Cell-penetrating peptides, conjugates thereof, and methods of use thereof

Abstract

Disclosed are peptides and conjugates of peptides linked covalently or via a linker to a therapeutic or diagnostic molecule, wherein the peptide comprises at least one cationic domain and at least one hydrophobic domain.

Description

[Technical Field]

[0001] The present invention relates to cell-penetrating peptides, conjugates thereof, compositions containing them, and methods of using them. [Background technology]

[0002] The delivery of exogenous biomolecules, such as therapeutic and diagnostic molecules, through cell membranes is essential for these biomolecules to exert their intended effects within cells. Because the cell membrane acts as a biochemical barrier, it can be difficult for exogenous biomolecules to reach their targets. Certain methods for delivering biomolecules into cells can result in toxicity, reduced specificity, reduced stability, immunogenicity, reduced efficacy, and reduced delivery efficiency. Therefore, there is a need to develop new methods for safely and efficiently delivering biomolecules to target cells.

[0003] Nucleic acid medicines are genomic medicines with the potential to transform human healthcare. Research has shown that these therapeutics may be applicable to a wide range of disease areas, including genetic disorders such as certain neuromuscular and neurological disorders. Oligonucleotide therapeutics are nucleic acid-based gene therapy drugs designed to target the underlying causes of many diseases by modulating RNA expression and processing. These drugs' mechanisms of action include disrupting gene expression, degrading toxic RNA species, altering gene translation, interfering with RNA interactions with other nucleic acids or proteins, endogenous human adenosine deaminases acting on RNA (ADARs), site-specific RNA editing, and modulating gene splicing, each of which can have profound biological effects. The application of antisense oligonucleotides to modulate pre-mRNA splicing in the neuromuscular disease Duchenne muscular dystrophy (DMD) has placed this monogenic disease at the forefront of advances in precision medicine. Further examples of genetic diseases treatable with oligonucleotide therapy include facioscapulohumeral muscular dystrophy (FSHD), myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), Charcot-Marie-Tooth disease type 1a (CMT1a), Charcot-Marie-Tooth disease type 2a (CMT2a), Fuchs corneal dystrophy (FCD), Friedreich's ataxia (FA), and spinal muscular atrophy (SMA).

[0004] However, the therapeutic development of these promising antisense oligonucleotide therapeutics has been hindered by poor cell penetration and distribution characteristics—challenges further accentuated by the large volume and dispersity of the muscle tissue matrix in DMD and other neuromuscular diseases, and the difficulty of accessing affected central and peripheral nervous systems in neurological diseases.

[0005] DMD affects approximately 1 in 3,500 newborn boys. This severe, X-linked recessive disorder is caused by mutations in the DMD gene, which encodes the dystrophin protein. The disease is characterized by progressive muscle degeneration and atrophy, leading to respiratory failure, cardiac complications, and ultimately premature death. The majority of mutations underlying DMD are genomic out-of-frame deletions that cause premature truncation of the open reading frame, resulting in the absence of dystrophin protein.

[0006] Exon skipping therapy uses splice-switching antisense oligonucleotides (SSOs) to target specific regions of the DMD transcript, inducing the elimination of individual exons and restoring the abnormal reading frame, resulting in the production of an internally deleted but partially functional dystrophin protein. Despite the undeniable potential of antisense oligonucleotide-based exon skipping therapy for DMD, its successful application is currently limited by relatively poor oligonucleotide uptake by skeletal muscle and insufficient targeting of single-stranded oligonucleotides to other affected tissues, such as the heart. In September 2016, the US Food and Drug Administration (FDA) granted accelerated approval to eteplirsen, a modulator of exon 51 splicing. This marked the first FDA-approved splicing-modulating oligonucleotide and the first approved treatment for DMD. However, the developer reported minimal dystrophin restoration in patients, approximately 1% of normal levels. Comparison with the allelic disease Becker muscular dystrophy (BMD) and experiments in standard mdx mice showed that homogeneous sarcolemmal dystrophin expression, at least 10–15% of wild-type, is required to protect muscles from exercise-induced damage.

[0007] Therefore, new methods for intracellular delivery of therapeutic and diagnostic molecules are needed, such as the delivery of antisense oligonucleotide-based therapeutics for severe genetic disorders such as DMD and other neuromuscular and neurological disorders. Summary of the Invention

[0008] Typically, the present invention provides conjugates of a peptide and a therapeutic or diagnostic molecule covalently linked via a covalent bond or linker. In some embodiments, the therapeutic or diagnostic molecule is an oligonucleotide that may be complementary to a target sequence within a transcript, the altered processing, recognition, or steric hindrance of which results in a beneficial therapeutic effect. In some embodiments, the target sequence is associated with a genetic disorder, such as a neuromuscular or neurological disease (e.g., DMD, BMD, DM1, DM2, CMT1a, CMT2a, FCD, FA, or SMA). For example, the oligonucleotide may be complementary to a target sequence within or near dystrophin, i.e., any one of exons 8-55 (e.g., exon 8, exon 23, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53, or exon 55) of the DMD transcript (e.g., the human dystrophin transcript). In another example, the oligonucleotide may be complementary to a target sequence within or near the r(CUG) region in the 3'-untranslated region of the DMPK transcript. exp (For example, the oligonucleotide may have at least 9 contiguous nucleobases complementary to the CUG repeat sequence, see also below.) In another example, the oligonucleotide may be complementary to the r(CCUG) repeat in the CNBP transcript. exp Alternatively, the oligonucleotide may be complementary to a target sequence located within or near intron 7 of the human motor neuron survival factor 2 (SMN2) transcript, or a target sequence located within the DUX4 transcript, PMP22 transcript, MFN2 transcript, TCF4 transcript, or FXN transcript.

