465] 88. The method of any one of paragraphs 1 to 84, wherein the frequency is every three months.

[0466] 89. 40 mg / kg to 60 mg / kg, 30 mg / kg to 50 mg / kg, 30 mg / kg to 40 mg / kg, 40 mg / kg to 50 mg / kg, 50 mg / kg to 60 mg / kg, 35 mg / kg to 45 mg / kg, 45 mg / kg to 55 mg / kg, 35 mg / kg to 55 mg / kg, 30 mg / kg to 45 mg / kg, 35 mg / kg to 50 mg / kg, 40 mg / kg to 55 mg / kg, 45 mg / kg to 60 mg / kg, 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 5 mg / kg to 25 mg / kg, 10 mg / kg to 30 mg / kg, 1 mg / kg to 15 mg / kg, 5 mg / kg to 20 mg / kg, 10 mg / kg to 25 mg / kg, 15 mg / kg to 30 mg / kg, 1 The method of any one of the above paragraphs, wherein from 1 mg / kg to 10 mg / kg, 5 mg / kg to 15 mg / kg, 10 mg / kg to 20 mg / kg, 15 mg / kg to 25 mg / kg, 20 mg / kg to 30 mg / kg, 1 mg / kg to 25 mg / kg, 4 mg / kg to 20 mg / kg, 6 mg / kg to 15 mg / kg, or 8 mg / kg to 10 mg / kg of the conjugate is administered.

[0467] 90. The method of any one of the above paragraphs, wherein 1 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, or 60 mg / kg of the conjugate is administered.

[0468] 91. The method of paragraph 90, wherein 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg of the conjugate is administered.

[0469] 92. The method of any one of the above paragraphs, wherein the method is for use in treating the cardiac effects of DMD, including, for example, cardiomyopathy (e.g., dilated, hypertrophic, or restrictive cardiomyopathy), heart failure, and / or cardiac arrhythmias.

[0470] Other embodiments are within the scope of the claims.

Claims

1. 1. A pharmaceutical composition comprising a conjugate of an oligonucleotide and a peptide linked to said oligonucleotide via a covalent bond or a linker, for use in treating a subject having Duchenne muscular dystrophy, said treatment comprising administering from 1 mg / kg to 60 mg / kg of said conjugate; the peptide comprises at least one cationic domain comprising at least four amino acid residues and at least one hydrophobic domain comprising at least three amino acid residues, wherein the peptide comprises a total of 7 to 40 amino acid residues and the at least one cationic domain comprises β-alanine residues in combination with arginine and / or histidine residues; and A pharmaceutical composition, wherein the oligonucleotide comprises a total of 12 to 40 contiguous nucleobases, at least 12 contiguous nucleobases being complementary to a target sequence of a human dystrophin gene.

2. The oligonucleotide comprises: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), or 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO: 113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO: 114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), 2. The pharmaceutical composition of claim 1, comprising a sequence selected from the group consisting of 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), and thymine substituted analogs thereof.

3. The pharmaceutical composition of claim 1 , wherein each cationic domain has a length between 4 and 12 amino acid residues.

4. The pharmaceutical composition of claim 3 , wherein each cationic domain has a length of between 4 and 7 amino acid residues.

5. 5. The pharmaceutical composition of claim 1, wherein each cationic domain comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cationic amino acids.

6. 5. The pharmaceutical composition of claim 1, wherein each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% arginine and / or histidine residues.

7. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein each cationic domain comprises one of the following sequences: RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB[Hyp]B (SEQ ID NO:17), R[Hyp]H[Hyp]HB (SEQ ID NO:18), R[Hyp]RR[Hyp]R (SEQ ID NO:19), or any combination thereof.

8. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein each cationic domain comprises or consists of one of the following sequences: RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB[Hyp]B (SEQ ID NO:17), R[Hyp]H[Hyp]HB (SEQ ID NO:18), R[Hyp]RR[Hyp]R (SEQ ID NO:19), or any combination thereof.

9. The pharmaceutical composition of claim 1 , wherein the peptide comprises two cationic domains.

10. 5. The pharmaceutical composition according to claim 1, wherein each hydrophobic domain has a length of 3 to 6 amino acids, preferably each hydrophobic domain has a length of 5 amino acids.

