Methods and compositions for treating duchenne muscular dystrophy

Abstract

Disclosed are conjugates of an oligonucleotide and a peptide covalently bonded or linked to the oligonucleotide via a linker that target a human dystrophin gene, compositions including the same, and methods of use thereof.

Description

[0001] PATENT

[0002] Attorney Docket No.: 51558-023WO2

[0003] METHODS AND COMPOSITIONS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY

[0004] SEQUENCE LISITNG

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 19, 2025, is named “51558-023WO2_Sequence_Listing_11_19_25” and is 161 ,023 bytes in size.

[0006] TECHNICAL FIELD

[0007] This disclosure relates to peptide conjugates of antisense oligonucleotides, compositions containing them, and methods of their use.

[0008] BACKGROUND

[0009] Duchenne muscular dystrophy (DMD) affects approximately one in 3,500 live newborn boys. DMD is a rare disease and is the most common inherited myopathy affecting children. This severe, X- linked recessive disease results from mutations in the DMD gene that encodes dystrophin protein. Dystrophin protein is an essential cytoskeletal protein that plays a critical role in stabilizing the muscle membrane during muscle contraction and its absence disrupts muscle structure and function. The disorder is characterized by progressive muscle degeneration and wasting resulting in loss of ambulation, along with the emergence of respiratory failure and cardiac complications, ultimately leading to premature death by 20-30 years of age. Most mutations underlying DMD are genomic out- of-frame deletions that induce a premature truncation in the open reading frame resulting in the absence of the dystrophin protein.

[0010] Exon skipping therapy utilizes splice switching antisense oligonucleotides (SSOs) to target specific regions of the DMD transcript, inducing the exclusion of individual exons, leading to the restoration of aberrant reading frames and resulting in the production of an internally deleted, yet partially functional, dystrophin protein. This truncated dystrophin protein isoform, generated by exon skipping in DMD patients with an amenable mutation, is more like the shorter dystrophin protein isoform present in Becker muscular dystrophy (BMD) patients. Therefore, it is expected that exon skipping will result in DMD patients having less severe symptoms and become more similar to BMD, which presents with a milder disease profile / phenotype than DMD. Approximately 8% of all DMD patients may be treated by skipping exon 53 of the dystrophin (DMD) pre-mRNA, as this is the second most common DMD mutation amenable to an exon skipping therapeutic approach. Despite the undoubted potential of antisense oligonucleotide exon skipping therapy for DMD, the successful application of this approach is currently limited by the relatively poor delivery and uptake of oligonucleotides by skeletal muscle, as well as the inadequate delivery of single stranded oligonucleotides to other critically affected tissues such as the diaphragm and heart. In December 2019, the United States Food and Drug Administration (FDA) approved golodirsen, a modulator for DMD exon 53 skipping, for treatment of DMD. Despite this approval, levels of dystrophin restoration in patients were minimal, with only about 1% of normal dystrophin values being reported by the study sponsor. Similarly, viltolarsen, another modulator for DMD exon 53 skipping, was granted accelerated PATENT

[0011] Attorney Docket No.: 51558-023WO2 approval by the FDA in August 2020 but in May 2024 the sponsor reported that it failed to reach the primary endpoint in the confirmatory trial.

[0012] Therefore, there is an unmet medical need for new approaches with improved delivery of oligonucleotides for treating diseases such as DMD.

[0013] SUMMARY OF THE INVENTION

[0014] In general, the invention provides a conjugate of a peptide and an oligonucleotide covalently bonded or covalently linked via a linker.

[0015] In a first aspect, the invention features a conjugate including a peptide and an oligonucleotide covalently bonded or linked via a non-cationic linker to the peptide, wherein the peptide includes a total of one hydrophobic domain and a total of two cationic domains, further wherein the hydrophobic domain includes at least 5 amino acids, and each cationic domain includes at least one cationic amino acid; and wherein the oligonucleotide includes a sequence selected from the group consisting of:

[0016] (a) 5’-CTGAAGGTGTTCTTGTACTTCATCC-3’ (SEQ ID NO: 1);

[0017] (b) 5’-CAACTGTTGCCTCCGGTTCTGAAGGTGTTC-3’ (SEQ ID NO: 2);

[0018] (c) 5’-TTGCCTCCGGTTCTGAAGGTGTTCTTGTAC-3’ (SEQ ID NO: 3);

[0019] (d) 5’-CTGTTGCCTCCGGTTCTGAAGGTGTTCTTG-3’ (SEQ ID NO: 4);

[0020] (e) 5’-CATTCAACTGTTGCCTCCGGTTCTGAAGGTG-3’ (SEQ ID NO: 5) or a pharmaceutically acceptable salt thereof.

[0021] In some embodiments, the sequence is selected from the group consisting of (a)-(d).

[0022] In some embodiments, the target sequence includes a splice site for DMD exon 53.

[0023] In some embodiments, the peptide includes 40 or fewer amino acids (e.g., 40 or fewer, 39 or fewer, 38 or fewer, 37 or fewer, 36 or fewer, 35 or fewer, 34 or fewer, 33 or fewer, 32 or fewer, 31 or fewer, 30 or fewer, 29 or fewer, 28 or fewer, 27 or fewer, 26 or fewer, 25 or fewer, 24 or fewer, 23 or fewer, 22 or fewer, 21 or fewer, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, or 6 or fewer). In some embodiments, the peptide includes 5 to 30 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids).

[0024] In some embodiments, the peptide includes an amino acid sequence selected from the group consisting of:

[0025] (a) RBRRBRFQILYBRBR (SEQ ID NO: 9);

[0026] (b) RBRRBRRFQILYRBRBR (SEQ ID NO: 17);

[0027] (c) RBRRBRRYQFLIRBRBR (SEQ ID NO: 18);

[0028] (d) RBRRBRRILFQYRBRBR (SEQ ID NO: 19);

[0029] (e) RBRRBRRFQILYRBHBH (SEQ ID NO: 20);

[0030] (f) RBRRBRRFQILYHBHBR (SEQ ID NO: 21);

[0031] (g) RBRRBRFQILYRBHBH (SEQ ID NO: 22);

[0032] (h) RRRRR (SEQ ID NO: 23);

[0033] (i) RRRRRR (SEQ ID NO: 24);

[0034] 0) RRRRRRR (SEQ ID NO: 25); or PATENT

[0035] Attorney Docket No.: 51558-023WO2

[0036] (k) RRRRRRRR (SEQ ID NO: 26), wherein B is beta-alanine.

[0037] In some embodiments, the peptide has the sequence of RBRRBRFQILYBRBR (SEQ ID NO: 9). In some embodiments, the peptide has the sequence of RBRRBRRFQILYRBRBR (SEQ ID NO:

[0038] 17). In some embodiments, the peptide has the sequence of RBRRBRRYQFLIRBRBR (SEQ ID NO:

[0039] 18). In some embodiments, the peptide has the sequence of RBRRBRRILFQYRBRBR (SEQ ID NO:

[0040] 19). In some embodiments, the peptide has the sequence of RBRRBRRFQILYRBHBH (SEQ ID NO:

[0041] 20). In some embodiments, the peptide has the sequence of RBRRBRRFQILYHBHBR (SEQ ID NO:

[0042] 21). In some embodiments, the peptide has the sequence of RBRRBRFQILYRBHBH (SEQ ID NO:

[0043] 22). In some embodiments, the peptide has the sequence of RRRRR (SEQ ID NO: 23). In some embodiments, the peptide has the sequence of RRRRRR (SEQ ID NO: 24). In some embodiments, the peptide has the sequence of RRRRRRR (SEQ ID NO: 25). In some embodiments, the peptide has the sequence of RRRRRRRR (SEQ ID NO: 26).

[0044] In some embodiments, the peptide includes an artificial amino acid.

[0045] In some embodiments, the peptide includes an amino acid sequence selected from the group consisting of: RXRRBRRXRRBR (SEQ ID NO: 27); RXRRBRRXRRBRX (SEQ ID NO: 28); RXRRXRRXRRXRX (SEQ ID NO: 29); RXRRBRRFQILYRBRXR (SEQ ID NO: 30); RXRRBRRXRILFQYRXRBRXR (SEQ ID NO: 31); RXRRBRRXRILFQYRXRXRXR (SEQ ID NO: 32); RXRRXRILFQYRXRRXR (SEQ ID NO: 33); RBRRXRRBRILFQYRBRXRBR (SEQ ID NO: 34); RBRRXRRBRILFQYRXRBRXR (SEQ ID NO: 35); RBRRXRRBRILFQYRXRRXR (SEQ ID NO: 36); RBRRXRRBRILFQYRXRBRX (SEQ ID NO: 37); RXRRBRRXRILFQYRXRRXR (SEQ ID NO: 38); RXRRBRRXRILFQYRXRBRX (SEQ ID NO: 39); RXRRBRRXRYQFLIRXRBRXR (SEQ ID NO: 40); RXRRBRRXRIQFLIRXRBRXR (SEQ ID NO: 41); RXRRBRRXRQFLIRXRBRXR (SEQ ID NO: 42); RXRRBRRXRQFLRXRBRXR (SEQ ID NO: 43); RXRRBRRXYRFLIRXRBRXR (SEQ ID NO: 44); RXRRBRRXRFQILYRXRBRXR (SEQ ID NO: 45); RXRRBRRXYRFRLIXRBRXR (SEQ ID NO: 46); RXRRBRRXILFRYRXRBRXR (SEQ ID NO: 47); RXRRBRRXRIYQFLIRXRBRXR (SEQ ID NO: 48); YQFLIRBRRXRBRXBRXRBYQFLI (SEQ ID NO: 49); YQFLIRBRRBRBRBRRBYQFLI (SEQ ID NO: 50); or YQFLIRBRRBRBRBBRXRBYQFLI (SEQ ID NO: 51), wherein X is aminohexanoic acid and B is beta-alanine.

[0046] In some embodiments, the peptide has the sequence of RXRRBRRXRRBR (SEQ ID NO: 27). In some embodiments, the peptide has the sequence of RXRRBRRXRRBRX (SEQ ID NO: 28). In some embodiments, the peptide has the sequence of RXRRXRRXRRXRX (SEQ ID NO: 29). In some embodiments, the peptide has the sequence of RXRRBRRFQILYRBRXR (SEQ ID NO: 30). In some embodiments, the peptide has the sequence of RXRRBRRXRILFQYRXRBRXR (SEQ ID NO: 31). In some embodiments, the peptide has the sequence of RXRRBRRXRILFQYRXRXRXR (SEQ ID NO:

[0047] 32). In some embodiments, the peptide has the sequence of RXRRXRILFQYRXRRXR (SEQ ID NO:

[0048] 33). In some embodiments, the peptide has the sequence of RBRRXRRBRILFQYRBRXRBR (SEQ ID NO: 34). In some embodiments, the peptide has the sequence of RBRRXRRBRILFQYRXRBRXR (SEQ ID NO: 35). In some embodiments, the peptide has the sequence of RBRRXRRBRILFQYRXRRXR (SEQ ID NO: 36). In some embodiments, the peptide has the sequence of RBRRXRRBRILFQYRXRBRX (SEQ ID NO: 37). In some embodiments, the peptide has the PATENT

[0049] Attorney Docket No.: 51558-023WO2 sequence of RXRRBRRXRILFQYRXRRXR (SEQ ID NO: 38). In some embodiments, the peptide has the sequence of RXRRBRRXRILFQYRXRBRX (SEQ ID NO: 39). In some embodiments, the peptide has the sequence of RXRRBRRXRYQFLIRXRBRXR (SEQ ID NO: 40). In some embodiments, the peptide has the sequence of RXRRBRRXRIQFLIRXRBRXR (SEQ ID NO: 41). In some embodiments, the peptide has the sequence of RXRRBRRXRQFLIRXRBRXR (SEQ ID NO: 42). In some embodiments, the peptide has the sequence of RXRRBRRXRQFLRXRBRXR (SEQ ID NO: 43). In some embodiments, the peptide has the sequence of RXRRBRRXYRFLIRXRBRXR (SEQ ID NO: 44). In some embodiments, the peptide has the sequence of RXRRBRRXRFQILYRXRBRXR (SEQ ID NO: 45). In some embodiments, the peptide has the sequence of RXRRBRRXYRFRLIXRBRXR (SEQ ID NO: 46). In some embodiments, the peptide has the sequence of RXRRBRRXILFRYRXRBRXR (SEQ ID NO: 47). In some embodiments, the peptide has the sequence of RXRRBRRXRIYQFLIRXRBRXR (SEQ ID NO: 48). In some embodiments, the peptide has the sequence of YQFLIRBRRXRBRXBRXRBYQFLI (SEQ ID NO: 49). In some embodiments, the peptide has the sequence of YQFLIRBRRBRBRBRRBYQFLI (SEQ ID NO: 50). In some embodiments, the peptide has the sequence of YQFLIRBRRBRBRBBRXRBYQFLI (SEQ ID NO: 51).

[0050] In some embodiments, at least one cationic domain is arginine rich. In some embodiments, the cationic domain includes at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% arginine residues.

[0051] In some embodiments, at least one cationic domain is histidine rich. In some embodiments, the cationic domain includes at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% histidine residues.

[0052] In some embodiments, the hydrophobic domain includes the amino acid sequence FQILY (SEQ ID NO: 6), YQFLI (SEQ ID NO: 52), ILFQY (SEQ ID NO: 53), FQIY (SEQ ID NO: 54), WWW, WWPWW (SEQ ID NO: 55), WPWW (SEQ ID NO: 56), WWPW (SEQ ID NO: 57), ILFQ (SEQ ID NO: 58), ILIQ (SEQ ID NO: 59), IKILFQN (SEQ ID NO: 60), IHILFQN (SEQ ID NO: 61), IRILFQN (SEQ ID NO: 62), IILFQN (SEQ ID NO: 63), KILFQN (SEQ ID NO: 64), HILFQN (SEQ ID NO: 65), RILFQN (SEQ ID NO: 66), ILFQN (SEQ ID NO: 67), HLIQN (SEQ ID NO: 68), KILIQN (SEQ ID NO: 69), KILIQY (SEQ ID NO: 70), HILIQN (SEQ ID NO: 71), RILIQN (SEQ ID NO: 72), HILIQY (SEQ ID NO: 73), RILIQY (SEQ ID NO: 74), ILIQN (SEQ ID NO: 75), ILIQY (SEQ ID NO: 76).

[0053] In some embodiments, at least one of the cationic domains includes the amino acid sequence: RBRRBRR (SEQ ID NO: 77), RBRBR (SEQ ID NO: 78), RBRR (SEQ ID NO: 79), RBRRBR (SEQ ID NO: 7), RRBRBR (SEQ ID NO: 80), RBRRB (SEQ ID NO: 81), BRBR (SEQ ID NO: 8), RBHBH (SEQ ID NO: 82), HBHBR (SEQ ID NO: 83), RBRHBHR (SEQ ID NO: 84), RBRBBHR (SEQ ID NO: 85), RBRRBH (SEQ ID NO: 86), HBRRBR (SEQ ID NO: 87), HBHBH (SEQ ID NO: 88), BHBH (SEQ ID NO: 89), BRBSB (SEQ ID NO: 90), BRB[Hyp]B (SEQ ID NO: 91), R[Hyp]H[Hyp]HB (SEQ ID NO: 92), R[Hyp]RR[Hyp]R (SEQ ID NO: 93), RBR, RXR, XXR, XRR, RRX, BXR, RXB, XRB, RBB, BRB, BBR, RRB, BRR, and BRX, 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, wherein B is beta-alanine, X is aminohexanoic acid, and [Hyp] is hydroxyproline. In some embodiments, at least one of the cationic domains includes the amino acid sequence RBRRBR (SEQ ID NO: 7) or BRBR (SEQ ID NO: 8). PATENT

[0054] Attorney Docket No.: 51558-023WO2

[0055] In some embodiments, the linker includes at least one amino acid. In some embodiments, the amino acid is selected from glutamic acid, beta-alanine, glycine, delta-aminovaleric acid, and gamma- aminobutyric acid. In some embodiments, the glutamic acid linker is linked to the oligonucleotide via a gamma carboxylic acid group and has the following structure:

[0056] In some embodiments, the peptide is linked to the oligonucleotide, either directly or via a linker, through its C-terminus.

[0057] In some embodiments, the peptide is acylated at its N-terminus.

[0058] In some embodiments, the peptide is linked to the oligonucleotide, either directly or via a linker, through its N-terminus.

[0059] In some embodiments, the peptide is amidated at its C-terminus.

[0060] In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the oligonucleotide is a peptide nucleic acid (PNA).

[0061] In some embodiments, the oligonucleotide is PMO having a 5’ end group of the structure:

[0062] In a second aspect, the invention features a pharmaceutical composition including the conjugate of any one of the foregoing aspects and embodiments and a pharmaceutically acceptable excipient.

[0063] In a third aspect, the invention features method of treating a subject having Duchenne muscular dystrophy (DMD), the method including administering to the subject a therapeutically effective amount of the conjugate or the pharmaceutical composition of any one of the foregoing aspects and embodiments.

[0064] In some embodiments, the subject is determined to have a genotype that is amenable to exon 53 skipping in a human dystrophin (DMD) gene. In some embodiments, the genotype is selected from the following DMD mutations: a 3-52 deletion, a 4-52 deletion, a 5-52 deletion, a 6-52 deletion, a 9-52 deletion, a 10-52 deletion, an 1 1-52 deletion, a 13-52 deletion, a 14-52 deletion, a 15-52 deletion, a 16-52 deletion, a 17-52 deletion, a 19-52 deletion, a 21 -42 deletion, a 23-52 deletion, a 24-52 deletion, a 25-52 deletion, a 26-52 deletion, a 27-52 deletion, a 28-52 deletion, a 29-52 deletion, a 30-52 deletion, a 31 -52 deletion, a 32-52 deletion, a 33-52 deletion, a 34-52 deletion, a 35-52 deletion, a 36- 52 deletion, a 37-52 deletion, a 38-52 deletion, a 39-52 deletion, a 40-52 deletion, a 41-52 deletion, a 42-52 deletion, a 43-52 deletion, a 45-52 deletion, a 47-52 deletion, a 48-52 deletion, a 49-52 deletion, PATENT

[0065] Attorney Docket No.: 51558-023WO2 a 50-52 deletion, a 52 deletion, a 54-58 deletion, a 54-61 deletion, a 54-61 deletion, a 54-63 deletion, a 54-64 deletion, a 54-66 deletion, a 54-76 deletion, and a 54-77 deletion.

[0066] BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The accompanying drawings are included to illustrate embodiments of the disclosure and further an understanding of its implementations.

[0068] FIG. 1A is a graph showing levels of exon 53 skipping in patient-derived DMD del52 myotubes induced by peptide-conjugated phosphorodiamidate morpholino oligomer 1 (PPMO-1) (square) or R6Gly-control PMO (triangle) at the indicated concentrations.

[0069] FIG. 1B is a graph showing levels of exon 53 skipping in patient-derived DMD del52 myotubes induced by PPMO-3 (circle) or R6Gly-control PMO (triangle) at the indicated concentrations.

[0070] FIG. 2A is a graph showing levels of exon 53 skipping in patient-derived DMD del45-52 myotubes induced by PPMO-1 (square) or R6Gly-control PMO (triangle) at the indicated concentrations.

[0071] FIG. 2B is a graph showing levels of exon 53 skipping in patient-derived DMD del45-52 myotubes induced by PPMO-3 (circle) or R6Gly-control PMO (triangle) at the indicated concentrations.

[0072] FIG. 3 is a graph showing levels of exon 53 skipping in human-derived control (i.e., non-DMD) myotubes induced by PPMO-1 (circle) or R6Gly-control PMO (square) at the indicated concentrations.

[0073] FIG. 4A is a graph showing levels of exon 53 skipping in human-derived control (i.e., non- DMD) myotubes induced by the phosphorodiamidate morpholino oligomers (PMO) form of control PMO sequence (i.e., without a conjugated peptide) (square) and the corresponding PPMO R6Gly- control PMO (circle) at the indicated concentrations.