[0009] In one aspect, a conjugate includes a peptide and a therapeutic or diagnostic molecule (e.g., an oligonucleotide) linked to the peptide via a covalent bond (i.e., direct bond) or a non-cationic linker (e.g., via a linker comprising a total of one amino acid or an aliphatic dicarboxylic acid linker), wherein the peptide comprises a total of one hydrophobic domain and a total of one cationic domain, wherein the cationic domain comprises at least one cationic amino acid residue, and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise an artificial amino acid residue. In some embodiments, the amino acid residue of each cationic domain is selected from the group consisting of arginine, histidine, and beta-alanine.

[0010] In another aspect, a conjugate includes a peptide and a therapeutic or diagnostic molecule (e.g., an oligonucleotide) covalently (i.e., directly) linked to the peptide or via a non-cationic linker (e.g., via a linker comprising a total of one amino acid or an aliphatic dicarboxylic acid linker), wherein the peptide comprises a total of one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, at least one cationic domain comprising a total of one cationic amino acid residue, and the hydrophobic domain comprising at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise an artificial amino acid residue. In some embodiments, the amino acid residues in each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine. In some embodiments, at least one cationic domain comprises three or fewer amino acid residues.

[0011] In yet another aspect, a conjugate includes a peptide and a therapeutic or diagnostic molecule (e.g., an oligonucleotide) covalently (i.e., directly) or via a non-cationic linker (e.g., via a linker comprising a total of one amino acid or an aliphatic dicarboxylic acid linker) linked to the peptide, wherein the peptide comprises a total of one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, all cationic domains comprising a total of five or fewer cationic amino acid residues, and the hydrophobic domains comprising at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues. In some embodiments, the amino acid residues in each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine. In some embodiments, at least one cationic domain comprises three or fewer amino acid residues.

[0012] In yet another aspect, a conjugate includes a peptide and a therapeutic or diagnostic molecule (e.g., an oligonucleotide) covalently (i.e., directly) linked to the peptide or via a non-cationic linker (e.g., via a linker comprising a total of one amino acid or an aliphatic dicarboxylic acid linker), wherein the peptide comprises a total of one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, at least one-third of the amino acid residues in the N-terminal cationic domain are histidine, and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues. In some embodiments, the amino acid residues in each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine. In some embodiments, at least one cationic domain comprises three or fewer amino acid residues.

[0013] In some embodiments, each cationic domain comprises at least 40%, at least 45%, or at least 50% cationic amino acids. In some embodiments, each cationic domain comprises a majority of cationic amino acids, preferably at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cationic amino acids. In some embodiments, each cationic domain comprises arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues, preferably, each cationic domain consists of arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues, provided that at least one arginine or histidine is present. In some embodiments, each cationic domain is arginine-rich and / or histidine-rich, preferably each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% arginine and / or histidine residues.In some embodiments, at least one cationic domain (e.g., all cationic domains) is selected from the group consisting of RBRR (SEQ ID NO: 419), RBR, RB, R, RBRRBRR (SEQ ID NO: 420), RRBRR (SEQ ID NO: 421), BRR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRB, RBRBRB (SEQ ID NO: 424), RBRB (SEQ ID NO: 425), RB, HRHRHR (SEQ ID NO: 426), HRHR (SEQ ID NO: 427), H R, RRRRRR (SEQ ID NO: 428), RBRRBR (SEQ ID NO: 429), RBHBHB (SEQ ID NO: 430), RBHBH (SEQ ID NO: 431), BHBHB (SEQ ID NO: 432), BHBH (SEQ ID NO: 433), HBHB (SEQ ID NO: 434), HBH, BHB, BH, HB, H, RBHBHE (SEQ ID NO: 435), RBHBB (SEQ ID NO: 436), RBHB (SEQ ID NO: 437), RBB, HRBRHB (SEQ ID NO: 438), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRB, RBRBR (SEQ ID NO: 441), HRHRHRB (SEQ ID NO: 442), HRHRB (SEQ ID NO: 443), HRBRH (SEQ ID NO: 444), HRB, RRRRRRB (SEQ ID NO: 445), BRE, and BR, for example, RBRR (SEQ ID NO: 419), RBR, RB, R, RBRRBRR (SEQ ID NO: 420), RRBRR (SEQ ID NO: 421), BRR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRB, RBRBRB (SEQ ID NO: 424), RBRB (SEQ ID NO: 425), RB, HRHRHR (SEQ ID NO: 426), HRHR (SEQ ID NO: 427), HR, RRRRRR (SEQ ID NO: 428), RBRRBR (SEQ ID NO: 429), RBHBH (SEQ ID NO: 431), BHBH (SEQ ID NO: 433), HBH, BH, H, RBHB (SEQ ID NO: 437), HRBRH (SEQ ID NO: 444), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), RBRBR (SEQ ID NO: 441), and BR.In some embodiments, at least one cationic domain (e.g., all cationic domains) is selected from the group consisting of RBR, RBRB (SEQ ID NO: 425), RB, R, RBRRBRR (SEQ ID NO: 420), BRR, BR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRB, HRHRHR (SEQ ID NO: 426), HR, RRRRRR (SEQ ID NO: 428), ), RRRRRRB (SEQ ID NO: 445), RBHBH (SEQ ID NO: 431), BH, BHB, H, HB, RBH, and RBHB (SEQ ID NO: 437), e.g., RBR, R, RBRRBRR (SEQ ID NO: 420), BRR, BR, RR, HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRHRHR (SEQ ID NO: 426), HR, RRRRRR (SEQ ID NO: 428), RBHBH (SEQ ID NO: 431), BH, H, and RBH.