11. 5. The pharmaceutical composition of claim 1, wherein each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids.

12. 5. The pharmaceutical composition of claim 1 , wherein each hydrophobic domain comprises a phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residue, preferably each hydrophobic domain comprises or consists of a phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residue.

13. The pharmaceutical composition of claim 1 , wherein the peptide comprises one hydrophobic domain.

14. The pharmaceutical composition of any one of claims 1 to 4, wherein each hydrophobic domain comprises one of the following sequences: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26), or any combination thereof.

15. The pharmaceutical composition of any one of claims 1 to 4, wherein each hydrophobic domain comprises or consists of one of the following sequences: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26), or any combination thereof.

16. The pharmaceutical composition according to any one of claims 1 to 4, wherein the peptide comprises or consists of two cationic domains and one hydrophobic domain.

17. 5. The pharmaceutical composition of claim 1, wherein the peptide comprises or consists of a hydrophobic core domain flanked by two cationic arm domains.

18. The peptides are RBRRBRR (SEQ ID NO:1), RBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBHR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBH (SEQ ID NO:14), BHBH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB[Hyp]B (SEQ ID NO:17), 5. The pharmaceutical composition of claim 1, comprising or consisting of one hydrophobic domain comprising a sequence selected from YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), and WWPW (SEQ ID NO:26), flanked by two cationic arm domains each comprising a sequence selected from YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), and WWPW (SEQ ID NO:26).

19. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the peptide comprises or consists of one of the following sequences: RBRRBRRFQILYRBRBR (SEQ ID NO: 27), RBRRBRRYQFLIRBRBR (SEQ ID NO: 31), RBRRBRRILFQYRBRBR (SEQ ID NO: 32), RBRRBRFQILYBRBR (SEQ ID NO: 35), RBRRBRRFQILYRBHBH (SEQ ID NO: 37), RBRRBRRFQILYHBHBR (SEQ ID NO: 38), and RBRRBRFQILYRBHBH (SEQ ID NO: 44).

20. 5. The pharmaceutical composition of claim 1, wherein the peptide has the following amino acid sequence: RBRRBRFQILYBRBR (SEQ ID NO: 35).

21. 5. The pharmaceutical composition of claim 1, wherein the peptide has the following amino acid sequence: RBRRBRRFQILYRBHBH (SEQ ID NO: 37).

22. 5. The pharmaceutical composition of claim 1, wherein the peptide has the following amino acid sequence: RBRRBRFQILYRBHBH (SEQ ID NO: 44).

23. The pharmaceutical composition of claim 1 , wherein the peptide is linked to the remainder of the complex via its N-terminus.

24. The C-terminus of the peptide is -NH 2 24. The pharmaceutical composition of claim 23, wherein

25. The pharmaceutical composition of claim 1 , wherein the peptide is linked to the remainder of the complex via its C-terminus.

26. 26. The pharmaceutical composition of claim 25, wherein the peptide is acylated at its N-terminus.

27. 5. The pharmaceutical composition of claim 1, wherein the conjugate comprises or is of the following structure: 【Chemistry 1】

28. 5. The pharmaceutical composition of claim 1, wherein the conjugate comprises or is of the following structure: 【Chemistry 2】

29. 5. The pharmaceutical composition of claim 1, wherein the conjugate comprises or is of the following structure: 【Chemistry 3】