[0074] FIG. 4B is a graph showing levels of exon 53 skipping in human-derived control (i.e., non- DMD) myotubes induced by the PMO form of PPMO-1 (PMO-1) (i.e., without the conjugated peptide) (square) and PPMO-1 (circle) at the indicated concentrations.

[0075] FIG. 5 is a graph showing levels of exon 53 skipping in biceps tissue samples from nonhuman primates (NHP) on Day 8 (i.e., 7 days after initial dose) of the indicated doses of PPMO-3, PPMO-1 , R6Gly-control PMO.

[0076] FIG. 6 is a graph showing levels of exon 53 skipping in biceps tissue samples from NHP on day 64 (i.e., 7 days post final dose) following repeated administration (3 doses once every 4 weeks) of the indicated doses of PPMO-3, PPMO-1 , R6Gly-control PMO, or vehicle-control PBS. R6Gly-control PMO was administered only at 30 mg / kg doses. Asterisk (*) indicates a p-value <0.05, as determined by two-way ANOVA.

[0077] FIG. 7 is a graph showing levels of exon 53 skipping in diaphragm tissue samples from NHP on day 64 (i.e., 7 days post final dose) following repeated administration (3 doses once every 4 weeks) of the indicated doses of PPMO-3, PPMO-1 , R6Gly-control PMO, or vehicle-control PBS. R6Gly- control PMO was administered only at 30 mg / kg doses, ns indicates no significant difference. Asterisk (*) indicates a p-value <0.05.

[0078] FIG. 8 is a graph showing levels of exon 53 skipping in left ventricle tissue samples on day 64 (i.e., 7 days post final dose) following repeated administration (3 doses once every 4 weeks) of the PATENT

[0079] Attorney Docket No.: 51558-023WO2 indicated doses of PPMO-3, PPMO-1 , R6Gly-control PMO, or vehicle-control PBS. R6Gly-control PMO was administered only at 30 mg / kg doses, ns indicates no significant difference.

[0080] DEFINTIONS

[0081] Unless otherwise defined herein, scientific, and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and / or" unless stated otherwise. The use of the term "including," as well as other forms, such as "includes" and "included," is not limiting.

[0082] The term "about," as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0083] References to “B” throughout the disclosure, in the context of a peptide or protein sequence, denote the amino acid beta-alanine.

[0084] References to “Ac” throughout denote an acetyl group (CH3-C(O)-).

[0085] References to other capital letters throughout denote the relevant amino acid residues in accordance with the accepted alphabetic amino acid code.

[0086] The term “alkyl,” as used herein, refers to a straight or branched chain hydrocarbon group containing a total of one to twenty carbon atoms, unless otherwise specified (e.g., (1 -6C) alkyl, (1-4C) alkyl, (1-3C) alkyl, or (1-2C) alkyl). Non-limiting examples of alkyls are methyl, ethyl, 1 -methylethyl, propyl, 1 -methylbutyl, 1 -ethylbutyl, etc. Unless otherwise specified, alkyl may be optionally substituted by one, two, three, four, or five groups selected from the group consisting of carbocyclyl, aryl, heterocyclyl, heteroaryl, oxo, halogen, and hydroxyl.

[0087] The term "alkenyl," as used herein, refers to an aliphatic group containing one, two, or three carbon-carbon double bonds and containing a total of two to twenty carbon atoms, unless otherwise specified (e.g., (2-6C) alkenyl, (2-4C) alkenyl, or (2-3C) alkenyl). Non-limiting examples of alkenyl are vinyl, allyl, homoallyl, isoprenyl, etc. Unless otherwise specified, alkenyl may be optionally substituted by one, two, three, four, or five groups selected from the group consisting of carbocyclyl, aryl, heterocyclyl, heteroaryl, oxo, halogen, and hydroxyl.

[0088] The term “alkynyl,” as used herein, refers to an aliphatic group containing one, two, or three carbon-carbon triple bonds and containing a total of two to twenty carbon atoms, unless otherwise specified (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, etc. Unless otherwise specified, alkynyl may be optionally substituted by one, two, three, four, or five groups selected from the group consisting of carbocyclyl, aryl, heterocyclyl, heteroaryl, oxo, halogen, and hydroxyl.

[0089] 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. An unsubstituted aryl contains a total of 6 to 14 carbon atoms. The term aryl includes both monovalent species and divalent species. Examples of aryl groups PATENT

[0090] Attorney Docket No.: 51558-023WO2 include, but are not limited to, phenyl, naphthyl, indanyl, and the like. In particular embodiments, an optionally substituted aryl is optionally substituted phenyl.

[0091] By “bridged ring systems,” as used herein, are meant ring systems in which two rings share more than two atoms, see for example Advanced Organic Chemistry, by Jerry March, 4thEdition, Wiley Interscience, pages 131 -133, 1992. Examples of bridged heterocyclyl ring systems include, aza-bicydo[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, etc.

[0092] References made herein to “cationic” denote an amino acid or domain of amino acids having an overall positive charge at neutral pH, though positively charged and neutral forms may coexist at this pH. For example, at neutral pH, histidine is believed to have neutral and cationic forms in an equilibrium, as pKaof protonated histidine side chain is 6.3. Non-limiting examples of cationic amino acids include histidine, lysine, and arginine.

[0093] The term “artificial amino acid,” as used herein, refers to an abiogenic amino acid (e.g., non- proteinogenic). For example, artificial amino acids may include synthetic amino acids, modified amino acids (e.g., those modified with sugars), non-natural amino acids, man-made amino acids, spacers, and non-peptide bonded spacers. 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, beta-alanine (B) and hydroxyproline (Hyp) occur in nature and therefore are not artificial amino acids in the context of the present invention but are natural amino acids. Artificial amino acids may include, for example, 6- aminohexanoic acid (X), tetrahydroisoquinoline-3-carboxylic acid (TIC), 1- (amino)cyclohexanecarboxylic acid (Cy), 3-azetidine-carboxylic acid (Az), and 11-aminoundecanoic acid. For the avoidance of doubt, it is noted that the D enantiomer of a naturally occurring L-amino acid is not to be considered as an artificial amino acid.

[0094] By “arginine rich,” it is meant that at least 40% of the cationic domain is formed of arginine residues.

[0095] The term “complementary,” as used herein in reference to a nucleobase sequence, refers to the nucleobase sequence having a pattern of contiguous nucleobases that permits an oligonucleotide having the nucleobase sequence to hybridize to another oligonucleotide or nucleic acid to form a duplex structure under physiological conditions. Complementary sequences include Watson-Crick base pairs formed from natural and / or modified nucleobases. Complementary sequences can also include non-Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthineuracil, hypoxanthine-adenine, and hypoxanthine-cytosine) and Hoogsteen base pairs.

[0096] The term “cycloalkyl,” as used herein, refers to a saturated carbocyclic ring system containing one or two rings, and containing a total of 3 to 10 carbon atoms, unless otherwise specified. The two- ring cycloalkyls may be arranged as fused ring systems (two bridgehead carbon atoms are directly bonded to one another), bridged ring systems (two bridgehead carbon atoms are linked to one another via a covalent linker containing at least one carbon atom), and spiro-ring (two rings are fused at the same carbon atom) systems. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, etc.

[0097] “Dystrophin” is a rod-shaped cytoskeletal protein and a vital part of the dystrophin-associated protein complex (DAPC) that connects the cytoskeleton of a muscle fiber to the surrounding PATENT

[0098] Attorney Docket No.: 51558-023WO2 extracellular matrix through the cell membrane. Dystrophin contains multiple functional domains. For instance, dystrophin contains an actin binding domain at about amino acids 14-240 and a central rod domain at about amino acids 253-3040. This large central domain is formed by 24 spectrin-like triplehelical elements of about 109 amino acids, which have homology to alpha-actinin and spectrin. The repeats are typically interrupted by four proline-rich non-repeat segments, also referred to as hinge regions. Repeats 15 and 16 are separated by an 18 amino acid stretch that appears to provide a major site for proteolytic cleavage of dystrophin. The sequence identity between most repeats ranges from 10-25%. One repeat contains three alpha-helices: 1 , 2 and 3. Alpha-helices 1 and 3 are each formed by 7 helix turns, probably interacting as a coiled-coil through a hydrophobic interface. Alpha-helix 2 has a more complex structure and is formed by segments of four and three helix turns, separated by a glycine or proline residue. Each repeat is encoded by two exons, typically interrupted by an intron between amino acids 47 and 48 in the first part of alpha-helix 2. The other intron is found at different positions in the repeat, usually scattered over helix-3. Dystrophin also contains a cysteine-rich domain at about amino acids 3080-3360) including a cysteine-rich segment (i.e., 15 cysteines in 280 amino acids) showing homology to the C-terminal domain of the slime mold (Dictyostelium discoideum) alpha-actinin. The carboxy-terminal domain is at about amino acids 3361-3685.

[0099] The amino-terminus of dystrophin binds to F-actin and the carboxy-terminus binds to the DAPC at the sarcolemma acting as a bridge and provides stabilization of the sarcolemma membrane during contraction and relaxation. The DAPC includes, and is not limited to, the dystroglycans, sarcoglycans, integrins and caveolin, and mutations in any of these components cause autosomally inherited muscular dystrophies. The DAPC is destabilized when dystrophin is absent, which results in diminished levels of the member proteins, and in turn leads to muscle contraction induced progressive fiber damage and membrane leakage. In various forms of muscular dystrophy, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), muscle cells produce little to no dystrophin at all, or an altered and functionally defective form of dystrophin, respectively, mainly due to mutations in the gene sequence that lead to incorrect splicing. The predominant expression of the defective dystrophin protein, or the complete lack of dystrophin or a dystrophin-like protein, leads to rapid progression of muscle degeneration, as noted above. In this regard, a “defective” dystrophin protein may be characterized by the forms of dystrophin that are produced in certain subjects with DMD or BMD, as known in the art, or by the absence of detectable dystrophin.

[0100] An “exon” refers to a defined section of nucleic acid that encodes for a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after a portion of a pre- processed (or precursor) RNA has been removed by splicing. The mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as rRNA ortRNA. The human dystrophin (DMD) gene has 79 exons.

[0101] “Exon skipping” refers generally to the process by which an entire exon, or a portion thereof, is removed from a given pre-processed RNA, and is thereby excluded from being present in the mature RNA, such as the mature mRNA that is translated into a protein. Hence, the portion of the protein that is otherwise encoded by the skipped exon is not present in the expressed form of the protein, typically creating an altered, though still functional, form of the protein. In certain embodiments, the exon being skipped is an aberrant exon from the human dystrophin (DMD gene, which may contain a mutation or PATENT

[0102] Attorney Docket No.: 51558-023WO2 other alteration in its sequence that otherwise causes aberrant splicing. In certain embodiments, the exon being skipped is exon 53 of the human dystrophin gene.

[0103] The term “halo” or “halogen,” as used herein, refer to fluoro, chloro, bromo, and iodo.

[0104] The term “heteroalkyl,” as used herein, refers to a straight or branched chain hydrocarbon group containing a total of one to thirty carbon atoms, unless otherwise specified, and at least one heteroatom that is oxygen, nitrogen, or sulfur. For example, a heteroalkyl may be (2-20C) heteroalkyl, (2-12C) heteroalkyl, or (2-10C) heteroalkyl. Non-limiting examples of heteroalkyls include, e.g., PEG3.

[0105] By “histidine rich,” it is meant that at least 40% of the cationic domain is formed of histidine residues.

[0106] The term “heteroaryl,” as used herein, refers to a ring system containing one, two, or three rings, at least one of which is aromatic and containing one to four (e.g., one, two, or three) heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. An unsubstituted heteroaryl group contains a total of one to nine carbon atoms. The term heteroaryl includes both monovalent species and divalent species. Examples of heteroaryl groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members. The heteroaryl group can be, for example, a 5- or 6-membered monocyclic ring or a 9- or 10- membered bicyclic ring, for example, a bicyclic structure formed from fused five and six membered rings or two fused six membered rings. Each ring may contain up to four heteroatoms typically selected from nitrogen, sulfur and oxygen. Typically, the heteroaryl ring will contain up to 3 heteroatoms, more usually up to 2, for example, a single heteroatom. In some embodiments, the heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl rings can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in the case of an indole or pyrrole nitrogen. In general, the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.

[0107] 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, isoquinolyl, 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]-o- oxazinyl, 1 H-pyrazolo[4,3-d]-oxazolyl, 4H-imidazo[4,5-d]thiazolyl, pyrazino[2,3-d]pyridazinyl, imidazo[2,1-b]thiazolyl, imidazo[1 ,2-b][1 ,2,4]triazinyl. “Heteroaryl” also covers partially aromatic bi- or polycyclic ring systems wherein at least one ring is an aromatic ring and one or more of the other ring(s) is a non-aromatic, saturated or partially saturated ring, provided 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-oxo-1 .2.3.4- tetrahydroquinolinyl, dihydrobenzothienyl, dihydrobenzofuranyl, 2,3-dihydro-benzo[1 ,4]dioxinyl, benzo[1 ,3]dioxolyl, 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-2H- pyrido[3,2-b][1 ,4]oxazinyl. Examples of five membered heteroaryl groups include but are not limited to pyrrolyl, furanyl, thienyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, PATENT

[0108] Attorney Docket No.: 51558-023WO2 isothiazolyl, pyrazolyl, triazolyl and tetrazolyl groups. Examples of six membered heteroaryl groups include but are not limited to pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl and triazinyl. A bicyclic heteroaryl group may be, for example, a group selected from: a benzene ring fused to a 5- or 6- membered ring containing 1 , 2 or 3 ring heteroatoms; a pyridine ring fused to a 5- or 6-membered ring containing 1 , 2 or 3 ring heteroatoms; a pyrimidine ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a pyrrole ring fused to a 5- or 6-membered ring containing 1 , 2 or 3 ring heteroatoms; a pyrazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a pyrazine ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an imidazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an oxazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an isoxazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a thiazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an isothiazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a thiophene ring fused to a 5- or 6-membered ring containing 1 , 2 or 3 ring heteroatoms; a furan ring fused to a 5- or 6-membered ring containing 1 , 2 or 3 ring heteroatoms; a cyclohexyl ring fused to a 5- or 6-membered heteroaromatic ring containing 1 , 2 or 3 ring heteroatoms; and a cyclopentyl ring fused to a 5- or 6-membered heteroaromatic ring containing 1 , 2 or 3 ring heteroatoms. Particular examples of bicyclic heteroaryl groups containing a six membered ring fused to a five membered ring include but are not limited to benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, indolinyl, isoindolinyl, purinyl (e.g., adeninyl, guaninyl), indazolyl, benzodioxolyl and pyrazolopyridinyl groups. Particular examples of bicyclic heteroaryl groups containing two fused six membered rings include but are not limited to quinolinyl, isoquinolinyl, chromanyl, thiochromanyl, chromenyl, isochromenyl, chromanyl, isochromanyl, benzodioxanyl, quinolizinyl, benzoxazinyl, benzodiazinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl and pteridinyl groups.

[0109] The terms “heterocyclyl,” as used herein, refer to a ring system containing one, two, or three rings, at least one of which containing one to four (e.g., one, two, or three) heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, provided that the ring system does not contain aromatic rings. An unsubstituted heterocyclyl group contains a total of two to nine carbon atoms. The term heterocyclyl includes both monovalent species and divalent species. Examples of heterocyclyl groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members. The heterocyclyl group can be, for example, a 5- or 6- membered monocyclic ring or a 9- or 10-membered bicyclic ring, for example, a bicyclic structure formed from fused five and six membered rings or two fused six membered rings. Each ring may contain up to four heteroatoms typically selected from nitrogen, sulfur and oxygen. Non-limiting examples of heterocyclyl groups include, e.g., 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. Unless otherwise specified, alkyl may be optionally substituted by one, two, three, four, or five groups selected from the group consisting of alkyl, carbocyclyl, aryl, heterocyclyl, heteroaryl, oxo, halogen, and hydroxyl. PATENT

[0110] Attorney Docket No.: 51558-023WO2

[0111] The term “increase” or “restore” or “improve” may relate generally to the ability of one or more compounds of the invention to “increase” a relevant physiological, cellular, or molecular response, which may be decreased in a disease state or condition as described herein, and as measured according to routine techniques in the diagnostic art. In some embodiments, the physiological or cellular response is in a subject, such as a human or non-human animal (e.g., a non-human primate), or is in cells, such as non-DMD cells. Relevant physiological or cellular responses ( / n vivo or in vitro) will be apparent to persons skilled in the art, and may include, for example, improvements in the symptoms or pathology of a neuromuscular or neurologic disease (e.g., DMD). An “increase” in a response may be statistically significant as compared to the response produced by no administration of a conjugate or produced by administration of a control composition, 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% increase, including all integers in between.

[0112] The term “morpholino,” as used herein in reference to a class of oligonucleotides, represents an oligomer of at least 10 morpholino subunits interconnected by morpholino internucleoside linkages. A morpholino includes a 5’ group and a 3’ group. For example, a morpholino may be of the following structure: where n is an integer of at least 10 (e.g., 12 to 30) indicating the number of morpholino subunits and associated groups L; each B is independently a nucleobase;

[0113] R1is a 5’ group (R1may be referred to herein as a 5’ terminus);

[0114] R2is a 3’ group (R2may be referred to herein as a 3’ terminus); and

[0115] L is (i) a morpholino internucleoside linkage or, (ii) if L is attached to R2, a covalent bond.

[0116] A 5’ group in morpholino may be, e.g., hydroxyl, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, phosphorodiamidate, a bond to a peptide, a bond to a peptide / linker combination, an endosomal escape moiety, a neutral organic polymer, or a group of the following structure:

[0117] R1R1y R2

[0118] O=P— N

[0119] 6 R2JW IV where each R1is independently alkyl optionally substituted with =O, -OH, and / or -NH2; heteroalkyl optionally substituted with =O, -OH, and / or -NH2; cycloalkyl; heterocyclyl; heteroaryl; or -L-R3; where L is a linker that is alkyl, cycloalkyl, heteroalkyl, heterocyclyl, or heteroaryl, and R3is a solid support, PATENT

[0120] Attorney Docket No.: 51558-023WO2 provided that no more than one R1is -L-R3; or two R1groups, together with the nitrogen atom to which they are attached, combine to form a heterocyclyl optionally substituted with alkyl optionally substituted with =O, -OH, and / or -NH2 or heteroalkyl optionally substituted with =O, -OH, and / or -NH2; and each R2is independently alkyl optionally substituted with =O, -OH, and / or -NH2; heteroalkyl optionally substituted with =O, -OH, and / or -NH2; cycloalkyl; heterocyclyl; or heteroaryl.

[0121] Preferably, a 5’ group is a hydroxyl or a group of the following structure:

[0122] A more preferred 5’ group is of the following structure:

[0123] A 3’ group in morpholino may be, e.g., hydrogen, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a bond to a peptide, a bond to a peptide / linker combination, a group for conjugation (e.g., a maleimide, an alkyl or aryl substituted with thiol, an alkynyl, or an alkyl substituted with azide), an endosomal escape moiety, or a neutral organic polymer.

[0124] In a conjugate of an oligonucleotide that is a morpholino and a peptide that is 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.

[0125] The term “morpholino internucleoside linkage,” as used herein, represents a divalent group of the following structure: where

[0126] Z is O or S;

[0127] X1is a bond, -CH2-, or -O-;

[0128] X2is a bond, -CH2-O-, or -O-; and

[0129] Y is -NR2, where each R is independently H or C1-6 alkyl (e.g., methyl), or both R combine together with the nitrogen atom to which they are attached to form a C2-9 heterocyclyl (e.g., N- piperazinyl); PATENT

[0130] Attorney Docket No.: 51558-023WO2 provided that one and only one of X1and X2is a bond. The morpholino internucleoside linkage is bonded through its phosphorus atom to the nitrogen atom of the morpholine ring in the morpholino subunit.

[0131] Preferably, the morpholino is a phosphorodiamidate morpholino (PMO), which has the morpholino internucleoside linkage of -P(O)(NMe2)O-.

[0132] The term “morpholino subunit,” as used herein, refers to the following structure: where B is a nucleobase.