[0014] In some embodiments, each hydrophobic domain has a length of 3-6 amino acids, preferably 5 amino acids. In some embodiments, each hydrophobic domain comprises a majority of hydrophobic amino acid residues, preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids. In some embodiments, each hydrophobic domain comprises phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and glutamine residues, preferably consisting of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues, provided that at least one of phenylalanine, leucine, isoleucine, tyrosine, and tryptophan is present. In some embodiments, the peptide comprises one hydrophobic domain. In some embodiments, the hydrophobic domain is FQILY (SEQ ID NO: 446).

[0015] In another embodiment, a conjugate comprises a peptide and a therapeutic or diagnostic molecule linked to the peptide (i.e., directly) via a covalent bond or a non-cationic linker (e.g., via a linker comprising a total of one amino acid or an aliphatic dicarboxylic acid linker), wherein the peptide is selected from the group consisting of RBRRFQILYRBHBH (SEQ ID NO: 447), RBRFQILYRBHBH (SEQ ID NO: 448), RBFQILYRBHBH (SEQ ID NO: 449), RFQILYRBHBH (SEQ ID NO: 450), FQILYRBHBH (SEQ ID NO: 451), SEQ ID NO: 451), RBRRBRRFQILYBHBHB (SEQ ID NO: 452), RBRRBRRFQILYHBH (SEQ ID NO: 453), RBRRBRRFQILYBH (SEQ ID NO: 454), RBRRBRRFQILYH (SEQ ID NO: 455), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBH (SEQ ID NO: 457), RRBRRFQILYRBHBH (SEQ ID NO: 458), BRRFQILYRBHBH (SEQ ID NO: 459), RRFQILYRBHBH (SEQ ID NO: 460), RBRRBR RFQILYRBHB (SEQ ID NO: 461), RBRRBRRFQILYRBH (SEQ ID NO: 462), RBRRBRRFQILYRB (SEQ ID NO: 463), RBRRBRRFQILYR (SEQ ID NO: 464), HRBHRBHRBFQILYHRBRH (SEQ ID NO: 465), HRBHRBHRBFQILYHRBHRBHR (SEQ ID NO: 466), HRBHRBFQILYHRBHR (SEQ ID NO: 467), HRBFQILYHR (SEQ ID NO: 468), RBRBRBFQILYRBRBR (SEQ ID NO: 469), RBRBF QILYRBR (SEQ ID NO: 470), RBFQILYR (SEQ ID NO: 471), HRHRHRFQILYHRHRHR (SEQ ID NO: 472), HRHRFQILYHRHR (SEQ ID NO: 473), HRFQILYHR (SEQ ID NO: 474), RRRRRRFQILY (SEQ ID NO: 475), FQILYRRRRRR (SEQ ID NO: 476), RRRRRRFQILYRRRRRR (SEQ ID NO: 477), RBRRBRFQILYBR (SEQ ID NO: 478), and RBRRBRFQILY (SEQ ID NO: 479).In another embodiment, a conjugate comprises a peptide and a therapeutic or diagnostic molecule linked to the peptide (i.e., directly) via a covalent bond or a non-cationic linker (e.g., via a linker comprising a total of 1 amino acid or an aliphatic dicarboxylic acid linker), wherein the peptide is rBrrBrfqilyBrBr (SEQ ID NO: 510), where lowercase letters indicate D-amino acids.

[0016] In some embodiments, the peptide of the complex is (RBRRBRFQILYBR (SEQ ID NO: 478)). In some embodiments, the peptide of the complex is (RBRRBRFQILY (SEQ ID NO: 479)). In some embodiments, the peptide of the complex is (RBRRBRRFQILY (SEQ ID NO: 456)). In some embodiments, the peptide of the complex is (RRFQILYRBHBH (SEQ ID NO: 460)).