30. 5. The pharmaceutical composition according to claim 1, wherein each linker is independently represented by formula (I): T 1 -(CR 1 R 2 ) n -T 2 (I) (In the formula, T 1 is a divalent group for attaching to the peptide and is selected from the group consisting of -NH- and carbonyl; T 2 is a divalent group for attachment to an oligonucleotide and is selected from the group consisting of -NH- and carbonyl; n is 1, 2 or 3; Each R 1 is independently -Y 1 -X 1 -Z 1 and Where: Y 1 does not exist or is -(CR A1 R A2 ) m -, m is 1, 2, 3 or 4, R A1 and R A2 are each independently hydrogen, OH, or (1-2C)alkyl; X 1 is absent or -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR A3 )-, -N(R A3 )-, -N(R A3 )-C(O)-, -N(R A3 )-C(O)O-, -C(O)-N(R A3 )-, -N(R A3 )C(O)N(R A3 )-, -N(R A3 )C(NR A3 )N(R A3 )-, -SO-, -S-, -SO 2 -, -S(O) 2 N(R A3 )- or -N(R A3 )SO 2 - and each R A3 is independently selected from hydrogen and methyl; and Z 1 is further an oligonucleotide or is hydrogen, (1-6C) alkyl, (2-6C) alkenyl, (2-6C) alkynyl, aryl, (3-6C) cycloalkyl, (3-6C) cycloalkenyl, or heteroaryl; wherein each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl is selected from the group consisting of (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR A4 R A5 and (1-4C)alkoxy; R A4 and R A5 are each independently selected from the group consisting of hydrogen and (1-4C)alkyl, and Each R 2 is independently -Y 2 -X 2 -Z 2 where: Y 2 does not exist or is not a B1 R B2 ] m -, m is an integer selected from 1, 2, 3, or 4, R B1 and R B2 are each independently selected from hydrogen, OH or (1-2C)alkyl; X 2 is absent or -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR B3 )-, -N(R B3 )-, -N(R B3 )-C(O)-, -N(R B3 )-C(O)O-, -C(O)-N(R B3 )-, -N(R B3 )C(O)N(R B3 )-, -N(R B3 )C(NR B3 )N(R B3 )-, -SO-, -S--SO 2 -, -S(O) 2 N(R B3 )- or -N(R B3 )SO 2 - and R B3 is independently selected from hydrogen or methyl, and Z 2 is selected from hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl, each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl being selected from (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR B4 R B5 and (1-4C)alkoxy; R B4 and R B5 are each independently hydrogen or (1-2C)alkyl, provided that n=1 and T 1 and T 2 are different from each other, R 1 and R 2 are not H, n=1, and T 1 and T 2 are different from each other, and R 1 and R 2 When one of the groups is H, R 1 and R 2 The other is not methyl, or n=2 and R 1 and R 2 Each occurrence of is H when T 1 and T 2 are both -C(O)- or both -NH-.

31. T 2 The pharmaceutical composition of claim 30, wherein is -C(O)-.

32. Each R 1 is independently -Y 1 -X 1 -Z 1 and Y 1 does not exist or is -(CR A1 R A2 ) m -, m is 1, 2, 3 or 4, R A1 and R A2 are each hydrogen or (1-2C)alkyl, X 1 is absent or -O-, -C(O)-, -C(O)O-, -N(R A3 )-, -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, -N(R A3 )C(O)N(R A3 )-, -N(R A3 )C(NR A3 )N(R A3 )- or -S-, and each R A3 are independently hydrogen or methyl; and Z 1 is further an oligonucleotide or is hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl, each of which is (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, and heteroaryl is (1-4C)alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR A4 R A5 and (1-4C)alkoxy; R M and R A5 The pharmaceutical composition of claim 30, wherein each is independently hydrogen or (1-2C)alkyl.

33. Each R 1 is independently -Y 1 -X 1 -Z 1 and Y 1 does not exist or is -(CR A1 R A2 ) m -, m is 1, 2, 3, or 4, and R A1 and R″ are each independently hydrogen or (1-2C)alkyl; X 1 does not exist or is -O-, -C(O)-, -C(O)O-, -N(R A3 )-, -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, -N(R A3 )C(O)N(R A3 )-, -N(R A3 )C(NR A3 )N(R A3 )- or -S-, and each R A3 is independently hydrogen or methyl; and Z 1 is further an oligonucleotide or is hydrogen, (1-6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, each of which (1-6C)alkyl, aryl, (3-6C)cycloalkyl, and heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituents selected from the group consisting of (1-4C)alkyl, halo, and hydroxy.

34. Each R 1 is independently -Y 1 -X 1 -Z 1 and Y 1 does not exist or the formula -(CR A1 R A2 ) m -, m is 1, 2, 3 or 4, R A1 and R A2 are each independently hydrogen or (1-2C)alkyl; X 1 does not exist or is -C(O)-, -C(O)O-, -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, and each R A3 is hydrogen or methyl, and Z 1 is further an oligonucleotide or is hydrogen, (1-6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, each of which (1-6C)alkyl, aryl, (3-6C)cycloalkyl, and heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituents selected from the group consisting of (1-4C)alkyl, halo, and hydroxy.