[0133] The term “nucleobase,” as used herein, represents a nitrogen-containing heterocyclic ring found at the T position of the ribofuranose / 2’-deoxyribofuranose of a nucleoside. Nucleobases are 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 0-6 substituted purines, as well as synthetic and natural nucleobases, e.g., 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-alkyl (e.g., 6-methyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil, 5- propynyl cytosine, 5-trifluoromethyl uracil, 5-trifluoromethyl cytosine, 7-methyl guanine, 7-methyl adenine, 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, e g., 5-substituted pyrimidines; 6-azapyrimidines; N2-, N6-, and / or O6-substituted purines. Nucleic acid duplex stability can be enhanced using, e.g., 5-methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl ( — CEC — CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6- azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo-, 8-amino-, 8-thiol-, 8-thioalkyl-, 8- hydroxyl-, 8-aza- and other 8-substituted purines, 5-halo-, particularly 5-bromo-, or 5-trifluoromethyl- pyrimidines,e.g., 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-diazaphenoxazine-2-one, 1 ,3-diazaphenothiazine-2-one and 9-(2- aminoethoxy)-1 ,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7- deazaadenine, 7- deazaguanine, 2-aminopyridine, or 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch PATENT

[0134] Attorney Docket No.: 51558-023WO2 et al., Angewandte Chemie, International Edition, 1991 , 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.

[0135] The term “nucleoside,” as used herein, represents 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 ribofuranose having an anomeric carbon bond to an 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. A nucleobase modification is a replacement of an unmodified nucleobase with a modified nucleobase. A sugar modification may be, e.g., a 2’-substitution, locking, carbocyclization, or unlocking. A 2’-substitution is a replacement of 2’-hydroxyl in ribofuranose, e.g., with 2’-fluoro, 2’-methoxy, or 2’-(2-methoxy)ethoxy. Alternatively, a 2’-substitution may be a 2’-(ara) substitution, which corresponds to the following structure: where B is a nucleobase, and R is a 2’-(ara) substituent (e.g., fluoro). 2’-(ara) substituents are known in the art and can be same as other 2’-substituents described herein. In some embodiments, 2’-(ara) substituent is a 2’-(ara)-F substituent (R is fluoro). A locking modification is an incorporation of a bridge between 4’-carbon atom and 2’-carbon atom of ribofuranose. Nucleosides having a locking modification are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene- bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides. A “nucleoside” may also refer to a morpholino subunit.

[0136] The term “nucleotide,” as used herein, represents a nucleoside bonded to an internucleoside linkage or a monovalent group of the following structure -X1-P(X2)(R1)2, where X1is O, S, or NH, and X2is absent, =O, or =S, and each R1is independently -OH, -N(R2)2, or -O-CH2CH2CN, where each R2is independently an optionally substituted alkyl, or both R2groups, together with the nitrogen atom to which they are attached, combine to form an optionally substituted heterocyclyl.

[0137] The term “oligonucleotide,” as used herein, represents a structure containing 10 or more contiguous nucleosides covalently bound together by internucleoside linkages; a morpholino containing 10 or more morpholino subunits; or a peptide nucleic acid containing 10 or more peptide nucleic acid subunits. Preferably, an oligonucleotide is a morpholino. In some embodiments, the oligonucleotide includes phosphorothioate (PS) internucleotide linkages. In some embodiments, the oligonucleotide includes 2’-O-alkyl nucleotides, e.g., a 2’-O-methyl nucleotides. In some embodiments, the oligonucleotide includes 2’-O-alkyl, e.g., 2’-O-methyl, nucleotides and phosphorothioate internucleotide linkages. In some embodiments, the oligonucleotide is a peptide nucleic acid. PATENT

[0138] Attorney Docket No.: 51558-023WO2

[0139] The term “peptide nucleic acid (PNA),” as used herein in reference to a class of oligonucleotides, represents an oligomer of at least 10 nucleobases interconnected by 2- aminoethylglycine units. The 2-aminoethylglycine units may be substituted, e.g., y-PNA.

[0140] The term “pharmaceutically acceptable,” as used herein, refers to those compounds, materials, compositions, and / or dosage forms, which are suitable for contact with the tissues of an individual (e.g., a human), without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit / risk ratio.

[0141] The term “pharmaceutically acceptable salt,” as used herein, means any pharmaceutically acceptable salt of a conjugate, oligonucleotide, or peptide disclosed herein. Pharmaceutically acceptable salts of any of the compounds described herein may include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, or allergic response and are 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 in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group 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-hydroxy-ethanesulfonate, 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 salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0142] As used herein, the term “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than active agents (e.g., as described herein) present in pharmaceutical compositions and having the properties of being substantially nontoxic and non-inflammatory in subjects. In some embodiments, pharmaceutically acceptable excipients are vehicles capable of suspending and / or stabilizing active agents. Excipients may include, for example: antiadherents, antioxidants, binders, dis integrants, emollients, emulsifiers, fillers (diluents), film formers or coatings, glidants (flow enhancers), preservatives, sorbents, suspending or dispersing agents, and waters of hydration. Excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, PATENT

[0143] Attorney Docket No.: 51558-023WO2 povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and / or xylitol.

[0144] The term “pharmaceutical composition,” as used herein, represents a composition containing an oligonucleotide described herein, formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a subject.

[0145] The term “reduce” or “inhibit” may relate generally to the ability of one or more compounds of the invention to “decrease” a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic art. In some embodiments, the physiological or cellular response is in a subject, such as a human or non-human animal (e.g., a non-human primate), or is in cells, such as non-DMD cells. Relevant physiological or cellular responses ( / n vivo or in vitro) will be apparent to persons skilled in the art, and may include, for example, reductions in the symptoms or pathology of a neuromuscular or neurologic disease. A “decrease” in a response may be statistically significant as compared to the response produced by no administration of a conjugate or produced by administration of a control composition, 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% decrease, including all integers in between.

[0146] The term “subject,” as used herein, represents a human or non-human animal (e.g., a mammal, such as a non-human primate) that is suffering from, or is at risk of, disease, disorder, or condition, as determined by a qualified professional (e.g., a doctor or a nurse practitioner) with or without known in the art laboratory test(s) of sample(s) from the subject. In some embodiments, a subject has Duchenne muscular dystrophy (DMD). In some embodiments, a subject has DMD and is one who is amenable to a therapy that induces skipping of exon 53 of the DMD transcript (i.e., a therapy including administration of a conjugate that induces exon 53 skipping or pharmaceutical composition including the same).

[0147] “Treatment” and “treating,” as used herein, refer to the medical management of a subject with the intent to improve, ameliorate, or stabilize a disease, disorder, or condition (e.g., DMD). This term includes active treatment (treatment directed to improve DMD); palliative treatment (treatment designed for the relief of symptoms of DMD); and supportive treatment (treatment employed to supplement another therapy).

[0148] As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., therapeutic agent) that is sufficient when administered to a subject suffering from or susceptible to a disease, disorder, or condition, to treat, improve the symptoms of, alleviate, ameliorate, and / or delay the onset of the disease, disorder, or condition. In some embodiments, a therapeutically effective amount is provided in a single dose. In some embodiments, a therapeutically effective amount is administered in a dosage regimen including a plurality of doses. Those skilled in the art will appreciate that in some embodiments, a unit dosage form may be considered to include a therapeutically effective amount of a particular agent or entity if it is in an amount that is effective when administered as part of such a dosage regimen. PATENT

[0149] Attorney Docket No.: 51558-023WO2

[0150] All references to “conjugates” also refer to salts thereof. As will be appreciated by one of skill in the art, the conjugates disclosed herein may include multiple ionizable and / or protonatable groups. Accordingly, a conjugate of the invention may be used in a salt form, such as acid addition salt, or in a substantially neutral form.

[0151] All references to “oligonucleotides” also refer to salts thereof.

[0152] Unless otherwise specified, all peptides are shown herein in N-terminus to C-terminus direction (left to right). Unless otherwise specified, all oligonucleotides are shown herein in 5’ to 3’ direction (left to right).

[0153] Features, integers, characteristics, compounds, chemical moieties, or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0154] DETAILED DESCRIPTION

[0155] In general, the invention provides a conjugate, or a pharmaceutically acceptable salt thereof, of an oligonucleotide (e.g., a morpholino, such as a phosphorodiamidate morpholino (PMO)) covalently bonded or covalently linked via a linker to a peptide. The oligonucleotide is complementary to a target sequence within or proximal to exon 53 of a human dystrophin (DMD) gene. The peptide includes at least one positively charged domain and at least one hydrophobic domain. Without wishing to be bound by theory, the peptide may act as a cell-penetrating peptide to enhance the activity of the conjugated oligonucleotide, e.g., by improving intracellular delivery of the conjugated oligonucleotide to tissues. Advantageously, as described in the Examples herein, the conjugates disclosed herein exhibit enhanced cellular uptake and intracellular delivery thereby yielding enhanced activity of the conjugated oligonucleotide.

[0156] Oligonucleotides

[0157] In some embodiments, oligonucleotides used in the conjugates disclosed herein may be those complementary to a target site within a dystrophin transcript. Without wishing to be bound by theory, it is believed that the oligonucleotide induces the skipping of exon 53 during the dystrophin pre-mRNA splicing, thereby ameliorating Duchenne muscular dystrophy (DMD).

[0158] In some embodiments, the oligonucleotide sequence includes the sequence 5’-CTGAAGGTGTTCTTGTACTTCATCC-3’ (SEQ ID NO: 1).

[0159] In some embodiments, the oligonucleotide includes the sequence 5’-CAACTGTTGCCTCCGGTTCTGAAGGTGTTC-3’ (SEQ ID NO: 2). In some embodiments, the oligonucleotide sequence includes the sequence 5’-TTGCCTCCGGTTCTGAAGGTGTTCTTGTAC-3’ (SEQ ID NO: 3).

[0160] In some embodiments, the oligonucleotide sequence includes the sequence 5’-CTGTTGCCTCCGGTTCTGAAGGTGTTCTTG-3’ (SEQ ID NO: 4). PATENT

[0161] Attorney Docket No.: 51558-023WO2

[0162] In some embodiments, the oligonucleotide sequence includes the sequence 5’-CATTCAACTGTTGCCTCCGGTTCTGAAGGTG-3’ (SEQ ID NO: 5).

[0163] In some embodiments, one or more thymines or all thymines in an oligonucleotide sequence are replaced with uracils.

[0164] In some embodiments, the oligonucleotides are morpholinos, e.g., phosphorodiamidate morpholino oligomers (PMOs). In some embodiments, the oligonucleotides are phosphorothioates, e.g., as described herein. In some embodiments, the oligonucleotides include 2’-O-alkyl nucleotides, e.g., 2’-O-methyl nucleotides and / or 2’-O-alkyl phosphorothioates, such as 2’-O-methyl phosphorothioates. In some embodiments, the oligonucleotides are peptide nucleic acids (PNAs).

[0165] In some embodiments, the oligonucleotide includes the following group as its 5’ terminus:

[0166] In some embodiments, the oligonucleotide includes the following group as its 5’ terminus:

[0167] In some embodiments, the oligonucleotide includes hydroxyl as its 5’ terminus.

[0168] Peptides

[0169] The peptide has a sequence that is a contiguous single molecule. Therefore, the domains of the peptide are contiguous. In some embodiments, the peptide includes several domains in a linear arrangement between the N-terminus and the C-terminus. In some embodiments, the domains are selected from cationic domains and hydrophobic domains described herein. In some embodiments, the peptide consists of cationic domains and hydrophobic domains wherein the domains are as defined herein.

[0170] Each domain has common sequence characteristics as described herein, but the exact sequence of each domain is capable of variation and modification. Thus, a range of sequences is possible for each domain. The combination of each possible domain sequence yields a range of peptide structures, each of which form part of the present invention. Features of the peptide structures are described herein.

[0171] In some embodiments, the peptide includes a total of one hydrophobic domain and at least one cationic domain, the cationic domain including at least 1 cationic amino acid residue, and the hydrophobic domain including at least 3 amino acid residues, provided that the peptide includes a total PATENT

[0172] Attorney Docket No.: 51558-023WO2 of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues and, in some embodiments, does not include any artificial amino acid residues.

[0173] In some embodiments, the peptide includes a total of one hydrophobic domain and a total of one or two cationic domains flanking the hydrophobic domain, each cationic domain including at least one cationic amino acid residue, at least one of the cationic domains including a total of one cationic amino acid residue, and the hydrophobic domain including at least 3 amino acid residues, provided that the peptide includes a total of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues and, in some embodiments, does not include any artificial amino acid residues.

[0174] In some embodiments, the peptide includes a total of one hydrophobic domain and a total of one or two cationic domains flanking the hydrophobic domain, each cationic domain including at least 1 cationic amino acid residue, all cationic domains collectively including a total of five or fewer cationic amino acid residue, and the hydrophobic domain including at least 3 amino acid residues, provided that the peptide includes a total of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues and, in some embodiments, does not include any artificial amino acid residues.

[0175] In some embodiments, the peptide includes a total of one hydrophobic domain and a total of one or two cationic domains flanking the hydrophobic domain, each cationic domain including at least 1 cationic amino acid residue, at least one third of amino acid residues in the N-terminal cationic domain are histidines, and the hydrophobic domain including at least 3 amino acid residues, provided that the peptide includes a total of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues and, in some embodiments, does not include any artificial amino acid residues.

[0176] 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.

[0177] In some embodiments, the peptide includes a total of one or two hydrophobic domains and a total of one or two cationic domains. In some embodiments, the peptide includes a total of one or two cationic domains flanked by two hydrophobic domains, provided that the peptide includes a total of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues.

[0178] In some embodiments, the peptide contains artificial amino acid residues. In some embodiments the peptide contains aminohexanoic acid residues. In some embodiments, the peptide contains at least one aminohexanoic acid residue. In some embodiments, the peptide contains between 1 and 6 (e.g., 1 to 3 or 3 to 6) aminohexanoic acid residues.

[0179] In some embodiments, the peptide contains only natural amino acid residues and therefore consists of natural amino acid residues.

[0180] In some embodiments, there is provided a peptide having a total length of 40 amino acid residues or less, the peptide including: two or more cationic domains each including at least 4 amino acid residues; and one or more hydrophobic domains each including at least 3 amino acid residues; wherein at least one cationic domain includes histidine residues. In some embodiments, wherein at least one cationic domain is histidine rich.

[0181] In some embodiments, the peptide includes L-amino acids, e.g., all chiral amino acids are L- amino acids. In some embodiments, the peptide includes D-amino acids, e.g., all chiral amino acids are D-amino acids. In some embodiments, the peptide includes L- and D-amino acids. PATENT

[0182] Attorney Docket No.: 51558-023WO2

[0183] In some embodiments, the peptides include at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 D-amino acid(s). In some embodiments, in a peptide including D-amino acids, the remainder of the amino acids in such a peptide, if any, are L-amino acids or achiral amino acids.

[0184] In some embodiments, the peptide is N-terminally modified.

[0185] In some embodiments, the peptide is N-acetylated, N-methylated, N-trifluoroacetylated, N- trifluoromethylsulfonylated, or N-methylsulfonylated. In some embodiments, the peptide is N- acetylated.

[0186] In other embodiments, the N-terminus of the peptide is unmodified.

[0187] In some embodiments, the peptide is C-terminal modified.

[0188] In some embodiments, the peptide includes a C-terminal modification selected from: thioacid-, aminooxy-, hydrazino-, thioester-, azide, strained alkyne, strained alkene, aldehyde-, thiol or haloacetyl-group.

[0189] Advantageously, the C-terminal modification provides a means for linkage of the peptide to the oligonucleotide.

[0190] In some embodiments, the peptide includes a C-terminal carboxyl group.

[0191] The peptide of the present invention may have a total length of 40 amino acid residues or fewer.

[0192] In some embodiments, the peptide has a total length of 3-30 amino acid residues, e.g., of 5-25 amino acid residues, of 10-25 amino acid residues, of 13-23 amino acid residues, or of 15-20 amino acid residues.

[0193] In some embodiments, the peptide has a total length of at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 amino acid residues.

[0194] In some embodiments, the peptide has a total length of at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 amino acid residues.

[0195] In some embodiments, the peptide includes an amino acid sequence selected from the group consisting of RBRRBRFQILYBRBR (SEQ ID NO: 9); RBRRBRRFQILYRBRBR (SEQ ID NO: 17); RBRRBRRYQFLIRBRBR (SEQ ID NO: 18); RBRRBRRILFQYRBRBR (SEQ ID NO: 19); RBRRBRRFQILYRBHBH (SEQ ID NO: 20); RBRRBRRFQILYHBHBR (SEQ ID NO: 21); RBRRBRFQILYRBHBH (SEQ ID NO: 22); RRRRR (SEQ ID NO: 23); RRRRRR (SEQ ID NO: 24); RRRRRRR (SEQ ID NO: 25); or RRRRRRRR (SEQ ID NO: 26).

[0196] In some embodiments, the peptide includes an amino acid sequence selected from the group consisting of: RXRRBRRXRRBR (SEQ ID NO: 27); RXRRBRRXRRBRX (SEQ ID NO: 28); RXRRXRRXRRXRX (SEQ ID NO: 29); RXRRBRRFQILYRBRXR (SEQ ID NO: 30); RXRRBRRXRILFQYRXRBRXR (SEQ ID NO: 31); RXRRBRRXRILFQYRXRXRXR (SEQ ID NO: 32); RXRRXRILFQYRXRRXR (SEQ ID NO: 33); RBRRXRRBRILFQYRBRXRBR (SEQ ID NO: 34); RBRRXRRBRILFQYRXRBRXR (SEQ ID NO: 35); RBRRXRRBRILFQYRXRRXR (SEQ ID NO: 36); RBRRXRRBRILFQYRXRBRX (SEQ ID NO: 37); RXRRBRRXRILFQYRXRRXR (SEQ ID NO: 38); RXRRBRRXRILFQYRXRBRX (SEQ ID NO: 39); RXRRBRRXRYQFLIRXRBRXR (SEQ ID NO: 40); RXRRBRRXRIQFLIRXRBRXR (SEQ ID NO: 41); RXRRBRRXRQFLIRXRBRXR (SEQ ID NO: 42); RXRRBRRXRQFLRXRBRXR (SEQ ID NO: 43); RXRRBRRXYRFLIRXRBRXR (SEQ ID NO: 44); PATENT

[0197] Attorney Docket No.: 51558-023WO2

[0198] RXRRBRRXRFQILYRXRBRXR (SEQ ID NO: 45); RXRRBRRXYRFRLIXRBRXR (SEQ ID NO: 46); RXRRBRRXILFRYRXRBRXR (SEQ ID NO: 47); RXRRBRRXRIYQFLIRXRBRXR (SEQ ID NO: 48); YQFLIRBRRXRBRXBRXRBYQFLI (SEQ ID NO: 49); YQFLIRBRRBRBRBRRBYQFLI (SEQ ID NO: 50); or YQFLIRBRRBRBRBBRXRBYQFLI (SEQ ID NO: 51),

[0199] In some embodiments, the peptide is bonded to the rest of the conjugate through its N- terminus. In some embodiments, the C-terminus of the peptide is amidated, i.e., terminated with -NH2. In some embodiments, the peptide is bonded to the rest of the conjugate through its C-terminus. In some embodiments, the peptide is acylated at its N-terminus. In some embodiments, the peptide is covalently bonded (i.e., directly) to the oligonucleotide.

[0200] Peptides of the invention may be produced by any standard protein synthesis method (e.g., solid-phase peptide synthesis), for example, chemical synthesis or semi-chemical synthesis.

[0201] Cationic Domain

[0202] In the peptides described herein, each cationic domain includes at least 1 cationic amino acid residue. In some embodiments, each cationic domain includes at least one cationic amino acid residue, and at least one of the cationic domains includes a total of one cationic amino acid residue. In some embodiments, all cationic domains collectively include a total of seven or fewer (e.g., six or fewer) cationic amino acid residues.

[0203] In some embodiments, the peptide includes a total of one or two cationic domains flanking the hydrophobic domain, each cationic domain including at least 1 cationic amino acid residue.