[0017] In another embodiment, a conjugate comprises a peptide and a therapeutic or diagnostic molecule linked to the peptide (i.e., directly) via a covalent bond or a non-cationic linker (e.g., via a linker comprising a total of one amino acid or an aliphatic dicarboxylic acid linker), wherein the peptide is selected from the group consisting of RBRRFQILYRBHBHB (SEQ ID NO: 481), RBRFQILYRBHBHB (SEQ ID NO: 482), RBFQILYRBHBHB (SEQ ID NO: 483), RFQILYRBHBHB (SEQ ID NO: 484), FQILYRBHBHB (SEQ ID NO: 485), RBRR BRRFQILYBHBHB (SEQ ID NO: 452), RBRRBRRFQILYHBHB (SEQ ID NO: 486), RBRRBRRFQILYBHB (SEQ ID NO: 487), RBRRBRRFQILYHB (SEQ ID NO: 488), RBRRBRRFQILYB (SEQ ID NO: 489), RBRRBRRFQILYE (SEQ ID NO: 416), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBHB (SEQ ID NO: 490), RRBRRFQILYRBHBHB (SEQ ID NO: 491), BRRFQILYRBHBHB (SEQ ID NO: 492), RRF QILYRBHBHB (SEQ ID NO: 493), RRFQILYRBHBHE (SEQ ID NO: 417), RBRRBRRFQILYRBHBB (SEQ ID NO: 494), RBRRBRRFQILYRBHB (SEQ ID NO: 461), RBRRBRRFQILYRBB (SEQ ID NO: 495), RBRRBRRFQILYRB (SEQ ID NO: 463), HRBHRBHRBFQILYHRBRHB (SEQ ID NO: 496), HRBHRBHRBFQILYHRBHRBHRB (SEQ ID NO: 497), HRBHRBFQILYHRBHRB (SEQ ID NO: 498), HRBFQILYHRB (SEQ ID NO: 499), RBRRBFQILYRBRBRB (SEQ ID NO: 500), RBRBFQILYRBRB (SEQ ID NO: 501), RBFQILYRB (SEQ ID NO: 502), HRHRHRFQILYHRHRHRB (SEQ ID NO: 503), HRHRFQILYHRHRB (SEQ ID NO: 504), HRFQILYHRB (SEQ ID NO: 505), RRRRRRFQILYB (SEQ ID NO: 506), FQILYRRRRRRB (SEQ ID NO: 507), RRRRRRFQILYRRRRRRB (SEQ ID NO: 508), RBRRBRFQILYBRE (SEQ ID NO: 415),and RBRRBRFQILYE (SEQ ID NO: 509). In another embodiment, a conjugate comprises a peptide and a therapeutic or diagnostic molecule linked to the peptide (i.e., directly) via a covalent bond or a non-cationic linker (e.g., via a linker comprising a total of one amino acid or an aliphatic dicarboxylic acid linker), wherein the peptide is rBrrBrfqilyBrBre (SEQ ID NO: 405), where lowercase letters indicate D-amino acids. In another embodiment, a conjugate comprises a peptide as described above in this paragraph, but with the C-terminal amino acid removed, or removed and replaced with, for example, a linker described herein (e.g., a B linker or an E linker).

[0018] In some embodiments, the peptide of the complex is DPep1.9b-del2 (RBRRBRFQILYBRE) (SEQ ID NO: 415). In some embodiments, the peptide of the complex is Dpep1.9b-del4 (RBRRBRFQILYE (SEQ ID NO: 509)). In some embodiments, the peptide of the complex is peptide E5-E (RBRRBRRFQILYE (SEQ ID NO: 416)). In some embodiments, the peptide of the complex is peptide G5-E (RRFQILYRBHBHE (SEQ ID NO: 417)).

[0019] In some embodiments, the peptide is attached to the remainder of the complex through its N-terminus. In some embodiments, the C-terminus of the peptide is amidated, i.e., terminated with -NH. In some embodiments, the peptide is attached to the remainder of the complex through its C-terminus. In some embodiments, the peptide is acylated at the N-terminus. In some embodiments, the peptide is covalently attached (i.e., directly attached) to a therapeutic or diagnostic molecule (e.g., an oligonucleotide). In some embodiments, the peptide is covalently linked to a therapeutic or diagnostic molecule (e.g., an oligonucleotide) via a linker comprising a total of one amino acid. In some embodiments, the linker is selected from the group consisting of glutamic acid (e.g., linked via its gamma-carboxyl group), beta-alanine, glycine, delta-aminovaleric acid, and gamma-aminobutyric acid. In some embodiments, the peptide is covalently attached to a therapeutic or diagnostic molecule (e.g., an oligonucleotide) via an aliphatic dicarboxylic acid linker (e.g., succinyl).

[0020] In some embodiments, the linker has the following structure: [ka]

[0021] In some embodiments, the linker has the following structure: [ka]

[0022] In some embodiments, the linker has the following structure: [ka]

[0023] In some embodiments, the linker has the following structure: [ka]

[0024] In some embodiments, the linker has the following structure: [ka]

[0025] In some embodiments, the complex is of the following structure: [ka]

[0026] In some embodiments, the complex is of the following structure: [ka]

[0027] In some embodiments, the complex is of the following structure: [ka]

[0028] In some embodiments, the complex is of the following structure: [ka]

[0029] In some embodiments, the complex is of the following structure: [ka]

[0030] In some embodiments, the therapeutic or diagnostic molecule is an oligonucleotide, optionally attached at its 3' end to the linker or the peptide.

[0031] In some embodiments, the oligonucleotide comprises a sequence complementary to a target sequence, and targeting of the target sequence by the oligonucleotide alters the processing or recognition of the target sequence, and optionally, the targeting can be used to treat, ameliorate, or prevent one or more symptoms of a genetic disease.