35. Each R １ is independently -Y 1 -X 1 -Z 1 and Y 1 does not exist or is -(CH 2 )- or -(CH 2 CH 2 )-and X 1 does not exist or is -N(R A3 )-C(O)-, -C(O)-N(R A3 )-, and each R A3 are independently hydrogen or methyl; and Z 1 The pharmaceutical composition of claim 30, wherein is hydrogen or (1-2C)alkyl.

36. Each R 2 is independently -Y 2 -Z 2 and Y 2 does not exist or is -(CR B1 R B2 ) m -, m is 1, 2, 3 or 4, R B1 and R B2 are each independently hydrogen or (1-2C)alkyl; and Z 2 The pharmaceutical composition of claim 30, wherein is hydrogen or (1-6C)alkyl.

37. Each R 2 The pharmaceutical composition of claim 30, wherein is hydrogen.

38. 31. The pharmaceutical composition of claim 30, wherein n is 2 or 3.

39. 31. The pharmaceutical composition of claim 30, wherein n is 1.

40. The pharmaceutical composition according to any one of claims 1 to 4, wherein the linker is an amino acid residue selected from the group consisting of glutamic acid, succinic acid, and γ-aminobutyric acid residues.

41. The pharmaceutical composition of claim 1 , wherein the linker has the following structure: 【Chemistry 4】

42. The pharmaceutical composition of claim 1 , wherein the linker has the following structure: 【Chemistry 5】

43. The pharmaceutical composition of claim 1 , wherein the linker has the following structure: 【Chemistry 6】

44. The pharmaceutical composition of claim 1 , wherein the linker has the following structure: 【Chemistry 7】

45. The pharmaceutical composition of claim 1 , wherein the linker has the following structure: 【Chemistry 8】

46. 5. The pharmaceutical composition of claim 1, wherein the conjugate comprises or is of the following structure: 【Chemistry 9】

47. 5. The pharmaceutical composition of claim 1, wherein the conjugate comprises or is of the following structure: 【Chemistry 10】

48. 5. The pharmaceutical composition of claim 1, wherein the conjugate comprises or is of the following structure: 【Chemistry 11】

49. 5. The pharmaceutical composition of claim 1, wherein the conjugate comprises or is of the following structure: 【Chemistry 12】

50. 5. The pharmaceutical composition of claim 1, wherein the conjugate comprises or is of the following structure: 【Chemistry 13】

51. The pharmaceutical composition of claim 1 , wherein the oligonucleotide is linked to the linker or the peptide at its 3' end.

52. The conjugates include the following: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCAUUU CUAG-3' (SEQ ID NO:111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof, wherein if a free -COOH is present in said glutamic acid residue, it is replaced with -CONH 2 The pharmaceutical composition of claim 1 , wherein the compound is substituted with

53. The conjugate comprises or consists of a peptide Ac-RBRRBRFQILYBRBR having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide, 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), where if there is a free -COOH in the glutamic acid residue, then it is replaced by -CONH 2 53. The pharmaceutical composition of claim 52, wherein said compound is substituted with:

54. The conjugate comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109) having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRBRFQILYBRBR, or a thymine substituted analog thereof, wherein if there is a free -COOH in the glutamic acid residue, it is replaced by -CONH 2 53. The pharmaceutical composition of claim 52, wherein said compound is substituted with:

55. The conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115) having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR, or a thymine-substituted analog thereof, wherein if there is a free -COOH in the glutamic acid residue, it is replaced by -CONH 2 53. The pharmaceutical composition of claim 52, wherein said compound is substituted with:

56. The complex comprises the peptide RBRRBRFQILYBRBR-NH 2 and 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 111), 1), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116) or a thymine substituted analog thereof, wherein if a free -COOH is present in said glutamic acid residue, it is replaced with -CONH 2 The pharmaceutical composition of claim 1 , wherein the compound is substituted with

57. The complex comprises the peptide RBRRBRFQILYBRBR-NH 2 and having a 3' end covalently linked to the N-terminus of said amino acid through a glutamic acid residue, 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), wherein if a free -COOH is present in said glutamic acid residue, it is replaced by -CONH 2 57. The pharmaceutical composition of claim 56, wherein said compound is substituted with:

58. The complex comprises the peptide RBRRBRFQILYBRBR-NH 2 or a thymine-substituted analog thereof, having a 3' terminus covalently linked to the N-terminus of said amino acid through a glutamic acid residue, 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), wherein if a free -COOH is present in said glutamic acid residue, it is replaced with -CONH 2 57. The pharmaceutical composition of claim 56, wherein said compound is substituted with:

59. The complex comprises the peptide RBRRBRFQILYBRBR-NH 2 or a thymine-substituted analog thereof, having a 3' terminus covalently linked to the N-terminus of said amino acid through a glutamic acid residue, 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115), wherein if a free -COOH is present in said glutamic acid residue, it is replaced by -CONH 2 57. The pharmaceutical composition of claim 56, wherein said compound is substituted with:

60. The conjugates include 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 111), 5'-ACAUCAAGG 2. The pharmaceutical composition of claim 1, comprising or consisting of AAGAUGGCAUUUCUAG-3' (SEQ ID NO:111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof.

61. 61. The pharmaceutical composition of claim 60, wherein the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' end covalently linked via a gamma-aminobutyric acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH.

62. 61. The pharmaceutical composition of claim 60, wherein the conjugate comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109) having its 3' end covalently linked via a γ-aminobutyric acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine substituted analog thereof.

63. 61. The pharmaceutical composition of claim 60, wherein the conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115) having its 3' end covalently linked via a γ-aminobutyric acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine substituted analog thereof.

64. The conjugates include the following peptides: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCAUUU CUAG-3' (SEQ ID NO:111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof, wherein if a free -COOH is present in said glutamic acid residue, it is replaced with -CONH 2 The pharmaceutical composition of claim 1 , wherein the compound is substituted with

65. The conjugate comprises or consists of a peptide Ac-RBRRBRFQILYRBHBH having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide, 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), where if there is a free -COOH in the glutamic acid residue, it is replaced by -CONH 2 65. The pharmaceutical composition of claim 64, wherein said compound is substituted with:

66. The conjugate comprises or consists of the peptide Ac-RBRBRFQILYRBHBH having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), or a thymine substituted analog thereof, wherein if there is a free -COOH in the glutamic acid residue, it is replaced by -CONH 2 65. The pharmaceutical composition of claim 64, wherein said compound is substituted with:

67. The conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115) having its 3' end covalently linked via a glutamic acid residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine-substituted analog thereof, wherein if there is a free -COOH in the glutamic acid residue, it is replaced by -CONH 2 65. The pharmaceutical composition of claim 64, wherein said compound is substituted with:

68. 70. The pharmaceutical composition of any one of claims 1-4 or 52-67, wherein the linker is of the following structure: 【Chemistry 14】

69. The conjugates include 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGA 2. The pharmaceutical composition of claim 1, comprising or consisting of 5'-AGAUGGCAUUUCUAG-3' (SEQ ID NO:111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof.

70. 70. The pharmaceutical composition of claim 69, wherein the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR.

71. 70. The pharmaceutical composition of claim 69, wherein the conjugate comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109) having its 3' end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR, or a thymine substituted analog thereof.

72. 70. The pharmaceutical composition of claim 69, wherein the conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115) having its 3' end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRRBRFQILYBRBR, or a thymine-substituted analog thereof.

73. The conjugates include the following peptides: 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106), 5'-ACCAGAGUAACAGUCUGAGUAGGAGC-3' (SEQ ID NO: 107), 5'-CUCAUACCUUCUGCUUGAUGAUC-3' (SEQ ID NO: 108), 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 110), 5'-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3' (SEQ ID NO: 111), 5'-ACAUCAAGG 2. The pharmaceutical composition of claim 1, comprising or consisting of 5'-AAGAUGGCAUUUCUAG-3' (SEQ ID NO:111), 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO:112), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAG-3' (SEQ ID NO:113), 5'-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3' (SEQ ID NO:114), 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO:115), or 5'-AUCAUCUCGUUGAUAUCCUCAA-3' (SEQ ID NO:116), or a thymine substituted analog thereof.

74. 74. The pharmaceutical composition of claim 73, wherein the conjugate comprises or consists of 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 106) having its 3' end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH.