[0204] In some embodiments, the peptide includes up to 4 cationic domains, e.g., up to 3 cationic domains.

[0205] In some embodiments, the peptide includes 2 cationic domains, e.g., a total of two cationic domains.

[0206] In some embodiments, the peptide includes two or more cationic domains each having a length of at least 4 amino acid residues.

[0207] In some embodiments, each cationic domain has a length of between 4 to 12 amino acid residues, e.g., a length of between 4 to 7 amino acid residues.

[0208] In some embodiments, each cationic domain has a length of 4, 5, 6, or 7 amino acid residues.

[0209] In some embodiments, each cationic domain is of similar length, e.g., each cationic domain is the same length.

[0210] In some embodiments, each cationic domain includes cationic amino acids and may also contain polar and / or nonpolar amino acids.

[0211] Non-polar amino acids may be selected from: alanine, beta-alanine, proline, glycine, cysteine, valine, leucine, isoleucine, methionine, tryptophan, and phenylalanine.

[0212] 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 have a negative charge.

[0213] Cationic amino acids may be selected from: arginine, histidine, and lysine. PATENT

[0214] Attorney Docket No.: 51558-023WO2

[0215] In some embodiments, each cationic domain does not include anionic or negatively charged amino acid residues. In some embodiments, each cationic domain includes arginine, histidine, betaalanine, hydroxyproline, and / or serine residues.

[0216] In some embodiments, each cationic domain consists of arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues.

[0217] In some embodiments, each cationic domain includes at least 30%, at least 40%, at least 45%, or at least 50% cationic amino acids.

[0218] In some embodiments, each cationic domain includes a majority of cationic amino acids (e.g., greater than 50% cationic amino acids). In some embodiments, each cationic domain includes 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.

[0219] In some embodiments, each cationic domain has an isoelectric point (pl) 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.

[0220] In some embodiments, each cationic domain has an isoelectric point (pl) of at least 10.0.

[0221] In some embodiments, each cationic domain has an isoelectric point (pl) of between 10.0 and 13.0

[0222] In some embodiments, each cationic domain has an isoelectric point (pl) of between 10.4 and 12.5.

[0223] In some embodiments, the isoelectric point of a cationic domain is calculated at physiological pH by any suitable means available in the art. In some embodiments, by using the I PC (www.isoelectric.org) a web-based algorithm developed by Lukasz Kozlowski, Biol Direct. 2016; 11 : 55. doi: 10.1186 / s 13062-016-0159-9.

[0224] 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.

[0225] In some embodiments, each cationic domain includes a majority of arginine and / or histidine residues.

[0226] In some embodiments, each cationic domain includes at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, at least 70% arginine and / or histidine residues. In some embodiments, a cationic domain may include at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, at least 70% arginine residues.

[0227] In some embodiments, a cationic domain may include at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, at least 70% histidine residues.

[0228] In some embodiments, a cationic domain may include between 1-5 arginine residues. In some embodiments, a cationic domain may include between 1-5 histidine residues. In some embodiments, a cationic domain may include a total of between 1-5 histidine and 1-5 arginine residues. In some embodiments, a cationic domain may include a total of between 2-5 histidine and 3-5 arginine residues. In some embodiments, a cationic domain may include between 3-5 arginine residues. In some embodiments, a cationic domain may include between 2-5 histidine residues.

[0229] In some embodiments, each cationic domain includes one or more beta-alanine residues. In some embodiments, each cationic domain may include a total of between 2-5 beta-alanine residues, PATENT

[0230] Attorney Docket No.: 51558-023WO2 e.g., a total of 2 or 3 beta-alanine residues. In some embodiments, each cationic domain does not include any beta-alanine residues.

[0231] In some embodiments, a cationic domain may include one or more hydroxyproline residues or serine residues.

[0232] In some embodiments, a cationic domain may include between 1-2 hydroxyproline residues. In some embodiments, a cationic domain may include between 1-2 serine residues.

[0233] In some embodiments, all of the cationic amino acids in a given cationic domain may be arginine. Alternatively, all of the cationic amino acids in a given cationic domain may be histidine.

[0234] In some embodiments, the peptide may include at least one arginine rich cationic domain. In some embodiments, the peptide may include at least one histidine rich cationic domain.

[0235] In some embodiments, the peptide may include at least one arginine rich cationic domain and at least one histidine rich cationic domain.

[0236] In some embodiments, the peptide includes two arginine rich cationic domains.

[0237] In some embodiments, the peptide includes two histidine rich cationic domains.

[0238] In some embodiments, the peptide includes two arginine and histidine rich cationic domains.

[0239] In some embodiments, the peptide includes one arginine rich cationic domain and one histidine rich cationic domain. In some embodiments, each cationic domain includes no more than 3 contiguous arginine residues, e.g., no more than 2 contiguous arginine residues.

[0240] In some embodiments, each cationic domain includes no contiguous histidine residues.

[0241] In some embodiments, each cationic domain includes arginine, histidine and / or beta-alanine residues. In some embodiments, each cationic domain includes a majority of arginine, histidine and / or beta-alanine residues. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100% of the amino acid residues in each cationic domain are arginine, histidine and / or beta-alanine residues. In some embodiments, each cationic domain consists of arginine, histidine and / or beta-alanine residues.

[0242] In some embodiments, the peptide includes a first cationic domain including arginine and betaalanine residues and a second cationic domain including arginine and beta-alanine residues.

[0243] In some embodiments, the peptide includes a first cationic domain including arginine and betaalanine residues, and a second cationic domain including arginine, beta-alanine, and, optionally, histidine residues.

[0244] In some embodiments, the peptide includes a first cationic domain including arginine and betaalanine residues, and a second cationic domain including arginine and beta-alanine residues.

[0245] In some embodiments, the peptide includes a first cationic domain consisting of arginine and beta-alanine residues and a second cationic domain consisting of histidine, and beta-alanine residues.

[0246] In some embodiments, the peptide includes a first cationic domain consisting of arginine and beta-alanine residues and a second cationic domain consisting of arginine, histidine, and beta-alanine residues.

[0247] In some embodiments, the peptide includes at least two cationic domains, e.g., these cationic domains form the arms of the peptide. In some embodiments, the cationic domains are located at the N-and C-termini of the peptide. In some embodiments, therefore, the cationic domains may be known as the cationic arm domains. PATENT

[0248] Attorney Docket No.: 51558-023WO2

[0249] In some embodiments, the peptide includes two cationic domains, wherein one is located at the N-terminus of the peptide, and one is located at the C-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, with the exception of other groups such as a terminal modification, linker, and / or oligonucleotide. For the avoidance of doubt, such other groups may be present in addition to “the peptide” described and claimed herein. In some embodiments, therefore each cationic domain forms the terminus of the peptide. In some embodiments, this does not preclude the presence of a further linker group as described herein.

[0250] In some embodiments, the peptide may include up to 4 cationic domains. In some embodiments, the peptide includes two cationic domains.

[0251] In some embodiments, the peptide includes two cationic domains that are both arginine rich. In some embodiments, the peptide includes one cationic domain that is arginine rich.

[0252] In some embodiments, each cationic domain in the peptide may be identical or different. In some embodiments, each cationic domain in the peptide is different.

[0253] In some embodiments, each cationic domain may further include an N- or C-terminal modification. In some embodiments, the cationic domain at the C-terminus includes a C-terminal modification. In some embodiments, the cationic domain at the N-terminus includes an N-terminal modification. In some embodiments, the cationic domain at the C-terminus includes a linker group. In some embodiments, the cationic domain at the C-terminus includes a C-terminal beta-alanine. In some embodiments, the cationic domain at the N-terminus is N-acetylated.

[0254] Suitably, the cationic domains comprise amino acid units selected from the following: RBR, RXR, XXR, XRR, RRX, BXR, RXB, XRB, RBB, BRB, BBR, RRB, BRR, and BRX, 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. In some embodiments, one cationic domain includes the amino acid sequence RBRRBR (SEQ ID NO: 7). In some embodiments, one cationic domain includes the amino acid sequence BRBR (SEQ ID NO: 8). In some embodiments, each cationic domain includes one of the following sequences: RBRRBRR (SEQ ID NO: 77), RBRBR (SEQ ID NO: 78), RBRR (SEQ ID NO: 79), RBRRBR (SEQ ID NO: 7), RRBRBR (SEQ ID NO: 80), RBRRB (SEQ ID NO: 81), HBHBR (SEQ ID NO: 83), RBRHBHR (SEQ ID NO: 84), RBRBBHR (SEQ ID NO: 85), RBRRBH (SEQ ID NO: 86), HBRRBR (SEQ ID NO: 87), HBHBH (SEQ ID NO: 88), BHBH (SEQ ID NO: 89), BRBSB (SEQ ID NO: 90), BRB[Hyp]B (SEQ ID NO: 91), R[Hyp]H[Hyp]HB (SEQ ID NO: 92), R[Hyp]RR[Hyp]R (SEQ ID NO: 93) or any combination thereof; preferably where each cationic domain consists of one the following sequences: RBRRBRR (SEQ ID NO: 77), RBRBR (SEQ ID NO: 78), RBRR (SEQ ID NO: 79), RBRRBR (SEQ ID NO: 7), RRBRBR (SEQ ID NO: 80), RBRRB (SEQ ID NO: 81), BRBR (SEQ ID NO: 8), RBHBH (SEQ ID NO: 82), HBHBR (SEQ ID NO: 83), RBRHBHR (SEQ ID NO: 84), RBRBBHR (SEQ ID NO: 85), RBRRBH (SEQ ID NO: 86), HBRRBR (SEQ ID NO: 87), HBHBH (SEQ ID NO: 88), BHBH (SEQ ID NO: 89), BRBSB (SEQ ID NO: 90), BRB[Hyp]B (SEQ ID NO: 91), R[Hyp]H[Hyp]HB (SEQ ID NO: 92), R[Hyp]RR[Hyp]R (SEQ ID NO: 93) or any combination thereof. PATENT

[0255] Attorney Docket No.: 51558-023WO2

[0256] Hydrophobic Domain

[0257] In some embodiments, the peptide includes 1 hydrophobic domain, e.g., a total of 1 hydrophobic domain. In some embodiments, the peptide includes up to 3 hydrophobic domains, up to 2 hydrophobic domains.

[0258] The peptide may include one or more hydrophobic domains each having a length of at least 3 amino acid residues.

[0259] In some embodiments, each hydrophobic domain has a length of between 3-6 amino acids. In some embodiments, each hydrophobic domain has a length of 5 amino acids.

[0260] In some embodiments, each hydrophobic domain may include nonpolar, polar, and hydrophobic amino acid residues.

[0261] Hydrophobic amino acid residues may be selected from: alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, methionine, and tryptophan.

[0262] Non-polar amino acid residues may be selected from: proline, glycine, cysteine, alanine, valine, leucine, isoleucine, tryptophan, phenylalanine, and methionine.

[0263] Polar amino acid residues may be selected from: serine, asparagine, hydroxyproline, histidine, arginine, threonine, tyrosine, and glutamine.

[0264] In some embodiments, the hydrophobic domains do not include hydrophilic amino acid residues.

[0265] In some embodiments, each hydrophobic domain includes a majority of hydrophobic amino acid residues. In some embodiments, each hydrophobic domain includes at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100% hydrophobic amino acids. In some embodiments, each hydrophobic domain includes only hydrophobic amino acid residues.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] In some embodiments, each hydrophobic domain has a hydrophobicity of between 0.4 and 1.4

[0270] In some embodiments, each hydrophobic domain has a hydrophobicity of between 0.45 and 0.48.

[0271] In some embodiments, each hydrophobic domain has a hydrophobicity of between 1.27 and 1.39

[0272] In some embodiments, hydrophobicity is as measured by White and Wimley: W.C. Wimley and S.H. White, “Experimentally determined hydrophobicity scale for proteins at membrane interfaces” Nature Struct Biol. 3:842 (1996).

[0273] In some embodiments, each hydrophobic domain includes at least 3 or at least 4 hydrophobic amino acid residues. PATENT

[0274] Attorney Docket No.: 51558-023WO2

[0275] In some embodiments, each hydrophobic domain includes phenylalanine, leucine, Isoleucine, tyrosine, tryptophan, proline, and glutamine residues. In some embodiments, each hydrophobic domain consists of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues.

[0276] In some embodiments, each hydrophobic domain includes phenylalanine, leucine, isoleucine, tyrosine and / or glutamine residues. In some embodiments, each hydrophobic domain consists of phenylalanine, leucine, isoleucine, tyrosine, and / or glutamine residues.

[0277] In some embodiments, each hydrophobic domain includes tryptophan and / or proline residues.

[0278] In some embodiments, the peptide includes two cationic domains flanking a hydrophobic core domain.

[0279] In some embodiments, the peptide consists of two cationic arm domains flanking a hydrophobic core domain.

[0280] In some embodiments, each hydrophobic domain includes FQILY (SEQ ID NO: 6), YQFLI (SEQ ID NO: 52), ILFQY (SEQ ID NO: 53), FQIY (SEQ ID NO: 54), WWW, WWPWW (SEQ ID NO: 55), WPWW (SEQ ID NO: 56), WWPW (SEQ ID NO: 57), ILFQ (SEQ ID NO: 58), ILIQ (SEQ ID NO: 59), IKILFQN (SEQ ID NO: 60), IHILFQN (SEQ ID NO: 61), IRILFQN (SEQ ID NO: 62), IILFQN (SEQ ID NO: 63), KILFQN (SEQ ID NO: 64), HILFQN (SEQ ID NO: 65), RILFQN (SEQ ID NO: 66), ILFQN (SEQ ID NO: 67), HLIQN (SEQ ID NO: 68), KILIQN (SEQ ID NO: 69), KILIQY (SEQ ID NO: 70), HILIQN (SEQ ID NO: 71), RILIQN (SEQ ID NO: 72), HILIQY (SEQ ID NO: 73), RILIQY (SEQ ID NO: 74), ILIQN (SEQ ID NO: 75), ILIQY (SEQ ID NO: 76) .

[0281] In some embodiments, each hydrophobic domain consists of FQILY (SEQ ID NO: 6).

[0282] In some embodiments, each hydrophobic domain in the peptide may have the same sequence or a different sequence.

[0283] In some embodiments, a hydrophobic domain separates any two cationic domains. In some embodiments, each hydrophobic domain is flanked by cationic domains on either side thereof.

[0284] In some embodiments, no cationic domain is contiguous with another cationic domain.

[0285] In some embodiments, the peptide includes one hydrophobic domain flanked by two cationic domains in the following arrangement:

[0286] [cationic domain] - [hydrophobic domain] - [cationic domain].

[0287] In some embodiments, the peptide has two cationic domains and one hydrophobic domain.

[0288] In some embodiments, the peptide has one hydrophobic core domain flanked by two cationic domains.

[0289] In some embodiments, the peptide has one hydrophobic core domain including the amino acid sequence FQILY (SEQ ID NO: 6).

[0290] In some embodiments, the peptide has one hydrophobic core domain that has the amino acid sequence FQILY (SEQ ID NO: 6).

[0291] Linkers

[0292] In some embodiments, the peptides of the conjugates are linked to an oligonucleotide via a linker. PATENT

[0293] Attorney Docket No.: 51558-023WO2

[0294] A suitable linker forms a click reaction or formation of a morpholino linkage with a basic amino acid or a carboxylic acid moiety covalently conjugated to an amino group to form a carboxamide linkage. Other suitable linkers include, for example, a cysteine residue that permits formation of a disulfide, thioether, orthiol-maleimide linkage or an aldehyde to form an oxime.

[0295] The linker may be a non-cationic linker, e.g., a linker including a total of 1-5 amino acids (e.g., a linker including a total of one amino acid) or an aliphatic dicarboxylic acid linker. The peptide may be attached to the linker at the side chain (e.g., to the side chain of an amino acid having a functional group amenable to covalent attachment, such as glutamic acid side chain, lysine side chain, or aspartic acid side chain). When side chains are employed, an unbound amino or carboxylic acid group may be modified, e.g., by acylation or amidation.

[0296] In some embodiments, the linker is glutamic acid (e.g., via the gamma carboxyl group), betaalanine, glycine, delta-aminovaleric acid, or gamma-aminobutyric acid.

[0297] The linker may be at the N-terminus or the C-terminus of the peptide.

[0298] In some embodiments, the linker may include any linker that is known in the art.

[0299] In some embodiments, the linker may be a polymer, such as PEG.

[0300] In some embodiments, the linker is beta-alanine.

[0301] In some embodiments, the peptide is conjugated to an oligonucleotide, through a carboxamide linkage.

[0302] In some embodiments, the peptide is covalently bonded to an oligonucleotide via an aliphatic dicarboxylic acid linker (e.g., succinyl).

[0303] The peptide may be chemically conjugated to the oligonucleotide. Chemical linkage may be via a disulfide, alkenyl, alkynyl, aryl, ether, thioether, triazole, amide, carboxamide, urea, thiourea, semicarbazide, carbazide, hydrazine, oxime, phosphate, phosphoramidate, thiophosphate, boranophosphate, iminophosphates, orthiol-maleimide linkage, for example.

[0304] In some embodiments, cysteine may be added at the terminus of an oligonucleotide to allow for disulfide bond formation to the peptide, or the terminus may undergo bromoacetylation for thioether conjugation to the peptide.

[0305] Examples of linkers that can be used in the conjugates of the invention are noted herein. Additional examples of linkers that can be used are described in WO 2020 / 115494 and WO 2020 / 030927, the contents of each of which are incorporated herein by reference.

[0306] In some embodiments, the linker is of the following structure:

[0307] In some embodiments, the linker is of the following structure: PATENT

[0308] Attorney Docket No.: 51558-023WO2

[0309] In some embodiments, the linker is of the following structure:

[0310] In some embodiments, the linker is of the following structure:

[0311] In some embodiments, the linker is of the following structure:

[0312] In some embodiments, the linker is of the following structure:

[0313] Conjugates

[0314] The peptides of the invention may be covalently linked to an oligonucleotide to provide a conjugate.

[0315] In some embodiments, the peptides of the conjugates are covalently linked directly to an oligonucleotide. Preferably, the oligonucleotide is a morpholino (more preferably, a phosphorodiamidate morpholino oligomer.

[0316] In some embodiments, the conjugate is capable of penetrating into cells and tissues, e.g., into the nucleus of cells, e.g., cells of muscle tissues.

[0317] In some embodiments, the conjugate has the following structure:

[0318] [peptide]-[linker]-[oligonucleotide].

[0319] In some embodiments, the conjugate is of the following structure:

[0320] [oligonucleotide]

[0321] In some embodiments, the conjugate is of the following structure:

[0322] [peptide] [oligonucleotide] PATENT

[0323] Attorney Docket No.: 51558-023WO2

[0324] In some embodiments, the conjugate is of the following structure:

[0325] 0

[0326] [peptide]N [oligonucleotide] H

[0327] In some embodiments, the conjugate is of the following structure:

[0328] O

[0329] [oligonucleotide]

[0330] [peptide]

[0331] O

[0332] In some embodiments, the conjugate is of the following structure:

[0333] O [peptide] [oligonucleotide]

[0334] O O

[0335] In some embodiments, the conjugate is of a peptide and an oligonucleotide covalently bonded (i.e., directly) or linked via a non-cationic linker (e.g., via a linker including a total of one amino acid) or via an aliphatic dicarboxylic acid linker to the peptide, the peptide including a total of one hydrophobic domain and at least one cationic domain, the cationic domain including at least 1 cationic amino acid residue, and the hydrophobic domain including at least 3 amino acid residues, provided that the peptide includes a total of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues.

[0336] In some embodiments, the conjugate is of a peptide and an oligonucleotide covalently bonded (i.e., directly) or linked via a non-cationic linker (e.g., via a linker including a total of one amino acid) or via an aliphatic dicarboxylic acid linker to the peptide, the peptide including a total of one hydrophobic domain and a total of two cationic domains flanking the hydrophobic domain, and the hydrophobic domain including at least 3 amino acid residues, provided that the peptide includes a total of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues.