[0032] In some embodiments, the genetic disease is optionally a neuromuscular disease selected from the group consisting of muscular dystrophy (eg, DMD, BMD, or FSHD), DM1, DM2, and SMA.

[0033] In some embodiments, the target sequence is present within a transcript of a DMD, DMPK, DUX4, CNBP, or SMN2 gene.

[0034] In some embodiments, the genetic disease is optionally a neurological disease selected from the group consisting of CMT1a, CMT2a, and FA.

[0035] In some embodiments, the genetic disease optionally includes an FCD and affects other organ systems of the body.

[0036] In some embodiments, the target sequence is present in a transcript of the PMP22, MFN2, TF4, or FXN gene.

[0037] In some embodiments, the oligonucleotide comprises at least 12 contiguous nucleobases complementary to a target exon sequence within a DMD gene (e.g., human dystrophin gene) transcript. In some embodiments, the target sequence is located within or near any one of exons 8 to 55 (e.g., exons 8, 23, 44, 45, 50, 51, 52, 53, or 55) of the gene (e.g., human dystrophin gene) transcript.

[0038] In some embodiments, the oligonucleotide comprises or consists of a sequence of a table herein (Table 1, Table 2, Table 3, Table 4, Table 5, or Table 8), optionally, in which one or more (e.g., all) uracils are substituted with thymines, or one or more (e.g., all) thymines are substituted with uracils.

[0039] In some embodiments, the target sequence includes the exon 45 splice site or is located within 150 nucleobases of the exon 45 splice site.

[0040] In some embodiments, the oligonucleotide comprises at least 12 contiguous nucleobases from any one of the sequences in Table 1, and thymine substituted versions thereof. In some embodiments, the oligonucleotide comprises any one of the sequences in Table 1 or thymine substituted versions thereof. In some embodiments, the sequence in Table 1 is 5'-GCTGCCCAATGCCATCCTGGAGTTCCTGTAA-3' (sequence number 276).

[0041] In some embodiments, the sequence in Table 1 above is 5'-CAATGCCATCCTGGAGTTCCTG-3' (sequence number 275).

[0042] In some embodiments, the sequence in Table 1 above is Selected from the group consisting of 5'-TTGCCGCTGCCCAATGCCATCCTGGAGTTC-3' (SEQ ID NO: 270), 5'-CAGTTTGCCGCTGCCCAATGCCATCCTGGA-3' (SEQ ID NO: 271), 5'-CCAAUGCCAUCCUGGAGUUCCUGUAA-3' (SEQ ID NO: 272), 5'-CCAATGCCATCCTGGAGTTCCTGTA-3' (SEQ ID NO: 273), and 5'-CTGACAACAGTTTGCCGCTGCCCAA-3' (SEQ ID NO: 274).

[0043] In some embodiments, the target sequence includes the exon 51 splice site or is located within 150 nucleobases of the exon 51 splice site.

[0044] In some embodiments, the oligonucleotide comprises at least 12 contiguous nucleobases from any one of the sequences in Table 2, and thymine substituted forms thereof.

[0045] In some embodiments, the oligonucleotide comprises any one of the sequences in Table 2, or a thymine substituted version thereof.

[0046] In some embodiments, the sequence in Table 2 above is 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 282).

[0047] In some embodiments, the sequence in Table 2 above is 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 287).

[0048] In some embodiments, the target sequence includes the exon 53 splice site or is located within 150 nucleobases of the exon 53 splice site.

[0049] In some embodiments, the oligonucleotide comprises at least 12 contiguous nucleobases from any one of the sequences in Table 3.

[0050] In some embodiments, the oligonucleotide comprises any one of the sequences in Table 3.

[0051] In some embodiments, the sequence in Table 3 above is 5'-CCTCCGGTTCTGAAGGTGTTCT-3' (sequence number 316).

[0052] In some embodiments, the sequence in Table 3 above is 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (sequence number 325).

[0053] In some embodiments, the sequence in Table 3 above is 5'-CTGTTGCCTCCGGTTCTGAAGGTGTTCTTG-3' (SEQ ID NO: 327), 5'-CAACTGTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 328), 5'-TTGCCTCCGGTTCTGAAGGTGTTCTTGTAC-3' (SEQ ID NO: 329), 5'-CTGAAGGTGTTCTTGTACTTCATCC-3' (SEQ ID NO: 330), and 5'-CATTCAACTGTTGCCTCCGGTTCTGAAGGTG-3' (SEQ ID NO: 331).

[0054] In some embodiments, the target sequence comprises the splice site of exon 44 or is located within 150 nucleobases of the splice site of exon 44. In some embodiments, the oligonucleotide comprises at least 12 contiguous nucleobases from any one of the sequences in Table 4. In some embodiments, the oligonucleotide comprises any one of the sequences in Table 4.

[0055] In some embodiments, the sequence in Table 4 above is Selected from the group consisting of 5'-TGAAAACGCCGCCATTTCTCAACAGATCTG-3' (SEQ ID NO: 335), 5'-CATAATGAAAACGCCGCCATTTCTCAACAG-3' (SEQ ID NO: 336), 5'-TGTTCAGCTTCTGTTAGCCACTGATTAAAT-3' (SEQ ID NO: 337), 5'-CGCCGCCATTTCTCAACAG-3' (SEQ ID NO: 338), and 5'-ATCTGTCAAATCGCCTGCAG-3' (SEQ ID NO: 339).