75. 74. The pharmaceutical composition of claim 73, wherein the conjugate comprises or consists of 5'-UUCUGUCCAAGCCCGGUUGAAAUC-3' (SEQ ID NO: 109) having its 3' end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine substituted analog thereof.

76. 74. The pharmaceutical composition of claim 73, wherein the conjugate comprises or consists of 5'-CACCCACCAUCACCCUCUGUG-3' (SEQ ID NO: 115) having its 3' end covalently linked via a β-alanine residue to the C-terminus of the peptide Ac-RBRRBRFQILYRBHBH, or a thymine substituted analog thereof.

77. 80. The pharmaceutical composition of any one of claims 1-4, 52-67, and 69-76, wherein the oligonucleotide is a morpholino.

78. All morpholino internucleoside linkages are -P(O)(NMe 2 78. The pharmaceutical composition of claim 77, wherein:

79. 79. The pharmaceutical composition of claim 78, wherein the nucleotide comprises the following group at its 5' end: 【Chemistry 15】

80. 77. The pharmaceutical composition of any one of claims 1 to 4, 52 to 67, and 69 to 76, wherein the nucleotide comprises the following group at its 5' end: 【Chemistry 16】

81. 80. The pharmaceutical composition of any one of claims 1-4, 52-67, and 69-76, wherein the complex is administered intraperitoneally.

82. 82. The pharmaceutical composition of claim 81, wherein the complex is administered by injection.

83. 83. The pharmaceutical composition of claim 82, wherein the conjugate is administered by intravenous injection.

84. The pharmaceutical composition of any one of claims 1 to 4, 52 to 67, and 69 to 76, wherein the subject is a candidate for exon 51 skipping.

85. 80. The pharmaceutical composition of any one of claims 1-4, 52-67, and 69-76, wherein the conjugate is administered to the subject at a frequency ranging from weekly to every three months.

86. 80. The pharmaceutical composition of any one of claims 1-4, 52-67, and 69-76, wherein the conjugate is administered to the subject on a weekly to monthly basis.

87. 87. The pharmaceutical composition of claim 86, wherein the frequency is weekly, biweekly, or monthly.

88. 80. The pharmaceutical composition of any one of claims 1-4, 52-67, and 69-76, wherein the frequency is every three months.

89. 40 mg / kg to 60 mg / kg, 30 mg / kg to 50 mg / kg, 30 mg / kg to 40 mg / kg, 40 mg / kg to 50 mg / kg, 50 mg / kg to 60 mg / kg, 35 mg / kg to 45 mg / kg, 45 mg / kg to 55 mg / kg, 35 mg / kg to 55 mg / kg, 30 mg / kg to 45 mg / kg, 35 mg / kg to 50 mg / kg, 40 mg / kg to 55 mg / kg, 45 mg / kg to 60 mg / kg, 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 5 mg / kg to 25 mg / kg, 10 mg / kg to 30 mg / kg, 1 mg / kg to 15 mg / kg, 5 mg / kg to 20 mg / kg, 10 mg / kg to 25 mg / kg, 15 mg / kg to 30 mg / kg, 1 mg / kg to 10 77. The pharmaceutical composition of any one of claims 1 to 4, 52 to 67, and 69 to 76, wherein the complex is administered at a dose of 5 mg / kg, 5 mg / kg to 15 mg / kg, 10 mg / kg to 20 mg / kg, 15 mg / kg to 25 mg / kg, 20 mg / kg to 30 mg / kg, 1 mg / kg to 25 mg / kg, 4 mg / kg to 20 mg / kg, 6 mg / kg to 15 mg / kg, or 8 mg / kg to 10 mg / kg.

90. 77. The pharmaceutical composition of any one of claims 1-4, 52-67, and 69-76, wherein 1 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, or 60 mg / kg of the conjugate is administered.

91. 91. The pharmaceutical composition of claim 90, wherein 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg of the conjugate is administered.

92. The pharmaceutical composition of any one of claims 1 to 4, 52 to 67, and 69 to 76, wherein the pharmaceutical composition is for use in treating the cardiac effects of DMD, including, for example, cardiomyopathy (e.g., dilated, hypertrophic, or restrictive cardiomyopathy), heart failure, and / or cardiac arrhythmias.

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