[0337] Pharmaceutical Compositions

[0338] The conjugate of the invention may be formulated into a pharmaceutical composition.

[0339] In some embodiments, the pharmaceutical composition includes a conjugate of the invention or a pharmaceutically acceptable salt thereof.

[0340] In some embodiments, the pharmaceutical composition may further include a pharmaceutically acceptable diluent, adjuvant, or carrier.

[0341] Suitable pharmaceutically acceptable diluents, adjuvants and carriers are well known in the art.

[0342] It should be understood that the pharmaceutical compositions of the present disclosure can further include additional known therapeutic agents, drugs, modifications of compounds into prodrugs, and the like for alleviating, mediating, preventing, and treating the diseases, disorders, and conditions described herein under medical use.

[0343] 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. All features PATENT

[0344] Attorney Docket No.: 51558-023WO2 described herein in relation to medical treatment using the conjugate apply to the pharmaceutical composition.

[0345] Accordingly, in a further aspect of the invention there is provided a pharmaceutical composition for use as a medicament. In a further aspect, there is provided a method of treating a subject for a disease condition including administering an effective amount of a pharmaceutical composition disclosed herein.

[0346] Methods of Treatment

[0347] The conjugate including a peptide, such as a conjugate including a peptide linked (e.g., via a covalent linkage) to an oligonucleotide or a composition containing the same (e.g., a pharmaceutical composition) may be used as a medicament for the treatment of a disease or condition. In some embodiments, the conjugate is for use in the treatment of diseases caused by splicing deficiencies. In such embodiments, the oligonucleotide may include an oligonucleotide capable of preventing or correcting the splicing defect and / or increasing the production of correctly spliced mRNA molecules. In some embodiments, the conjugate is for use in the treatment of diseases of the neuromuscular system. In some embodiments, the disease or condition is Duchenne muscular dystrophy (DMD).

[0348] A method of treatment of a patient or subject in need of treatment for a disease or condition includes administering a therapeutically effective amount of the conjugate or pharmaceutical composition including the conjugate to a subject or patient in need thereof (e.g., a subject having the disease or condition). In some embodiments, the treatment requires delivery of an oligonucleotide (i.e., an oligonucleotide of the conjugate) into a cell or organelle in a cell (e.g., a nucleus).

[0349] In some embodiments, there is provided a conjugate according to the second aspect for use in the treatment of DMD. In some embodiments, the oligonucleotide of the conjugate is operable to increase expression of the dystrophin protein. In some embodiments, the oligonucleotide of the conjugate is operable to increase the expression of functional dystrophin protein.

[0350] In some embodiments, the conjugate increases dystrophin expression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% relative to no treatment. In some embodiments, the conjugate increases dystrophin expression by up to 50% relative to no treatment. In some embodiments, the conjugate restores dystrophin protein expression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% relative to no treatment. In some embodiments, the conjugate restores dystrophin protein expression by up to 50% relative to no treatment.

[0351] In some embodiments, the conjugate restores dystrophin protein function by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% relative to no treatment. In some embodiments, the conjugate restores dystrophin protein function by up to 50% relative to no treatment.

[0352] In some embodiments, the oligonucleotide of the conjugate is operable to do so by causing skipping of one or more exons during dystrophin transcription.

[0353] In some embodiments, the oligonucleotide of the conjugate causes 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% skipping of one or more exons of the dystrophin gene. In some embodiments, the oligonucleotide of the conjugate causes up to 50% skipping of one or more exons of the dystrophin gene. In some embodiments, the oligonucleotide of the conjugate causes up to 80% of one or more exons of the dystrophin gene. PATENT

[0354] Attorney Docket No.: 51558-023WO2

[0355] In some embodiments, the exon skipped is exon 53.

[0356] 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.

[0357] In some embodiments, the patient or subject to be treated may be any age. In some embodiments, the patient or subject to be treated is aged between 0-40 years, e.g., 0-30 years, 0-25 years, or 0-20 years of age.

[0358] In some embodiments, the patient or subject is one who is amenable to a therapy that induces exon 53 skipping of the DMD gene (i.e., a therapy including administration of a conjugate that induces exon 53 skipping or pharmaceutical composition including the same).

[0359] In some embodiments, the patient or subject is amenable to a therapy that induces exon 53 skipping of DMD and has been determined to have any one of the following DMD gene mutations: a 3- 52 deletion, a 4-52 deletion, a 5-52 deletion, a 6-52 deletion, a 9-52 deletion, a 10-52 deletion, an 11- 52 deletion, a 13-52 deletion, a 14-52 deletion, a 15-52 deletion, a 16-52 deletion, a 17-52 deletion, a 19-52 deletion, a 21-42 deletion, a 23-52 deletion, a 24-52 deletion, a 25-52 deletion, a 26-52 deletion, a 27-52 deletion, a 28-52 deletion, a 29-52 deletion, a 30-52 deletion, a 31-52 deletion, a 32-52 deletion, a 33-52 deletion, a 34-52 deletion, a 35-52 deletion, a 36-52 deletion, a 37-52 deletion, a 38- 52 deletion, a 39-52 deletion, a 40-52 deletion, a 41-52 deletion, a 42-52 deletion, a 43-52 deletion, a 45-52 deletion, a 47-52 deletion, a 48-52 deletion, a 49-52 deletion, a 50-52 deletion, a 52 deletion, a 54-58 deletion, a 54-61 deletion, a 54-61 deletion, a 54-63 deletion, a 54-64 deletion, a 54-66 deletion, a 54-76 deletion, or a 54-77 deletion.

[0360] In some embodiments, the conjugate is for administration to a subject systemically for example by intramedullary, intrathecal, intraventricular, intravitreal, enteral, parenteral, intravenous, intra-arterial, intramuscular, intratumoral, subcutaneous, oral, or nasal routes.

[0361] In some embodiments, the conjugate is for administration to a subject intravenously.

[0362] In some embodiments, the conjugate is for administration to a subject intravenously by injection or infusion.

[0363] The amount of conjugate administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Decisions on dosage are within the responsibility of general practitioners and other medical doctors. Examples of the techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins. Exemplary doses may be between 0.01 mg / kg and 50 mg / kg, 0.05 mg / kg and 40 mg / kg, 0.1 mg / kg and 30 mg / kg, 0.5 mg / kg and 18 mg / kg, 1 mg / kg and 16 mg / kg, 2 mg / kg and 15 mg / kg, 5 mg / kg and 10 mg / kg, 10 mg / kg and 20 mg / kg, 12 mg / kg and 18 mg / kg, or 13 mg / kg and 17 mg / kg.

[0364] In some embodiments, the conjugate is for administration to a subject in a dose volume between 1 mL / kg and 10 mL / kg. Exemplary dose volumes may be between 1 mL / kg and 3 mL / kg, 2 mL / kg and 5 mL / kg, 4 mL / kg and 7 mL / kg, 5 mL / kg and 8 mL / kg, 6 mL / kg and 9 mL / kg, or 7 mL / kg and 10 mL / kg.

[0365] Advantageously, the dosage of the conjugates of the present invention may be lower, e.g., an order or magnitude lower, than the dosage required to see any effect from the oligonucleotide alone. PATENT

[0366] Attorney Docket No.: 51558-023WO2

[0367] In some embodiments, after administration of the conjugates of the present invention, one or more markers of toxicity are significantly reduced compared to prior conjugates using currently available peptide carriers.

[0368] In some embodiments, the conjugate is administered as a monotherapy or in combination with one or more therapeutic interventions for the treatment of the disease or condition.

[0369] EXAMPLES

[0370] Example 1 : MATERIALS AND METHODS

[0371] Reagents and General Methods

[0372] Peptides were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) protected L-amino acids, Fmoc-p-Ala-OH, and rink amide-MBHA resin with (0.53 mmol / g substitution).

[0373] Peptide SEQ ID NO: 9, PMOs (SEQ ID NO: 1 and SEQ ID NO:3), and PPMOs (PPMO-1 and PPMO-3) were manufactured at Wuxi STA Process, R&D lab, China. The preparation procedures of these three types of compounds are as follows.

[0374] A. Peptide synthesis procedure for SEQ ID NO: 9

[0375] Peptide synthesis was performed at 53 mmol scale according to the standard solid-phase peptide synthesis (SPPS) Fmoc chemistry starting from 100 g of rink amide-MBHA resin. Fmoc protecting group was removed using 20% piperdine in DMF. The Fmoc cleavage / deprotection was performed by using 20% piperidine in DMF for 5 min. The subsequent peptide coupling reactions were performed with Fmoc-protected amino acid using DIC as a coupling reagent (2.273.3 equivalents) in the presence of 2.0 / 3.0 equivalents of oxyma for 10-15 min. In case of incomplete coupling, coupling reagent was changed to DIEA (4.0 equivalents) including oxyma (1.0 equivalent), and HBTU (2.0 equivalents) to accelerate the reaction for 30-60 min. The washing steps were performed with DMF for 5-6 times in order to remove the residual amino acids. The progress / completeness of each coupling reaction was monitored by the standard ninhydrin (Kaiser) test.

[0376] After assembly, N-terminal acetylation (capping) was performed using the mixed solution of AC2O / NMM / DMF (5 / 10 / 85) for 30 min. After washing 6 times with 10 vol DMF for 5 min per time, the peptidyl resin was dried in a vacuum oven, affording 330.24 g resin. 80 g of peptidyl resin was taken forward for deprotection. Global deprotection of peptidyl resin was performed using 10 vol TFA / TIS / DTT / H2O = 92.5 / 2.5 / 2.5 / 2.5 as the global deprotection solution by stirring at 15 ± 5 °C for 2.5- 3.5 h. The deprotected peptide was then precipitated by adding the deprotection filtrate into methyl tert butyl ether (MTBE), affording 22.80 g of crude Peptide SEQ ID NO 9 with 74.93% purity. The crude peptide was purified as follows.

[0377] Peptide purification: The crude peptide was dissolved in 8M aqueous urea solution (600 ml). The resulting solution was filtered with Whatman Glass 1 .7 pm microfiber filter and purified by reversephase purification using preparative YMC Triart 200 A C18 column using mobile phases A (50 mM triethylammonium phosphate (TEAP) and B (ACN). The preparative column was equilibrated with 10% mobile phase B. The crude peptide solution was loaded on the column and purified using a gradient of 10% B to 16% B over 75.0 min at 250 ml / min (Gradient steps: 0.0 min, 10%B, 1.0 min 10%B, 57.7 min 16%B, 75.0 min 16%B). The fractions were monitored at UV (214 and 280 nm) and collected. The PATENT

[0378] Attorney Docket No.: 51558-023WO2 main fractions starting with >1000 mAu at 214 nm and finishing at UV (214 nm) < 500 mAu were pooled. The peptide was converted to the corresponding chloride salt by the following procedure.

[0379] Salt conversion procedure: 1 .5 Column volume (CV) H2O was added into the pooled fractions and mixed well. The preparative column was equilibrated with 2% ACN in water. The diluted sample solution was loaded onto the column and flushed with a series of aqueous phase with 2% ACN in water (250 ml / min, 20 min), 2% ACN in mobile phase (0.2M aqueous NaCI 250 ml / min, 30 min), 2% ACN in water. (250 ml / min, 20 min). Final elution was performed with 30% ACN in 0.02% aqueous HCI by monitoring UV at 214 and 280 nm. The pooled peptide fractions as their chloride salt were frozen and lyophilized, affording peptide as white lyophilized powder. The yield and analytical data are as follows.

[0380] Peptide SEQ ID NO 9: Yield 9.39 g (35.27%); Purity, 99.9%, LCMS (692.90 [M+3H]3+, 520.10 [M+4H]4+(target molecular weight ± 2Da).

[0381] B. General synthesis procedure for PMOs SEQ ID NOs: 1-5

[0382] The PMOs were prepared under the non-GMP conditions at 20 g scale. The PMOs SEQ ID NOs: 1-5 were synthesized by manual solid-phase assembly using a 2L jacket column reactor with a filter, and three chemical reactions detritylation, neutralization, and coupling per cycle were conducted to successively incorporate respective PMO subunits onto the solid support from 5' to 3' direction.

[0383] PMOs SEQ ID NOs: 1-5 were synthesized at 12 mmol scale starting from 29.21 g of sarcosinate modified aminomethyl resin with 411 .3 mol / g loading. Before assembling the PMO monomers onto the solid support, all the solutions were prepared in advance as listed below.

[0384] Wash solution: 30% TFE / DCM: 30% Trifluoroethanol in Dichloromethane (3:7, v / v) Detritylation solution: 2% 4-cyanopyridinium trifluoroacetate (CYTFA) (w / v) in 20% TFE / dichloromethane with 1% ethanol

[0385] Neutralization solution: 5% diisopropylethylamine in 25% isopropanol / dichloromethane

[0386] Coupling solution: 0.36M PMO subunits solution in 1 ,3-dimethylimidazolidinone (DMI) and 0.8M N- ethylmorpholine (NEM) in DMI.

[0387] Monomer Solution: The morpholino subunits solution (0.36M) in DMI were treated with molecular sieve over 4 h prior to coupling to reduce the water content.

[0388] PMO solid-phase assembly procedure

[0389] To a reactor with plates was charged the PMO support followed by 1-methyl-2-pyrrolidinone (NMP 10-20 ml / mmol resin), and the suspension was allowed to stir for 30-60 min at 15-25°C. Then, NMP was evacuated, and the resin was washed with DCM three times before detritylation. To assemble each PMO subunit onto the support, four steps were conducted.

[0390] (i) Firstly, to remove the trityl group on the support, 2% 4-cyanopyridinium trifluoroacetate (CYTFA) (w / v) solution was charged into the column reactor. The mixture was stirred for 3-5 min at 15- 25°C and then evacuated to remove the solution. This operation was repeated to 5-15 times until IPC showed almost complete removal of trityl group. The resin was washed once with DCM (500 PATENT

[0391] Attorney Docket No.: 51558-023WO2 mL) and NEM (VDCM:V NEM = 50:3) for 3-5 min.

[0392] (ii) Secondly, after detritylation, the resin was neutralized with 5% DIPEA in IPA / DCM (1 / 3) for 3-5 min.

[0393] (iii) Thirdly, the coupling was conducted by charging the morpholino subunits solution and NEM solution in DMI into the reactor, and the reaction was stirred at 35-40 °C for 1 .5-2.0 h. After assembling the morpholino subunit onto the support, the reaction mixture was evacuated and washed with DCM.

[0394] (iv) In a fourth step, the resin was washed twice with 30% TFE / DCM for 10 min.

[0395] These four steps were repeated until the target sequence was completely assembled. After complete successive rounds (depending on the number of nucleobases in the sequence) of PMO subunits coupling, the PMO bound resin with target sequence was obtained, which was then subjected to cleavage and deprotection to yield the crude PMO solution, which were further purified.

[0396] Cleavage and deprotection procedure of PMO bound solid support: The cleavage and de-protection of PMO bound resin were carried out to yield the target PMO. Three steps of cleavage & de-protection were conducted to cleave the PMO from solid support as well as remove the protecting group on base moiety to yield the crude PMO solution.

[0397] Step 1 : PMO was swelled with NMP for 0.5 h at 35°C; the protected PMO was cleaved from the PMO bound resin using concentrated ammonium hydroxide with 10% (v / v) NMP mixture at 40- 50°C. After filtration, the resulting aqueous PMO solution was obtained. Then, ultrafiltration and diafiltration were performed to remove NH3 H2O, followed by lyophilization to produce the protected PMO crude product.

[0398] Step 2: The protected PMO crude product from step 1 was re-dissolved into NMP and charged into DBU (0.5 mol / L) / DTT (1 mol / L) solution in NMP slowly to remove the nitro-phenylethyl (NPE) group on guanosine at 30-40 °C.

[0399] Step 3: A second aminolysis with 25-28% NH3.H2O at 40-50°C for 16-24 h was conducted to completely isobutyryl-butyl groups on guanosine to give target PMO, and crude PMO solution was obtained by UF&DF to remove small molecules.

[0400] PMO purification procedure: The crude fully deprotected PMO solution was subjected to anion exchange (AEX) chromatography purification using AKTA avant 150-2 instrument from GE. The AEX purification parameters are as follows: Purification Column: FineLINE 100; Purification Resin: Nano Q30L; Bead size 30 pm; Mobile Phase A: 25 mM NaOH, Mobile Phase B: 25 mM NaOH with 0.5 M NaCI; Gradient: Equilibration: 1% B, 2CV, 1-60% 24 CV, 60-100% 2 CV, 1% 2 CV. The AEX fractions with product were pooled and desalted by ultrafiltration and diafiltration (UF&DF). The desalted PMO solution was frozen and lyophilized.

[0401] PMO quantification: PMOs were dissolved in RNase free water and quantified by diluting with 0.1 N HCI and measuring UV absorbance at 265 nm. The concentration was determined using Beer-Lambert law c = (A265) / (E26SI). The yields and detailed analyses of PMOs SEQ ID NOs: 1 and 3 are as follows.

[0402] SEQ ID NO: 1 Yield: 25.39 g (25%); purity 91.4%; Identity by MW: 8420.20 Da (Molecular weight: 8420.20 g / mol); water content 1 .9%; endotoxin 0.12 EU / mg. PATENT

[0403] Attorney Docket No.: 51558-023WO2

[0404] SEQ ID NO: 3 Yield: 31.686 g (26%); purity: 89.2%; Identity by MW: 10120.40 Da (Molecular weight: 10120.40 g / mol); water content: 2.6%; endotoxin: 0.63 EU / mg.

[0405] C. General PPMO conjugation procedure:

[0406] PPMOs 1-5 were synthesized using EDCI / HOBt / DIPEA as condensation agents to conjugate PMO SEQ ID NOs: 1-5 and peptide SEQ ID NO: 9. The reaction mixture was diluted with water and purified by cation exchange (CEX) chromatography. The fractions with pure products were pooled together and desalted using UF & DF. The desalted PPMO solution was frozen and lyophilized.

[0407] General CEX purification procedure for PPMOs 1 -5: The crude PPMO solution was subjected to cation exchange (CEX) chromatography purification using AKTA avant 150-2 instrument from GE. The CEX purification parameters are as follows: Purification Column: FineLINE 100; Purification Resin: NanoSP30L; Bead size 30 pm; Mobile Phase A: 25% ACN, Mobile Phase B: 25% ACN with 0.5 M NaCI; Flowrate: 150 ml / min; Gradient: Equilibration: 15% B, 2CV, Elution: 15-50% 20 CV, 50% 2 CV, 50-100% 2 CV, 15% 2 CV. The CEX fractions with product were pooled and desalted by ultrafiltration and diafiltration (UF&DF). The desalted PPMO solution was frozen and lyophilized.

[0408] Synthesis of PPMO-1

[0409] PPMO-1 was obtained by conjugating PMO SEQ ID NO 1 with peptide SEQ ID NO: 9 following the general PPMO conjugation procedure followed by CEX purification and desalting. Yield: 21 .37 g (67%); purity: 96.5% (UV absorbance at 265 nm); Identity by MW: 10472.50 Da (Molecular Weight: 10474.63 g / mol); water content: 2.6%; endotoxin: <0.177 EU / mg.

[0410] Synthesis of PPMO-3

[0411] PPMO-3 was obtained by conjugating PMO SEQ ID NO: 3 with peptide SEQ ID NO: 9 as described in the general PPMO conjugation procedure followed by CEX purification and desalting. Yield: 23.44 g (61 %); purity: 96.8% (UV absorbance at 265 nm); Identity by MW (Da): 12174.59 da (Molecular weight: 12175.03 g / mol); water content: 3.4%; endotoxin: <0.174 EU / mg.