[0056] In some embodiments, the sequence is 5'-GGCCAAACCTCGGCTTACCTGAAAT-3' (SEQ ID NO: 288).

[0057] In some embodiments, the splice site is an acceptor splice site.

[0058] In some embodiments, the splice site is a donor splice site.

[0059] In some embodiments, the oligonucleotide comprises at least 9 contiguous nucleobases complementary to a CUG repeat sequence. n -3',5'-[AGC] n -3' and 5'-[GCA] n -3', where n is an integer from 5 to 8. In some embodiments, the oligonucleotide has a sequence selected from the group consisting of 5'-[CAG]5-3' (SEQ ID NO: 340), 5'-[CAG]6-3' (SEQ ID NO: 341), 5'-[CAG]7-3' (SEQ ID NO: 342), 5'-[CAG]8-3' (SEQ ID NO: 343), 5'-[AGC]5-3' (SEQ ID NO: 344), 5'-[AGC]6-3' (SEQ ID NO: 345), 5'-[AGC]7-3' (SEQ ID NO: 346), 5'-[AGC]8-3' (SEQ ID NO: 347), 5'-[GCA]5-3' (SEQ ID NO: 348), 5'-[GCA]6-3' (SEQ ID NO: 349), 5'-[GCA]7-3' (SEQ ID NO: 350), and 5'-[GCA]8-3' (SEQ ID NO: 351).

[0060] In some embodiments, the oligonucleotide comprises at least 8 consecutive nucleobases complementary to a repeat sequence of CCUG. In some embodiments, the oligonucleotide comprises 5'-[CAGG] n -3',5'-[AGGC] n -3',5'-[GGCA] n -3',5'-[GCAG]n and 5'-[CAGG]-3' (SEQ ID NO: 352), 5'-[CAGG]-3' (SEQ ID NO: 353), 5'-[CAGG]-3' (SEQ ID NO: 354), 5'-[CAGG]-3' (SEQ ID NO: 355), 5'-[CAGG]-3' (SEQ ID NO: 356), 5'-[AGGC]-3' (SEQ ID NO: 357), 5'-[AGGC]-3' (SEQ ID NO: 358), 5'-[AGGC]-3' (SEQ ID NO: 359), 5'-[AGGC]-3' (SEQ ID NO: 360), 5'-[AGGC]-3' (SEQ ID NO: 361), 5'-[AGGC]-3' (SEQ ID NO: 362), 5'-[AGGC]-3' (SEQ ID NO: 363), 5'-[AGGC]-3' (SEQ ID NO: 364), 5'-[AGGC]-3' (SEQ ID NO: 365), 5'-[AGGC]-3' (SEQ ID NO: 366), 5'-[AGGC]-3' (SEQ ID NO: 367), 5'-[AGGC]-3' (SEQ ID NO: 368), 5'-[AGGC]-3' (SEQ ID NO: 369), 5'-[AGGC]-3' (SEQ ID NO: 370), 5'-[AGGC]-3' (SEQ ID NO: 371), 5'-[AGGC]-3' (SEQ ID NO: 372), 5'-[AGGC]-3' (SEQ ID NO: 373), 5'-[AGGC]-3' (SEQ ID NO: 374), 5'- 61), 5'-[GGCA]4-3' (SEQ ID NO:362), 5'-[GGCA]5-3' (SEQ ID NO:363), 5'-[GGCA]6-3' (SEQ ID NO:364), 5'-[GGCA]7-3' (SEQ ID NO:365), 5'-[GGCA]8-3' (SEQ ID NO:366), 5'-[GCAG]4-3' (SEQ ID NO:367), 5'-[GCAG]5-3' (SEQ ID NO:368), 5'-[GCAG]6-3' (SEQ ID NO:369), 5'-[GCAG]7-3' (SEQ ID NO:370), and 5'-[GCAG]8-3' (SEQ ID NO:371).

[0061] In some embodiments, the oligonucleotide comprises at least 12 contiguous nucleobases complementary to a target sequence in a transcript of the SMN2 gene. In some embodiments, the target sequence is located in SMN2 intron 7. In some embodiments, the oligonucleotide comprises any one of the sequences in Table 5 and thymine-substituted forms thereof.

[0062] In some embodiments, the oligonucleotide comprises any one of the sequences in Table 8 and thymine substituted versions thereof.

[0063] In some embodiments, the oligonucleotide comprises the following group at its 5' end: [ka]

[0064] In some embodiments, the oligonucleotide comprises the following group at its 5' end: [ka]

[0065] In some embodiments, the oligonucleotide comprises a hydroxyl at its 5' end.

[0066] In another aspect, the present invention provides a pharmaceutical composition comprising a conjugate described herein and a pharmaceutically acceptable excipient.

[0067] In yet another aspect, the present invention provides a method of treating a subject having a genetic disease, comprising administering to the subject a therapeutically effective amount of a conjugate or pharmaceutical composition described herein.

[0068] In some embodiments, the genetic disease is optionally a neuromuscular disease selected from the group consisting of muscular dystrophy (eg, DMD, BMD, or FSHD), DM1, DM2, and SMA.