[0412] Example 2. In Vitro Pharmacologic Profiling of Conjugates that Target Dystrophin Gene

[0413] This Example describes the results of the in vitro pharmacological evaluation of dystrophin (DMD) gene targeting peptide-phosphorodiamidate morpholino oligonucleotide conjugates (PPMOs) in patient-derived DMD Del45-52 and DMD Del52 myotubes, and wild-type (WT) (i.e., nonDMD) control myotubes. Pharmacological activity was evaluated by the ability of the PPMOs to induce exon 53 skipping and compared to an unconjugated oligonucleotide, a control PMO (nucleotide sequence: 5’- GTTGCCTCCGGTTCTGAAGGTGTTC-3’ (SEQ ID NO: 94); references throughout this application to “control PMO” are to this PMO sequence) that has the peptide RBRRBRFQILYBRBR (SEQ ID NO: 9) conjugated to its 3’ end through the gamma carboxyl group of a glutamic acid linker residue (P- control PMO), or positive control PPMO R6Gly-control PMO (i.e., control PMO linked by its 3’ end to a peptide having the amino acid sequence RRRRRRG (SEQ ID NO: 10)). The PPMOs consisted of DMD exon 53-targeting oligonucleotide sequences and a peptide covalently linked by its C-terminus to the 3’ end of the oligonucleotide through the gamma carboxyl group of a glutamic acid linker residue. Each PMO PATENT

[0414] Attorney Docket No.: 51558-023WO2 included the group its 5’ terminus. Sequence details regarding the PPMOs investigated herein are provided in Table 1.

[0415] Table 1 : Structural information regarding PPMOs

[0416] Materials and Methods - DMD patient-derived muscle cells

[0417] Cell Culture

[0418] Wild-type (control) healthy volunteer myoblasts, DMD Del 45-52 myoblasts, and Del52 myoblasts were cultured in a DMD culture medium consisting of four parts high glucose DMEM with GlutaMAX™ (ThermoFisher Scientific, 31966-021) and one part Medium 199, supplemented with 20% heat-inactivated Fetal Bovine Serum, 25 pg / mL Bovine Fetuin, 5 ng / mL Human EGF Recombinant Protein, 0.5 ng / mL Human FGF-basic Recombinant Protein, 5 pg / mL Human Insulin Solution, 0.2 pg / mL Dexamethasone, and 50 pg / mL Gentamicin.

[0419] Cell cultures were maintained in uncoated 75 cm2or 175 cm2culture flasks. Cells were passaged 24 hours post-seeding and thereafter twice weekly when they reached 60-80% confluency by up to 8-minute incubation with pre-warmed 0.05% Trypsin-EDTA after a wash with pre-warmed PBS. Cells were never allowed to pass 80% confluency, as this would initiate differentiation. The flasks were incubated in a humidified incubator at 37°C and 5% CO2.

[0420] Plate Coating

[0421] A mixture of 0.5% MaxGel™ ECM (Merck, E0282-1 ML) thawed at room temperature and 1% Collagen Type I solution from rat tail pre-warmed at 37°C for 5 minutes in a 1 :1 ratio of PBS and high glucose, no glutamine, no phenol red DMEM was prepared. The coating mixture, 50 pL / well, was added to PhenoPlate 96-well Black Tissue Culture Plates (Perkin Elmer, 6055300) and incubated for 3 hours at 37°C and 5% CO2. Thereafter, plates were washed twice with 50 pL / well PBS and dried in a PATENT

[0422] Attorney Docket No.: 51558-023WO2 running LAF hood for 1 .5 to 2 hours. MaxGel™ / Collagen type l-coated plates were used for seeding immediately after drying.

[0423] Cell Seeding and Differentiation Initiation

[0424] On Day 1 , cells were dissociated using 0.05% Trypsin-EDTA, neutralized, collected, and counted using Countess 3 AMQAX2000 Automated Cell Counter (ThermoFisher Scientific™). The cell stock was diluted with DMD culture medium to a concentration of 60,000 cells / mL, and 100 pL / well (containing 6,000 cells) was manually seeded into black MaxGel™ / Collagen type l-coated 96-well plates. Microplates were then incubated in a humidified incubator at 37°C and 5% CO2.

[0425] On Day 2, approximately 20-24 hours after seeding, DMD culture medium was removed, and cells were carefully washed with 100 pL / well pre-warmed PBS using an electronic multichannel pipette at the lowest speed. After removing PBS, DMD differentiation medium consisting of high glucose DMEM with GlutaMAX™ supplemented with 20% heat-inactivated Fetal Bovine Serum, 5 pg / mL Human Insulin Solution, and 50 pg / mL Gentamicin was added to the cells at a volume of 150 pL / well to initiate differentiation.

[0426] PPMO Delivery for Exon Skipping Analysis

[0427] On Day 4 or about 48 hours after initiation of differentiation, PPMO delivery via gymnosis (i.e., in the absence of a transfection reagent) was performed following a concentration response curve (CRC) at concentrations of 30 pM to 0.31 pM, at concentrations of 20 pM to 0.5 pM, or at concentrations of 10 pM to 3 nM. Exon 53 skipping PPMOs were tested and compared to control PMO conjugated with the peptide RBRRBRFQILYBRBR (SEQ ID NO: 9) via the glutamic acid linker (P- control PMO) or exon 53 skipping comparator R6Gly-control PMO (positive control).

[0428] To initiate gymnosis, 50 pL / well of each serial dilution was added to each row of cells containing 150 pL DMD differentiation medium to reach the desired end concentration. DMD differentiation medium (for untreated wells), 50 pL / well, were added to all cell plates as controls. Exon 53 skipping PPMOs were added to both DMD donors Del45-52 and Del52. Two technical replicate plates with identical layout were prepared. Plates were swirled briefly to promote homogenization and subsequently incubated in a humidified incubator at 37°C and 5% CO2.

[0429] Cell Harvest and RNA Isolation

[0430] On Day 8, cell plates were harvested. Differentiation medium containing PPMOs was carefully removed, and cells were washed once with 100 pL / well PBS at room temperature using an electronic multichannel pipette at the lowest dispensing speed. After completely removing the PBS, the cell plates were frozen overnight at -70°C.

[0431] Following frozen storage overnight, cells were thawed at room temperature for approximately fifteen minutes. RNA isolation was performed using the PureLink™ Pro 96 Total RNA Purification Kit (Invitrogen™) according to the manufacturer’s protocol. Briefly, 70% ethanol was prepared by mixing absolute ethanol with RNase-free water. Cells were lysed by adding 150 pL 70% ethanol and 150 pL lysis buffer from the PureLink™ kit supplemented with 40 mM DTT. Lysates were transferred to PureLink™ filter plates, after which the plates were centrifuged for 1-2 minutes at >2,100 x g and the PATENT

[0432] Attorney Docket No.: 51558-023WO2 flowthrough discarded. Subsequently, an additional wash with 500 pL Wash Buffer I and two further washes with 750 pL Wash Buffer II from the PureLink™ kit were performed, whereby each wash step included a centrifugation for 1-2 minutes at >2,100 x g and the flowthrough was discarded. A final drying step was performed where plates were centrifuged for another 10 min at >2,100 x g. RNA elution is achieved by adding 45 pL RNase-free water to the plates, centrifuging for 1-2 minutes at >2,100 and using the eluate for another centrifugation step. The resulting RNA was transferred to 96- well PCR plates. DNase I treatment was performed on the samples by combining 10X DNase I Reaction Buffer with 10X DNase I AMP Grade 1 U / pL and nuclease-free water and adding 12 pL of this DNase I mixture to each sample. Plates were kept at room temperature for fifteen minutes and then incubated at 65°C for ten minutes. RNA concentration for a subset of samples was subsequently measured with the SimpliNano (GE Life Sciences) to confirm similarity in concentration and purity between samples. cDNA Synthesis and Amplification

[0433] RNA samples were kept on ice, and subsequent cDNA synthesis was performed using the Superscript IV First-Strand Synthesis System (ThermoFisher Scientific) according to manufacturer’s protocol. Briefly described, Primer Annealing mixture was prepared by adding a 1 :1 ratio of Random Hexamers to 10 mM dNTP Mix. The reverse transcription (RT) reaction mixture was prepared by adding an equal volume of 100 mM DTT, Ribonuclease Inhibitor and SSIV reverse transcriptase (SSIV RT), followed by addition of 5X SSIV Buffer. A no-RT control was included for each run, where the SSIV RT volume was replaced by nuclease-free water. The Primer Annealing mixture (2 pL) was added to 11 pL RNA for each sample and incubate the mixture at 65°C for 5 minutes. Samples were then cooled on ice for at least 1 minute. Subsequently, 7 pL of RT reaction mixture is added to each sample and plates were incubated in a thermal cycler, in which the samples were incubated at 23°C for ten minutes and then at 52°C for another ten minutes, inactivated at 80°C for ten minutes, and then held at 4°C until further use.

[0434] The resulting cDNA was amplified by PCR. One primer pair per DMD myoblast cell line was used for detection of exon skipping. The primer sequences are outlined in Table 2.

[0435] PATENT

[0436] Attorney Docket No.: 51558-023WO2

[0437] Table 2: Sequences of forward and reserve primers utilized for PCR amplification of cDNA prepared from WT and DMD RNA samples.

[0438] The PCR Master Mix was prepared by adding 25 pL ReadyMix Taq (2X) to 1 pL 100 pM forward and reverse primers (50X), and 13 pL nuclease-free water. Hereafter, 10 pL template DNA (5X) was added to obtain 50 pL per reaction. The PCR Master Mix was added to each treated and control cDNA sample and incubated in a thermal cycler under the conditions described in Table 3. The resulting PCR products were stored at -20° until further automatic electrophoresis using Tapestation was performed.

[0439] Table 3: Thermal cycling conditions for amplification of cDNA prepared from WT and DMD RNA samples.

[0440] Results

[0441] Wild-type patient controls and DMD patient-derived myoblasts were differentiated into myotubes for six days and then were administered varying concentrations of the PPMOs listed in Table 1 or control PMO covalently linked to the peptide RBRRBRFQILYBRBR (SEQ ID NO: 9) (P- control PMO) for 96 hours. Following exposure of myotubes to the PPMOs, the cells were harvested for RT-PCR and agarose gel analysis. The levels of exon skipping in patient-derived DMD Del45-52 myotubes compared to wild-type myotubes induced by the PPMOs of Table 1 and comparator R6Gly- control PMO are shown in Table 4. PPMO-1 and PPMO-2 had higher levels of exon 53 skipping compared to P-control PMO at the lowest concentration 0.31 pM. Similarly, PPMO-1 and PPMO-3 produced greater levels of exon 53 skipping in patient-derived DMD Del52 myotubes compared to P- control PMO at 0.31 pM (Table 5). PATENT

[0442] Attorney Docket No.: 51558-023WO2

[0443] Table 4: DMD exon 53 skipping in wild-type and DMD Del45-52 patient-derived myotubes

[0444] Table 5: DMD exon 53 skipping in wild-type and DMD Del52 patient-derived myotubes Comparisons of the maximum effect and potency of the PPMOs were performed based on data from the in vitro exon 53 skipping in DMD Del52 patient-derived myotubes. Such comparisons demonstrate that PPMO-1 and PPMO-3 exhibited superior exon 53 skipping compared to R6Gly- control PMO (Table 6). Table 6: Maximum effect and potency data of PPMOs PATENT

[0445] Attorney Docket No.: 51558-023WO2

[0446] Next, further evaluation of the efficiency and potency of PPMO-1 and PPMO-3 as compared to R6Gly-control PMO (i.e., control PMO linked by its 3’ end to a peptide having the amino acid sequence RRRRRRG (SEQ ID NO: 10)) were performed using lower concentrations of the PPMOs. PPMO-1 consistently induced higher levels of exon 53 skipping as compared to R6Gly-control PMO across PPMO concentrations ranging from 3 nM to 10 pM, and PPMO-3 induced higher or comparable levels of exon 53 skipping to R6Gly-control PMO in differentiated DMD patient-derived myotubes (Table 7, FIGS. 1-2). Additionally, in differentiated non-DMD patient-derived myotubes, PPMO-1 induced enhanced levels of exon 53 skipping compared to R6Gly- control PMO at all concentrations tested ranging from 0.5 pM to 20 pM, with up to about 16-times greater exon 53 skipping activity observed in myotubes treated with PPMO-1 compared to those treated with equal concentrations of R6Gly- control PMO (FIG. 3).

[0447] Table 7: Efficacy of exon 53 skipping of PPMO-1 and PPMO-3 in patient-derived myotubes

[0448] Example 3: In Vitro Pharmacologic Profiling of Peptides of Conjugates that Target DMD

[0449] This Example describes the results of the in vitro pharmacological evaluation of DMD exon 53- targeting PPMOs and corresponding PMOs (i.e., corresponding oligonucleotide sequence that is not linked to a peptide) in differentiated control, i.e., non-DMD, human-derived myotubes. Particularly, pharmacological activity was evaluated by levels of induced exon 53 skipping following administration of oligonucleotides that were either linked to a peptide (i.e., a carrier) or not linked to a peptide.

[0450] Materials and Methods - Human (non-DMD) primary muscle cells

[0451] Cell Culture

[0452] Control human primary myoblasts (Zen Bio) were cultured in a skeletal muscle cell growth medium (Zen Bio). Cell culture was maintained in uncoated 75 cm2or 175 cm2culture flasks. Cells were passaged 24-48 hours post-seeding and approximately twice weekly thereafter when they reached 60-80% confluency. Cells were never allowed to pass 80% confluency, as this would initiate differentiation. For cell splitting, cells were washed with pre-warmed PBS and detached from flask by PATENT

[0453] Attorney Docket No.: 51558-023WO2 incubation with pre-warmed 0.25% Trypsin / 2.21 mM EDTA for up to 5 minutes. The flasks were incubated in a humidified incubator at 37°C and 5% CO2.

[0454] Cell Seeding and Differentiation Initiation

[0455] On Day 1 , prior to cell seeding, cells were collected from culture flasks, counted using a Countess 3FL, (Invitrogen), and plated into 6-well plates with no adherence matrix with a cell concentration of 400,000 cells / well in skeletal muscle growth medium. The plates were incubated in a humidified incubator at 37°C and 5% CO2. On Day 2, approximately 20-24 hours after seeding, skeletal muscle cell growth medium was refreshed, and cells were allowed to proliferate for up to 4 days with a media refresh approximately every 48 hours. On Day 5, differentiation of myoblasts to myotubes was initiated by addition of differentiation media (Skeletal Muscle Cell Differentiation Medium, Zen Bio) to all wells, and media was refreshed approximately every 48 hours for a total of 6 days of differentiation.

[0456] PPMO Delivery for Exon Skipping Analysis

[0457] On Day 11 , approximately 6 days after initiation of differentiation, myotubes were dosed with PPMO or PMO via gymnosis (i.e., in the absence of a transfection reagent) across a concentration gradient between 0.5 to 20 pM. Exon 53 skipping PPMOs or PMOs were tested and compared to R6Gly- control PMO conjugated PPMO and unconjugated control PMO. Muscle cell differentiation medium was used to reach the desired final concentration of each PPMO or PMO. Muscle cell differentiation medium was used for untreated wells as a control. Three technical replicate plates with identical layout were prepared. Plates were subsequently incubated in a humidified incubator at 37°C and 5% CO2 for 48 hours.

[0458] Cell Harvest and RNA Isolation

[0459] Cells were harvested in accordance to manufacturer’s recommendation with homogenization buffer from Maxwell® RSC simplyRNA Tissue Kit (Promega). On Day 13, cells were harvested from dosed plates for exon skipping analysis. Media was removed and cells were washed with 3mL / well PBS at RT. After completely removing the PBS, cell detachment and homogenization was performed by adding 200 pL Maxwell simplyRNA Tissue Homogenisation Buffer (Promega), supplemented with 20 pL 1 -thioglycerol per 200 pL buffer (Promega). The cell plates were either used straight away or frozen at -80°C for further RNA isolation.

[0460] RNA isolation from cell pellets was performed by adding using 200 pL lysis buffer (Promega) to the pellet and extracting RNA with using a Maxwell® RSC simplyRNA Tissue Kit and a Maxwell RSC 48 extraction machine (Promega) according to manufacturer’s recommendations. Extracted RNA was eluted in up to 50 pL of nuclease-free water. The RNA concentration extracted from all cells pellets, as well as their purity (indicated by 260 / 280 ratio), were measured using a Clariostar plate reader (BMG LabTech). Subsequently, all extracted RNA samples were normalized to 50 ng / pL with nuclease-free water. PATENT

[0461] Attorney Docket No.: 51558-023WO2 cDNA Synthesis and Amplification

[0462] Reverse transcription was performed with extracted RNA from each sample, as well as a positive unskipped control (from primary human skeletal muscle cell culture samples), and a no template control (NTC, nuclease-free water). Briefly, RNA was reverse-transcribed using the High- Capacity cDNA Reverse Transcription Kit (Applied Biosystems), under the thermocycler conditions described in Table 8.

[0463] Table 8: Reverse transcription thermal cycling conditions for cDNA synthesis prepared from human (non-DMD) primary muscle cells.

[0464] RT-PCR and Agarose Gel Based Quantification of Exon Skipping

[0465] Amplification of synthesized cDNA was performed using the reverse transcribed cDNA template from tissue samples and a master mix containing 2X DreamTaq Green Master Mix (ThermoFisher Scientific) and forward and reverse primers (0.5 pM final concentration), and nuclease free water per sample reaction was used. Primers having nucleotide sequences 5’- CATCAAGCAGAAGGCAACAA-3’ (SEQ ID NO: 15) and 5’-GAAGTTTCAGGGCCAAGTCA-3’ (SEQ ID NO: 16) were used in the PCR temperature cycling conditions described in Table 9.

[0466] Table 9. PCR Thermal Cycling Conditions.

[0467] Gel Electrophoresis and Exon Skipping Analysis

[0468] Agarose gels (2%) were prepared using Midori Green Advance Stain (Nippon Genetics). A 50- bp DNA ladder and RT-PCR product were loaded on the agarose gel and run at 150 V for approximately 120 minutes or until an appropriate degree of band separation was achieved. Subsequently, gel image acquisition was performed on resolved gels using a ChemiDoc gel imaging system (BioRad). The expected fragment sizes for the unskipped amplicon are 413 bp and the skipped amplicon is 201 bp. PATENT

[0469] Attorney Docket No.: 51558-023WO2

[0470] Unskipped / native and skipped / Aex53 bands from RT-PCR gels were subjected to densitometry analysis using Imaged software. Densitometry values from band quantification were used to determine DMD exon53 skipping, using the formula:

[0471] Percent DMD exon53 skipping = ([area of skipped fragment] I [area of skipped fragment + area of unskipped fragment]) x 100.

[0472] Results

[0473] Exon 53 skipping in differentiated human myotubes was performed as described above. Equivalent concentrations of PPMO-1 (i.e., an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 1 that is linked to the peptide set forth in SEQ ID NO: 9) and its corresponding PMO (i.e., an oligonucleotide having the nucleic acid sequence SEQ ID NO: 1 without a linked peptide) were prepared. Similarly, R6Gly- control PMO (i.e., the PPMO form of control PMO) and its corresponding PMO (i.e., control PMO without the linked peptide) were prepared. The PPMOs and PMOs were administered to the differentiated myotubes and cultured for 48 hours prior to exon 53 skipping analyses via RT-PCR.

[0474] Both PPMO-1 and R6Gly- control PMO (i.e., PPMO forms) induced higher levels of DMD exon 53 skipping compared to their unconjugated PMO counterparts, indicating that linkage of a peptide sequence significantly improves pharmacokinetics of the oligonucleotide. R6Gly- control PMO induced about 1-fold to about 9-fold higher exon 53 skipping compared to the PMO control PMO counterpart (FIG. 4A). PPMO-1 induced about 8-fold to about 28-fold higher exon 53 skipping compared to its PMO counterpart (FIG. 4B). Moreover, consistent with exon 53 skipping data detailed in Example 2, PPMO-1 exhibited higher levels of exon 53 skipping in human-derived myotubes than R6Gly- control PMO at all concentrations tested.

[0475] Example 4: In Vivo Pharmacologic Profiling of Conjugates that Target DMD

[0476] This Example describes the results of the in vivo pharmacological evaluation of DMD-targeting PPMOs following multiple intravenous infusions. Particularly, pharmacological activity was evaluated by the ability of the PPMOs to induce exon 53 skipping in various tissue samples and compared to positive control PPMO R6Gly- control PMO (i.e., control PMO linked by its 3’ end to a peptide having the amino acid sequence RRRRRRG (SEQ ID NO: 10)).