[0069] In some embodiments, the target sequence is present in a transcript of a DMD, DMPK, CNBP, SMN2, or DUX4 gene.

[0070] In some embodiments, the genetic disease is optionally a neurological disease selected from the group consisting of CMT1a, CMT2a, and FA.

[0071] In some embodiments, the genetic disease is a disease that also affects other organ systems of the body, optionally including FCD.

[0072] In some embodiments, the target sequence is present in a transcript of the PMP22, MFN2, TF4, or RXN gene.

[0073] In some embodiments, the method is for treating a subject having DMD, BMD, SMA, DM1, or DM2, and the method comprises administering to the subject a therapeutically effective amount of a conjugate or pharmaceutical composition described herein, wherein the conjugate is for treating DMD or BMD, the conjugate is for treating DM1, the conjugate is for treating DM2, and the conjugate is for treating SMA.

[0074] In some embodiments, the subject has DMD. In some embodiments, the subject has DM1. In some embodiments, the subject has DM2. In some embodiments, the subject has SMA.

[0075] Preferably, the oligonucleotide is a morpholino (more preferably a morpholino in which all morpholino internucleoside linkages are -P(O)(Nme2)O-).

[0076] In some embodiments, the oligonucleotide is a phosphorothioate (PS) oligonucleotide.

[0077] In some embodiments, the oligonucleotide is a 2'-O-alkyl oligoribonucleotide, such as a 2'-O-methyl oligoribonucleotide.

[0078] In some embodiments, the oligonucleotide is a 2'-O-alkyl phosphorothioate, such as a 2'-O-methyl phosphorothioate.

[0079] In some embodiments, the oligonucleotide is a peptide nucleic acid (PNA).

[0080] In another aspect, the present invention provides a peptide comprising one hydrophobic domain and one cationic domain, wherein the cationic domain comprises at least one cationic amino acid residue and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues. In some embodiments, the amino acid residues in each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine.

[0081] In yet another aspect, the present invention provides a peptide comprising one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, wherein each cationic domain comprises at least one cationic amino acid residue, and the at least one cationic domain comprises one cationic amino acid residue, and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues. In some embodiments, the amino acid residues in each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine. In some embodiments, at least one cationic domain comprises three or fewer amino acid residues.

[0082] In yet another aspect, the present invention provides a peptide comprising one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, wherein each cationic domain comprises at least one cationic amino acid residue, all cationic domains comprise five or fewer cationic amino acid residues, and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues in total and does not comprise any artificial amino acid residues. In some embodiments, the amino acid residues in each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine. In some embodiments, at least one cationic domain comprises three or fewer amino acid residues.

[0083] In a further aspect, the present invention provides a peptide comprising one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, at least one-third of the amino acid residues in the N-terminal cationic domain being histidine, and the hydrophobic domain comprising at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues. In some embodiments, the amino acid residues in each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine. In some embodiments, at least one cationic domain comprises three or fewer amino acid residues.

[0084] In some embodiments, each cationic domain comprises at least 40%, at least 45%, or at least 50% cationic amino acids. In some embodiments, each cationic domain comprises a majority of cationic amino acids, preferably at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cationic amino acids. In some embodiments, each cationic domain comprises arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues, preferably, each cationic domain consists of arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues, provided that at least one arginine or histidine is present. In some embodiments, each cationic domain is arginine-rich and / or histidine-rich, preferably each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% arginine and / or histidine residues.

[0085] In some embodiments, at least one cationic domain is selected from the group consisting of RBRR (SEQ ID NO: 419), RBR, RB, R, RBRRBRR (SEQ ID NO: 420), RRBRR (SEQ ID NO: 421), BRR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRB, RBRBRB (SEQ ID NO: 424), RBRB (SEQ ID NO: 425), RB, HRHRHR (SEQ ID NO: 426), HRHR (SEQ ID NO: 427), HR, RRRRRR (SEQ ID NO: 4 28), RBRRBR (SEQ ID NO: 429), RBHBHB (SEQ ID NO: 430), RBHBH (SEQ ID NO: 431), BHBHB (SEQ ID NO: 432), BHBH (SEQ ID NO: 433), HBHB (SEQ ID NO: 434), HBH, BHB, BH, HB, H, RBHBHE (SEQ ID NO: 435), RBHBB (SEQ ID NO: 436), RBHB (SEQ ID NO: 437), RBB, HRBRHB (SEQ ID NO: 438), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRB, R The group consisting of BRBR (SEQ ID NO: 441), HRHRHRB (SEQ ID NO: 442), HRHRB (SEQ ID NO: 443), HRBRH (SEQ ID NO: 444), HRB, RRRRRRB (SEQ ID NO: 445), BRE, and BR, for example, RBRR (SEQ ID NO: 419), RBR, RB, R, RBRRBRR (SEQ ID NO: 420), RRBRR (SEQ ID NO: 421), BRR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRB, RBRBRB (SEQ ID NO: 4 24), RBRB (SEQ ID NO: 425), RB, HRHRHR (SEQ ID NO: 426), HRHR (SEQ ID NO: 427), HR, RRRRRR (SEQ ID NO: 428), RBRRBR (SEQ ID NO: 429), RBHBH (SEQ ID NO: 431), BHBH (SEQ ID NO: 433), HBH, BH, H, RBHB (SEQ ID NO: 437), HRBRH (SEQ ID NO: 444), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), RBRBR (SEQ ID NO: 441), and BR.In some embodiments, at least one cationic domain is selected from the group consisting of RBR, RBRB (SEQ ID NO: 425), RB, R, RBRRBRR (SEQ ID NO: 420), BRR, BR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRB, HRHRHR (SEQ ID NO: 426), HR, RRRRRR (SEQ ID NO: 428), RRRRRRB ( RBR, R, RBRRBRR (SEQ ID NO: 420), BRR, BR, RR, HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRHRHR (SEQ ID NO: 426), HR, RRRRRR (SEQ ID NO: 428), RBHBH (SEQ ID NO: 431), BH, H, and RBH.