[0477] Materials and Methods

[0478] Animals

[0479] Twenty-three (23) male cynomolgus monkeys (Macaca fascicuiaris) were used in the experiments described below.

[0480] PPMO Dosing and Administration

[0481] PPMO-1 , PPMO-3, and comparator R6Gly- control PMO were administered once every four weeks (Q4W) on Days 1 , 29 and 57 to male cynomolgous monkeys via intravenous infusions (over 60 minutes). PPMO-1 and PPMO-3 were repeatedly administered at doses of either 10 mg / kg, 30 mg / kg, PATENT

[0482] Attorney Docket No.: 51558-023WO2 or 60 mg / kg. R6Gly- control PMO was administered in a similar manner at a dose of 30 mg / kg. As a vehicle control, equal volumes of phosphate buffered saline was also administered.

[0483] Tissue Collection and RNA Extraction

[0484] Muscle biopsies were performed following the 1stand 2ndadministrations (Day 8 and Day 34, respectively). At 7 days post-first-dose on Day 8, the biceps brachii was biopsied from all dosed animals and at 5 days post-second dose on Day 34, the biceps brachii was biopsied from all 30 mg / kg dosed animals.

[0485] At necropsy, skeletal, smooth and cardiac muscle tissues were collected from all animals. All tissues were placed in RNAIater and stored frozen at <-20°C pending analysis of pharmacologic activity.

[0486] RNA was extracted from sampled tissues. Tissue samples were homogenized using steel- beaded homogenization tubes and a benchtop homogenizer. Homogenization was performed and RNA was extracted using a MAXWELL® RSC48 and Promega simplyRNA Tissue Kit according to manufacturer’s recommendations.

[0487] Extracted RNA was eluted in nuclease-free water. RNA was then qualified for 260 / 280 and quantified in duplicate from all tissue samples, and positive control total cynomolgus monkey RNA. Extracted RNA was normalized with nuclease-free water. cDNA Synthesis, Amplification, and Exon Skipping Analysis

[0488] Methods related to cDNA synthesis and amplification as well as exon skipping analyses via gel electrophoresis were performed as described in Example 3.

[0489] Results

[0490] Exon 53 skipping activity was observed in skeletal muscle tissue at all doses tested following three repeated 60-minute infusions of PPMO-1 , PPMO-3, and comparator R6Gly-control PMO. Exon skipping activity was not observed in animals treated with vehicle-control PBS. In general, a dosedependent increase in exon skipping activity was observed following administration of PPMO-1 and PPMO-3.

[0491] When comparing single versus repeated administration of PPMOs, there was generally no observed increase in exon skipping levels at 10 mg / kg for either PPMO-1 and PPMO-3. However, there was an increase in exon skipping levels following repeat administration of PPMO-1 and R6Gly- control PMO at >30 mg / kg, as compared to the first dose (i.e., single dose). PPMO-3, in contrast, did not show a dose-dependent response in exon 53 skipping between doses of 30 and 60 mg / kg on Day 8 following a single dose or between repeated doses of 30 mg / kg following the second and third doses.

[0492] On day 8, i.e., 7 days after infusion of the first dose of 30 mg / kg PPMO, biceps tissue exhibited exon 53 skipping at levels of 5% following administration of R6Gly- control PMO, 36.4% following administration of PPMO-1 , and 63.8% following administration of PPMO-3 (FIG. 5). On day 34, five days following the second dose of each PPMO, exon 53 skipping in biceps tissue was observed at levels of 18.5% from animals that received R6Gly- control PMO, at levels of 50.4% in PATENT

[0493] Attorney Docket No.: 51558-023WO2 animals that received PPMO-1 , and at levels of 72.5% in animals that received PPMO-3. Similarly, PPMO-1 and PPMO-3 induced higher levels of exon 53 on day 64, i.e., 7 days after the third administration, when compared to an equal dose of R6Gly- control PMO in tissue samples from the biceps and diaphragm (FIGS. 6-7).

[0494] Cardiac (left ventricle) muscle generally exhibited lower levels of exon 53 skipping following systemic infusion of PPMO-1 , PPMO-3, and R6Gly- control PMO, as compared to skeletal muscle. A dose-dependent increase in exon skipping was observed in left ventricle tissue biopsies following 3 Q4W infusions of PPMO-1 and PPMO-3 at day 64 of the treatment cycle, with exon skipping detected at doses of 30 mg / kg and higher for both PPMOs (PPMO-1 and PPMO-3) and comparator R6Gly- control PMO (FIG. 8). On day 64, left ventricle samples from animals that were administered repeated doses of PPMO-3 exhibited exon skipping levels of about 0% at a dose of 10 mg / kg, about 0.8% at a dose of 30 mg / kg, and about 27.6% at a dose of 60 mg / kg. Likewise, on day 64, animals that were administered repeated doses of PPMO-1 exhibited exon skipping levels of about 0.4% at a dose of 10 mg / kg, about 5.2% at a dose of 30 mg / kg, and about 46.1% at a dose of 60 mg / kg. Lastly, on day 64, left ventricle tissue samples from animals administered repeated doses of 30 mg / kg R6Gly- control PMO exhibited exon skipping levels of about 3.3%.

[0495] In conclusion, exon 53 skipping activity was detected at 10 mg / kg or higher in skeletal muscle, and relative levels of this exon skipping activity increased following repeat dosing at 30 mg / kg and above. The highest levels of exon skipping were observed in skeletal muscle and lower levels in cardiac muscle (left ventricle). Additionally, PPMO-1 and PPMO-3 showed higher levels of exon 53 skipping in muscle tissues compared to R6Gly- control PMO.

[0496] Specific Embodiments

[0497] Several non-limiting, exemplary embodiments of the disclosure are enumerated below. The below embodiments should not be construed to limit the scope of the invention, rather, the below are presented as some examples of the invention.

[0498] 1 . A conjugate, or a pharmaceutically acceptable salt thereof, comprising a peptide consisting of 40 or fewer amino acids, and an oligonucleotide covalently bonded or linked via a non-cationic linker to the peptide, wherein the peptide comprises a total of one hydrophobic domain and a total of two cationic domains flanking the hydrophobic domain, further wherein the hydrophobic domain comprises at least 5 amino acids, and each cationic domain independently comprises at least one cationic amino acid, wherein the peptide is optionally N-acetylated, N-methylated, N-trifluoroacetylated, N- trifluoromethylsulfonylated, or N-methylsulfonylated; and wherein the oligonucleotide comprises a sequence selected from the group consisting of:

[0499] (a) 5’-CTGAAGGTGTTCTTGTACTTCATCC-3’ (SEQ ID NO: 1);

[0500] (b) 5’-CAACTGTTGCCTCCGGTTCTGAAGGTGTTC-3’ (SEQ ID NO: 2);

[0501] (c) 5’-TTGCCTCCGGTTCTGAAGGTGTTCTTGTAC-3’ (SEQ ID NO: 3);

[0502] (d) 5’-CTGTTGCCTCCGGTTCTGAAGGTGTTCTTG-3’ (SEQ ID NO: 4); and

[0503] (e) 5’-CATTCAACTGTTGCCTCCGGTTCTGAAGGTG-3’ (SEQ ID NO: 5). PATENT

[0504] Attorney Docket No.: 51558-023WO2

[0505] 2. The conjugate or pharmaceutically acceptable salt of embodiment 1 , wherein the peptide is acetylated at its N-terminus.

[0506] 3. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence FQILY (SEQ ID NO: 6).

[0507] 4. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence YQFLI (SEQ ID NO: 52).

[0508] 5. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence ILFQY (SEQ ID NO: 53).

[0509] 6. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence FQIY (SEQ ID NO: 54).

[0510] 7. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence WWW.

[0511] 8. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence WWPWW (SEQ ID NO: 55).

[0512] 9. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence WPWW (SEQ ID NO: 56).

[0513] 10. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence WWPW (SEQ ID NO: 57).

[0514] 11 . The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence ILFQ (SEQ ID NO: 58).

[0515] 12. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence ILIQ (SEQ ID NO: 59).

[0516] 13. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence IKILFQN (SEQ ID NO: 60).

[0517] 14. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence IHILFQN (SEQ ID NO: 61).

[0518] 15. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence IRILFQN (SEQ ID NO: 62).

[0519] 16. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence IILFQN (SEQ ID NO: 63).

[0520] 17. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence KILFQN (SEQ ID NO: 64).

[0521] 18. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence HILFQN (SEQ ID NO: 65).

[0522] 19. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence RILFQN (SEQ ID NO: 66).

[0523] 20 The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence ILFQN (SEQ ID NO: 67).

[0524] 21 . The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence HLIQN (SEQ ID NO: 68). PATENT

[0525] Attorney Docket No.: 51558-023WO2

[0526] 22. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence KILIQN (SEQ ID NO: 69).

[0527] 23. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence KILIQY (SEQ ID NO: 70.

[0528] 24. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence HILIQN (SEQ ID NO: 71).

[0529] 25. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence RILIQN (SEQ ID NO: 72).

[0530] 26. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence HILIQY (SEQ ID NO: 73).

[0531] 27. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence RILIQY (SEQ ID NO: 74).

[0532] 28. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence ILIQN (SEQ ID NO: 75).

[0533] 29. The conjugate or pharmaceutically acceptable salt of embodiment 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence ILIQY (SEQ ID NO: 76).

[0534] 30. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -29, wherein at least one cationic domain is arginine rich.

[0535] 31 . The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -30, wherein at least one cationic domain is histidine rich.

[0536] 32. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -31 , wherein at least one of the cationic domains comprises the amino acid sequence RBRRBRR (SEQ ID NO: 77).

[0537] 33. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -32, wherein at least one of the cationic domains comprises the amino acid sequence RBRBR (SEQ ID NO: 78).

[0538] 34. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -33, wherein at least one of the cationic domains comprises the amino acid sequence RBRR (SEQ ID NO: 79).

[0539] 35. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -34, wherein at least one of the cationic domains comprises the amino acid sequence RBRRBR (SEQ ID NO: 7).

[0540] 36. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -35, wherein at least one of the cationic domains comprises the amino acid sequence RRBRBR (SEQ ID NO: 80).

[0541] 37. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -36, wherein at least one of the cationic domains comprises the amino acid sequence RBRRB (SEQ ID NO: 81).

[0542] 38. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -37, wherein at least one of the cationic domains comprises the amino acid sequence BRBR (SEQ ID NO: 8). PATENT

[0543] Attorney Docket No.: 51558-023WO2

[0544] 39. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -38, wherein at least one of the cationic domains comprises the amino acid sequence RBHBH (SEQ ID NO: 82).

[0545] 40. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -39, wherein at least one of the cationic domains comprises the amino acid sequence HBHBR (SEQ ID NO: 83).

[0546] 41 . The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -40, wherein at least one of the cationic domains comprises the amino acid sequence RBRHBHR (SEQ ID NO: 84).

[0547] 42. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-41 , wherein at least one of the cationic domains comprises the amino acid sequence RBRBBHR (SEQ ID NO: 85).

[0548] 43. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-42, wherein at least one of the cationic domains comprises the amino acid sequence RBRRBH (SEQ ID NO: 86).

[0549] 44. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-43, wherein at least one of the cationic domains comprises the amino acid sequence HBRRBR (SEQ ID NO: 87).

[0550] 45. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-44, wherein at least one of the cationic domains comprises the amino acid sequence HBHBH (SEQ ID NO: 88).

[0551] 46. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-45, wherein at least one of the cationic domains comprises the amino acid sequence BHBH (SEQ ID NO:

[0552] 89).

[0553] 47. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-46, wherein at least one of the cationic domains comprises the amino acid sequence BRBSB (SEQ ID NO:

[0554] 90).

[0555] 48. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -47, wherein at least one of the cationic domains comprises the amino acid sequence BRB[Hyp]B (SEQ ID NO: 91).

[0556] 49. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -48, wherein at least one of the cationic domains comprises the amino acid sequence R[Hyp]H[Hyp]HB (SEQ ID NO: 92).

[0557] 50. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -49, wherein at least one of the cationic domains comprises the amino acid sequence R[Hyp]RR[Hyp]R (SEQ ID NO: 93).

[0558] 51 . The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -50, wherein at least one of the cationic domains comprises the amino acid sequence RBR.

[0559] 52. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -51 , wherein at least one of the cationic domains comprises the amino acid sequence RXR. PATENT

[0560] Attorney Docket No.: 51558-023WO2

[0561] 53. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -52, wherein at least one of the cationic domains comprises the amino acid sequence XXR.

[0562] 54. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -53, wherein at least one of the cationic domains comprises the amino acid sequence XRR.

[0563] 55. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -54, wherein at least one of the cationic domains comprises the amino acid sequence RRX.

[0564] 56. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -55, wherein at least one of the cationic domains comprises the amino acid sequence BXR.

[0565] 57. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -56, wherein at least one of the cationic domains comprises the amino acid sequence RXB.

[0566] 58. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -57, wherein at least one of the cationic domains comprises the amino acid sequence XRB.

[0567] 59. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -58, wherein at least one of the cationic domains comprises the amino acid sequence RBB.

[0568] 60. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -59, wherein at least one of the cationic domains comprises the amino acid sequence BRB.

[0569] 61 . The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -60, wherein at least one of the cationic domains comprises the amino acid sequence BBR.

[0570] 62. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -61 , wherein at least one of the cationic domains comprises the amino acid sequence RRB.

[0571] 63. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -62, wherein at least one of the cationic domains comprises the amino acid sequence BRR.

[0572] 64. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -63, wherein at least one of the cationic domains comprises the amino acid sequence BRX.

[0573] 65. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -64, wherein at least one of the cationic domains comprises the amino acid sequence R.

[0574] 66. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -65, wherein at least one of the cationic domains comprises the amino acid sequence H.

[0575] 67. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -66, wherein at least one of the cationic domains comprises the amino acid sequence B.

[0576] 68. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -67, wherein at least one of the cationic domains comprises the amino acid sequence RR.

[0577] 69. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -68, wherein at least one of the cationic domains comprises the amino acid sequence HH.

[0578] 70. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -69, wherein at least one of the cationic domains comprises the amino acid sequence BB.

[0579] 71 . The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -70, wherein at least one of the cationic domains comprises the amino acid sequence RH.

[0580] 72. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -71 , wherein at least one of the cationic domains comprises the amino acid sequence HR. PATENT

[0581] Attorney Docket No.: 51558-023WO2

[0582] 73. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -72, wherein at least one of the cationic domains comprises the amino acid sequence RB.

[0583] 74. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -73, wherein at least one of the cationic domains comprises the amino acid sequence BR.

[0584] 75. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -74, wherein at least one of the cationic domains comprises the amino acid sequence HB.

[0585] 76. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -75, wherein at least one of the cationic domains comprises the amino acid sequence BH.

[0586] 77. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -76, wherein at least one of the cationic domains comprises the amino acid sequence RBR.

[0587] 78. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -77, wherein at least one of the cationic domains comprises the amino acid sequence RBB.

[0588] 79. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -78, wherein at least one of the cationic domains comprises the amino acid sequence BRR.

[0589] 80. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -79, wherein at least one of the cationic domains comprises the amino acid sequence BBR.

[0590] 81 . The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -80, wherein at least one of the cationic domains comprises the amino acid sequence BRB.

[0591] 82. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -81 , wherein at least one of the cationic domains comprises the amino acid sequence RBH.

[0592] 83. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -82, wherein at least one of the cationic domains comprises the amino acid sequence RHB.

[0593] 84. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -83, wherein at least one of the cationic domains comprises the amino acid sequence HRB.

[0594] 85. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -84, wherein at least one of the cationic domains comprises the amino acid sequence BRH.

[0595] 86. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -85, wherein at least one of the cationic domains comprises the amino acid sequence HRR.

[0596] 87. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -86, wherein at least one of the cationic domains comprises the amino acid sequence RRH.

[0597] 88. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -87, wherein at least one of the cationic domains comprises the amino acid sequence HRH.

[0598] 89. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -88, wherein at least one of the cationic domains comprises the amino acid sequence HBB.

[0599] 90. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -89, wherein at least one of the cationic domains comprises the amino acid sequence BBH.

[0600] 91 . The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -90, wherein at least one of the cationic domains comprises the amino acid sequence RHR.

[0601] 92. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -91 , wherein at least one of the cationic domains comprises the amino acid sequence BHB. PATENT

[0602] Attorney Docket No.: 51558-023WO2

[0603] 93. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1 -92, wherein at least one of the cationic domains comprises the amino acid sequence HBH.

[0604] 94. The conjugate or pharmaceutically acceptable salt of embodiment 1 , wherein the peptide comprises an artificial amino acid.

[0605] 95. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRRBR (SEQ ID NO: 27), wherein X is aminohexanoic acid.

[0606] 96. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRRBRX (SEQ ID NO: 28), wherein X is aminohexanoic acid.

[0607] 97. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRXRRXRRXRX (SEQ ID NO: 29), wherein X is aminohexanoic acid.

[0608] 98. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRFQILYRBRXR (SEQ ID NO: 30), wherein X is aminohexanoic acid.

[0609] 99. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRILFQYRXRBRXR (SEQ ID NO: 31), wherein X is aminohexanoic acid.

[0610] 100. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRILFQYRXRXRXR (SEQ ID NO: 32), wherein X is aminohexanoic acid.

[0611] 101 . The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRXRILFQYRXRRXR (SEQ ID NO: 33), wherein X is aminohexanoic acid.

[0612] 102. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RBRRXRRBRILFQYRBRXRBR (SEQ ID NO: 34), wherein X is aminohexanoic acid.

[0613] 103. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RBRRXRRBRILFQYRXRBRXR (SEQ ID NO: 35), wherein X is aminohexanoic acid.

[0614] 104. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RBRRXRRBRILFQYRXRRXR (SEQ ID NO: 36), wherein X is aminohexanoic acid.

[0615] 105. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RBRRXRRBRILFQYRXRBRX (SEQ ID NO: 37), wherein X is aminohexanoic acid.

[0616] 106. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRILFQYRXRRXR (SEQ ID NO: 38), wherein X is aminohexanoic acid. PATENT

[0617] Attorney Docket No.: 51558-023WO2

[0618] 107. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRILFQYRXRBRX (SEQ ID NO: 39), wherein X is aminohexanoic acid.

[0619] 108. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRYQFLIRXRBRXR (SEQ ID NO: 40), wherein X is aminohexanoic acid.

[0620] 109. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRIQFLIRXRBRXR (SEQ ID NO: 41), wherein X is aminohexanoic acid.

[0621] 110. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRQFLIRXRBRXR (SEQ ID NO: 42), wherein X is aminohexanoic acid.

[0622] 111. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRQFLRXRBRXR (SEQ ID NO: 43), wherein X is aminohexanoic acid.

[0623] 112. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXYRFLIRXRBRXR (SEQ ID NO: 44), wherein X is aminohexanoic acid.

[0624] 113. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRFQILYRXRBRXR (SEQ ID NO: 45), wherein X is aminohexanoic acid.

[0625] 114. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXYRFRLIXRBRXR (SEQ ID NO: 46), wherein X is aminohexanoic acid.

[0626] 115. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXILFRYRXRBRXR (SEQ ID NO: 47), wherein X is aminohexanoic acid.

[0627] 116. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of RXRRBRRXRIYQFLIRXRBRXR (SEQ ID NO: 48), wherein X is aminohexanoic acid.

[0628] 117. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of YQFLIRBRRXRBRXBRXRBYQFLI (SEQ ID NO: 49), wherein X is aminohexanoic acid.

[0629] 118. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of YQFLIRBRRBRBRBRRBYQFLI (SEQ ID NO: 50), wherein X is aminohexanoic acid.

[0630] 119. The conjugate or pharmaceutically acceptable salt of embodiment 94, wherein the peptide comprises an amino acid sequence of YQFLIRBRRBRBRBBRXRBYQFLI (SEQ ID NO: 51), wherein X is aminohexanoic acid.

[0631] 120. The conjugate or pharmaceutically acceptable salt of embodiment 1 , wherein the peptide consists of natural amino acid residues. PATENT

[0632] Attorney Docket No.: 51558-023WO2

[0633] 121 . The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RBRRBRFQILYBRBR (SEQ ID NO: 9).

[0634] 122. The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RBRRBRRFQILYRBRBR (SEQ ID NO: 17).

[0635] 123. The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RBRRBRRYQFLIRBRBR (SEQ ID NO: 18).

[0636] 124. The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RBRRBRRILFQYRBRBR (SEQ ID NO: 19).

[0637] 125. The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RBRRBRRFQILYRBHBH (SEQ ID NO: 20).

[0638] 126. The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RBRRBRRFQILYHBHBR (SEQ ID NO: 21).

[0639] 127. The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RBRRBRFQILYRBHBH (SEQ ID NO: 22).

[0640] 128. The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RRRRR (SEQ ID NO: 23).

[0641] 129. The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RRRRRR (SEQ ID NO: 24).

[0642] 130. The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RRRRRRR (SEQ ID NO: 25).

[0643] 131. The conjugate or pharmaceutically acceptable salt of embodiment 120, wherein the peptide comprises an amino acid sequence of RRRRRRRR (SEQ ID NO: 26).

[0644] 132. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-131 , wherein the linker comprises at least one amino acid selected from glutamic acid, beta-alanine, glycine, delta-aminovaleric acid, and gamma-aminobutyric acid.

[0645] 133. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-132, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0646] 134. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-132, wherein the oligonucleotide is a peptide nucleic acid (PNA).

[0647] 135. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-134, wherein the oligonucleotide comprises a sequence of 5’-CTGAAGGTGTTCTTGTACTTCATCC-3’ (SEQ ID NO: 1).

[0648] 136. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-134, wherein the oligonucleotide comprises a sequence of 5’- CAACTGTTGCCTCCGGTTCTGAAGGTGTTC-3’ (SEQ ID NO: 2).

[0649] 137. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-134, wherein the oligonucleotide comprises a sequence of 5’- TTGCCTCCGGTTCTGAAGGTGTTCTTGTAC-3’ (SEQ ID NO: 3).

[0650] 138. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-134, wherein the oligonucleotide comprises a sequence of 5’- CTGTTGCCTCCGGTTCTGAAGGTGTTCTTG-3’ (SEQ ID NO: 4). PATENT

[0651] Attorney Docket No.: 51558-023WO2

[0652] 139. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-138, wherein the oligonucleotide comprises the following group at its 5’ terminus:

[0653] 140. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-138, wherein the oligonucleotide comprises the following group at its 5’ terminus:

[0654] 141 . The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-140, wherein the linker is a glutamic acid linked to the oligonucleotide via a gamma carboxylic acid group and has the following structure:

[0655] 142. The conjugate or pharmaceutically acceptable salt of any one of embodiments 1-141 , wherein the conjugate is of the following structure:

[0656] [oligonucleotide]

[0657] 143. A pharmaceutical composition comprising the conjugate of any one of embodiments 1- 142, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0658] 144. A method of treating a human subject having a Duchenne muscular dystrophy (DMD) genotype that is amenable to exon 53 skipping in a human dystrophin (DMD) gene, the method comprising administering to the subject a therapeutically effective amount of the conjugate of any one of embodiments 1-142, or pharmaceutically acceptable salt thereof, or the pharmaceutical composition of embodiment 143.

[0659] 145. The method of embodiment 144, wherein the genotype is a 3-52 deletion DMD mutation.

[0660] 146. The method of embodiment 144, wherein the genotype is a 4-52 deletion DMD mutation.

[0661] 147. The method of embodiment 144, wherein the genotype is a 5-52 deletion DMD mutation.

[0662] 148. The method of embodiment 144, wherein the genotype is a 6-52 deletion DMD mutation. PATENT

[0663] Attorney Docket No.: 51558-023WO2

[0664] 149. The method of embodiment 144, wherein the genotype is a 9-52 deletion DMD mutation.

[0665] 150. The method of embodiment 144, wherein the genotype is a 10-52 deletion DMD mutation.

[0666] 151. The method of embodiment 144, wherein the genotype is an 11 -52 deletion DMD mutation.

[0667] 152. The method of embodiment 144, wherein the genotype is a 13-52 deletion DMD mutation.

[0668] 153. The method of embodiment 144, wherein the genotype is a 14-52 deletion DMD mutation.

[0669] 154. The method of embodiment 144, wherein the genotype is a 15-52 deletion DMD mutation.

[0670] 155. The method of embodiment 144, wherein the genotype is a 16-52 deletion DMD mutation.

[0671] 156. The method of embodiment 144, wherein the genotype is a 17-52 deletion DMD mutation.

[0672] 157. The method of embodiment 144, wherein the genotype is a 19-52 deletion DMD mutation.

[0673] 158. The method of embodiment 144, wherein the genotype is a 21-42 deletion DMD mutation.

[0674] 159. The method of embodiment 144, wherein the genotype is a 23-52 deletion DMD mutation.

[0675] 160. The method of embodiment 144, wherein the genotype is a 24-52 deletion DMD mutation.

[0676] 161 . The method of embodiment 144, wherein the genotype is a 25-52 deletion DMD mutation.

[0677] 162. The method of embodiment 144, wherein the genotype is a 26-52 deletion DMD mutation.

[0678] 163. The method of embodiment 144, wherein the genotype is a 27-52 deletion DMD mutation.

[0679] 164. The method of embodiment 144, wherein the genotype is a 28-52 deletion DMD mutation.

[0680] 165. The method of embodiment 144, wherein the genotype is a 29-52 deletion DMD mutation.

[0681] 166. The method of embodiment 144, wherein the genotype is a 30-52 deletion DMD mutation.

[0682] 167. The method of embodiment 144, wherein the genotype is a 31-52 deletion DMD mutation.

[0683] 168. The method of embodiment 144, wherein the genotype is a 32-52 deletion DMD mutation.

[0684] 169. The method of embodiment 144, wherein the genotype is a 33-52 deletion DMD mutation. PATENT

[0685] Attorney Docket No.: 51558-023WO2

[0686] 170. The method of embodiment 144, wherein the genotype is a 34-52 deletion DMD mutation.

[0687] 171. The method of embodiment 144, wherein the genotype is a 35-52 deletion DMD mutation.

[0688] 172. The method of embodiment 144, wherein the genotype is a 36-52 deletion DMD mutation.

[0689] 173. The method of embodiment 144, wherein the genotype is a 37-52 deletion DMD mutation.

[0690] 174. The method of embodiment 144, wherein the genotype is a 38-52 deletion DMD mutation.

[0691] 175. The method of embodiment 144, wherein the genotype is a 39-52 deletion DMD mutation.

[0692] 176. The method of embodiment 144, wherein the genotype is a 40-52 deletion DMD mutation.

[0693] 177. The method of embodiment 144, wherein the genotype is a 41-52 deletion DMD mutation.

[0694] 178. The method of embodiment 144, wherein the genotype is a 42-52 deletion DMD mutation.

[0695] 179. The method of embodiment 144, wherein the genotype is a 43-52 deletion DMD mutation.

[0696] 180. The method of embodiment 144, wherein the genotype is a 45-52 deletion DMD mutation.

[0697] 181. The method of embodiment 144, wherein the genotype is a 47-52 deletion DMD mutation.

[0698] 182. The method of embodiment 144, wherein the genotype is a 48-52 deletion DMD mutation.

[0699] 183. The method of embodiment 144, wherein the genotype is a 49-52 deletion DMD mutation.

[0700] 184. The method of embodiment 144, wherein the genotype is a 50-52 deletion DMD mutation.

[0701] 185. The method of embodiment 144, wherein the genotype is a 52 deletion DMD mutation.

[0702] 186. The method of embodiment 144, wherein the genotype is a 54-58 deletion DMD mutation.

[0703] 187. The method of embodiment 144, wherein the genotype is a 54-61 deletion DMD mutation.

[0704] 188. The method of embodiment 144, wherein the genotype is a 54-61 deletion DMD mutation.

[0705] 189. The method of embodiment 144, wherein the genotype is a 54-63 deletion DMD mutation.

[0706] 190. The method of embodiment 144, wherein the genotype is a 54-64 deletion DMD mutation. PATENT

[0707] Attorney Docket No.: 51558-023WO2

[0708] 191. The method of embodiment 144, wherein the genotype is a 54-66 deletion DMD mutation.

[0709] 192. The method of embodiment 144, wherein the genotype is a 54-76 deletion DMD mutation. 193. The method of embodiment 144, wherein the genotype is a 54-77 deletion DMD mutation.

[0710] Other Embodiments

[0711] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0712] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations following, in general, the principles and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0713] Other embodiments are within the claims.

Claims

PATENTAttorney Docket No.: 51558-023WO2CLAIMS1 . A conjugate, or a pharmaceutically acceptable salt thereof, comprising a peptide consisting of 40 or fewer amino acids, and an oligonucleotide covalently bonded or linked via a non-cationic linker to the peptide, wherein the peptide comprises a total of one hydrophobic domain and a total of two cationic domains flanking the hydrophobic domain, further wherein the hydrophobic domain comprises at least 5 amino acids, and each cationic domain independently comprises at least one cationic amino acid, wherein the peptide is optionally N-acetylated, N-methylated, N-trifluoroacetylated, N- trifluoromethylsulfonylated, or N-methylsulfonylated; and wherein the oligonucleotide comprises a sequence selected from the group consisting of:(a) 5’-CTGAAGGTGTTCTTGTACTTCATCC-3’ (SEQ ID NO: 1);(b) 5’-CAACTGTTGCCTCCGGTTCTGAAGGTGTTC-3’ (SEQ ID NO: 2);(c) 5’-TTGCCTCCGGTTCTGAAGGTGTTCTTGTAC-3’ (SEQ ID NO: 3);(d) 5’-CTGTTGCCTCCGGTTCTGAAGGTGTTCTTG-3’ (SEQ ID NO: 4); and(e) 5’-CATTCAACTGTTGCCTCCGGTTCTGAAGGTG-3’ (SEQ ID NO: 5).

2. The conjugate or pharmaceutically acceptable salt of claim 1 , wherein the peptide is acetylated at its N-terminus.

3. The conjugate or pharmaceutically acceptable salt of claim 1 or 2, wherein the hydrophobic domain comprises the amino acid sequence FQILY (SEQ ID NO: 6), YQFLI (SEQ ID NO: 52), ILFQY (SEQ ID NO: 53), FQIY (SEQ ID NO: 54), WWW, WWPWW (SEQ ID NO: 55), WPWW (SEQ ID NO: 56), WWPW (SEQ ID NO: 57), ILFQ (SEQ ID NO: 58), ILIQ (SEQ ID NO: 59), IKILFQN (SEQ ID NO: 60), IHILFQN (SEQ ID NO: 61), IRILFQN (SEQ ID NO: 62), IILFQN (SEQ ID NO: 63), KILFQN (SEQ ID NO: 64), HILFQN (SEQ ID NO: 65), RILFQN (SEQ ID NO: 66), ILFQN (SEQ ID NO: 67), HLIQN (SEQ ID NO: 68), KILIQN (SEQ ID NO: 69), KILIQY (SEQ ID NO: 70), HILIQN (SEQ ID NO: 71), RILIQN (SEQ ID NO: 72), HILIQY (SEQ ID NO: 73), RILIQY (SEQ ID NO: 74), ILIQN (SEQ ID NO: 75), or ILIQY (SEQ ID NO: 76).

4. The conjugate or pharmaceutically acceptable salt of any one of claims 1 -3, wherein at least one cationic domain is arginine rich.

5. The conjugate or pharmaceutically acceptable salt of any one of claims 1 -4, wherein at least one cationic domain is histidine rich.

6. The conjugate or pharmaceutically acceptable salt of any one of claims 1 -5, wherein at least one of the cationic domains comprises the amino acid sequence: RBRRBRR (SEQ ID NO: 77), RBRBR (SEQ ID NO: 78), RBRR (SEQ ID NO: 79), RBRRBR (SEQ ID NO: 7), RRBRBR (SEQ ID NO: 80), RBRRB (SEQ ID NO: 81), BRBR (SEQ ID NO: 8), RBHBH (SEQ ID NO: 82), HBHBR (SEQPATENTAttorney Docket No.: 51558-023WO2ID NO: 83), RBRHBHR (SEQ ID NO: 84), RBRBBHR (SEQ ID NO: 85), RBRRBH (SEQ ID NO: 86), HBRRBR (SEQ ID NO: 87), HBHBH (SEQ ID NO: 88), BHBH (SEQ ID NO: 89), BRBSB (SEQ ID NO: 90), BRB[Hyp]B (SEQ ID NO: 91), R[Hyp]H[Hyp]HB (SEQ ID NO: 92), R[Hyp]RR[Hyp]R (SEQ ID NO: 93), RBR, RXR, XXR, XRR, RRX, BXR, RXB, XRB, RBB, BRB, BBR, RRB, BRR, BRX, 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.

7. The conjugate or pharmaceutically acceptable salt of any one of claims 1 -6, wherein at least one of the cationic domains comprises the amino acid sequence RBRRBR (SEQ ID NO: 7) or BRBR (SEQ ID NO: 8).

8. The conjugate or pharmaceutically acceptable salt of claim 1 , wherein the peptide comprises an artificial amino acid.

9. The conjugate or pharmaceutically acceptable salt of claim 8, wherein the peptide comprises an amino acid sequence selected from the group consisting of:RXRRBRRXRRBR (SEQ ID NO: 27); RXRRBRRXRRBRX (SEQ ID NO: 28);RXRRXRRXRRXRX (SEQ ID NO: 29); RXRRBRRFQILYRBRXR (SEQ ID NO: 30);RXRRBRRXRILFQYRXRBRXR (SEQ ID NO: 31); RXRRBRRXRILFQYRXRXRXR (SEQ ID NO: 32); RXRRXRILFQYRXRRXR (SEQ ID NO: 33); RBRRXRRBRILFQYRBRXRBR (SEQ ID NO: 34);RBRRXRRBRILFQYRXRBRXR (SEQ ID NO: 35); RBRRXRRBRILFQYRXRRXR (SEQ ID NO: 36); RBRRXRRBRILFQYRXRBRX (SEQ ID NO: 37); RXRRBRRXRILFQYRXRRXR (SEQ ID NO: 38); RXRRBRRXRILFQYRXRBRX (SEQ ID NO: 39); RXRRBRRXRYQFLIRXRBRXR (SEQ ID NO: 40); RXRRBRRXRIQFLIRXRBRXR (SEQ ID NO: 41); RXRRBRRXRQFLIRXRBRXR (SEQ ID NO: 42); RXRRBRRXRQFLRXRBRXR (SEQ ID NO: 43); RXRRBRRXYRFLIRXRBRXR (SEQ ID NO: 44); RXRRBRRXRFQILYRXRBRXR (SEQ ID NO: 45); RXRRBRRXYRFRLIXRBRXR (SEQ ID NO: 46); RXRRBRRXILFRYRXRBRXR (SEQ ID NO: 47); RXRRBRRXRIYQFLIRXRBRXR (SEQ ID NO: 48); YQFLIRBRRXRBRXBRXRBYQFLI (SEQ ID NO: 49); YQFLIRBRRBRBRBRRBYQFLI (SEQ ID NO: 50); and YQFLIRBRRBRBRBBRXRBYQFLI (SEQ ID NO: 51), wherein X is aminohexanoic acid.

10. The conjugate or pharmaceutically acceptable salt of claim 1 , wherein the peptide consists of natural amino acid residues.11 . The conjugate or pharmaceutically acceptable salt of claim 10, wherein the peptide comprises an amino acid sequence selected from the group consisting of:(a) RBRRBRFQILYBRBR (SEQ ID NO: 9);(b) RBRRBRRFQILYRBRBR (SEQ ID NO: 17);(c) RBRRBRRYQFLIRBRBR (SEQ ID NO: 18);(d) RBRRBRRILFQYRBRBR (SEQ ID NO: 19);PATENTAttorney Docket No.: 51558-023WO2(e) RBRRBRRFQILYRBHBH (SEQ ID NO: 20);(f) RBRRBRRFQILYHBHBR (SEQ ID NO: 21);(g) RBRRBRFQILYRBHBH (SEQ ID NO: 22);(h) RRRRR (SEQ ID NO: 23);(i) RRRRRR (SEQ ID NO: 24);0) RRRRRRR (SEQ ID NO: 25); or(k) RRRRRRRR (SEQ ID NO: 26).

12. The conjugate or pharmaceutically acceptable salt of any one of claims 1-7, 10, and 11 , wherein the peptide comprises the amino acid sequence RBRRBRFQILYBRBR (SEQ ID NO: 9).

13. The conjugate or pharmaceutically acceptable salt of any one of claims 1-12, wherein the linker comprises at least one amino acid selected from glutamic acid, beta-alanine, glycine, delta- aminovaleric acid, and gamma-aminobutyric acid.

14. The conjugate or pharmaceutically acceptable salt of any one of claims 1-13, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

15. The conjugate or pharmaceutically acceptable salt of any one of claims 1-13, wherein the oligonucleotide is a peptide nucleic acid (PNA).

16. The conjugate or pharmaceutically acceptable salt of any one of claims 1-15, wherein the oligonucleotide comprises a sequence selected from the group consisting of:(a) 5’-CTGAAGGTGTTCTTGTACTTCATCC-3’ (SEQ ID NO: 1);(b) 5’-CAACTGTTGCCTCCGGTTCTGAAGGTGTTC-3’ (SEQ ID NO: 2);(c) 5’-TTGCCTCCGGTTCTGAAGGTGTTCTTGTAC-3’ (SEQ ID NO: 3); and(d) 5’-CTGTTGCCTCCGGTTCTGAAGGTGTTCTTG-3’ (SEQ ID NO: 4).

17. The conjugate or pharmaceutically acceptable salt of any one of claims 1-16, wherein the oligonucleotide comprises the following group at its 5’ terminus:

18. The conjugate or pharmaceutically acceptable salt of any one of claims 1-17, wherein the oligonucleotide comprises the following group at its 5’ terminus:PATENTAttorney Docket No.: 51558-023WO219. The conjugate or pharmaceutically acceptable salt of any one of claims 1-18, wherein the linker is a glutamic acid linked to the oligonucleotide via a gamma carboxylic acid group and has the following structure:

20. The conjugate or pharmaceutically acceptable salt of any one of claims 1-19, wherein the conjugate is of the following structure:[oligonucleotide]21 . A pharmaceutical composition comprising the conjugate of any one of claims 1 -20, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

22. A method of treating a human subject having a Duchenne muscular dystrophy (DMD) genotype that is amenable to exon 53 skipping in a human dystrophin (DMD) gene, the method comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1-20, or pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 21 .

23. The method of claim 22, wherein the genotype is selected from the following DMD mutations: a 3-52 deletion, a 4-52 deletion, a 5-52 deletion, a 6-52 deletion, a 9-52 deletion, a 10-52 deletion, an 11-52 deletion, a 13-52 deletion, a 14-52 deletion, a 15-52 deletion, a 16-52 deletion, a 17-52 deletion, a 19-52 deletion, a 21-42 deletion, a 23-52 deletion, a 24-52 deletion, a 25-52 deletion, a 26-52 deletion, a 27-52 deletion, a 28-52 deletion, a 29-52 deletion, a 30-52 deletion, a 31-52 deletion, a 32-52 deletion, a 33-52 deletion, a 34-52 deletion, a 35-52 deletion, a 36-52 deletion, a 37-52 deletion, a 38-52 deletion, a 39-52 deletion, a 40-52 deletion, a 41-52 deletion, a 42-52 deletion, a 43-52 deletion, a 45-52 deletion, a 47-52 deletion, a 48-52 deletion, a 49-52 deletion, a 50-52 deletion, a 52 deletion, a 54-58 deletion, a 54-61 deletion, a 54-61 deletion, a 54-63 deletion, a 54-64 deletion, a 54-66 deletion, a 54-76 deletion, and a 54-77 deletion.

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