[0086] In some embodiments, each hydrophobic domain has a length of 3 to 6 amino acid residues, and preferably, each hydrophobic domain has a length of 5 amino acid residues.

[0087] In some embodiments, each hydrophobic domain comprises a majority of hydrophobic amino acid residues, preferably, each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids.

[0088] In some embodiments, each hydrophobic domain comprises phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and glutamine residues, and preferably each hydrophobic domain consists of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues, provided that at least one of phenylalanine, leucine, isoleucine, tyrosine, and tryptophan is present.

[0089] In some embodiments, the hydrophobic domain is FQILY (SEQ ID NO: 446).

[0090] In yet another aspect, the present invention provides a method for the preparation of ribozymes comprising the steps of: RBRRFQILYRBHBH (SEQ ID NO: 447), RBRFQILYRBHBH (SEQ ID NO: 448), RBFQILYRBHBH (SEQ ID NO: 449), RFQILYRBHBH (SEQ ID NO: 450), FQILYRBHBH (SEQ ID NO: 451), RBRRBRRFQILYBHBHB (SEQ ID NO: 452), RBRRBRRFQILYHBH (SEQ ID NO: 453), RBRRBRRFQILYBH (SEQ ID NO: 454), RB RRBRRFQILYH (SEQ ID NO: 455), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBH (SEQ ID NO: 457), RRBRRFQILYRBHBH (SEQ ID NO: 458), BRRFQILYRBHBH (SEQ ID NO: 459), RRFQILYRBHBH (SEQ ID NO: 460), RBRRBRRFQILYRBHB (SEQ ID NO: 461), RBRRBRRFQILYRBH (SEQ ID NO: 462), RBRRBRRFQILYRB( SEQ ID NO: 463), RBRRBRRFQILYR (SEQ ID NO: 464), HRBHRBHRBFQILYHRBRH (SEQ ID NO: 465), HRBHRBHRBFQILYHRBHRBHR (SEQ ID NO: 466), HRBHRBFQILYHRBHR (SEQ ID NO: 467), HRBFQILYHR (SEQ ID NO: 468), RBRBRBFQILYRBRBR (SEQ ID NO: 469), RBRBFQILYRBR (SEQ ID NO: 470), RBFQILYR (SEQ ID NO: 471), The peptide is selected from the group consisting of HRHRHRFQILYHRHRHR (SEQ ID NO:472), HRHRFQILYHRHR (SEQ ID NO:473), HRFQILYHR (SEQ ID NO:474), RRRRRRFQILY (SEQ ID NO:475), FQILYRRRRRR (SEQ ID NO:476), RRRRRRFQILYRRRRRR (SEQ ID NO:477), RBRRBRFQILYBR (SEQ ID NO:478), and RBRRBRFQILY (SEQ ID NO:479). In another aspect, the invention provides a peptide that is rBrrBrfqilyBrBr (SEQ ID NO:510), where lowercase letters indicate D-amino acids.

[0091] In some embodiments, the peptide is (RBRRBRFQILYBR (SEQ ID NO: 478)). In some embodiments, the peptide is (RBRRBRFQILY (SEQ ID NO: 479)). In some embodiments, the peptide is (RBRRBRRFQILY (SEQ ID NO: 456)). In some embodiments, the peptide is (RRFQILYRBHBH (SEQ ID NO: 460)).

[0092] In yet another aspect, the present invention provides RBRRFQILYRBHBHB (SEQ ID NO: 481), RBRFQILYRBHBHB (SEQ ID NO: 482), RBFQILYRBHBHB (SEQ ID NO: 483), RFQILYRBHBHB (SEQ ID NO: 484), FQILYRBHBHB (SEQ ID NO: 485), RBRRBRRFQILYBHBHB (SEQ ID NO: 452), RBRRBRRFQILYHBHB (SEQ ID NO: 486), RBRRBRRFQILYBHB (SEQ ID NO: 487), RBRRBRRFQILYHB (SEQ ID NO: 488), RBRRBRRFQILYHB (SEQ ID NO: 489), RBRRBRRFQILYHB (SEQ ID NO: 490), RBRRBRRFQILYHB (SEQ ID NO: 491), RBRRBRRFQILYHB (SEQ ID NO: 492), RBRRBRRFQILYHB (SEQ ID NO: 493), RBRRBRRFQILYHB (SEQ ID NO: 494), RBRRBRRFQILYHB (SEQ ID NO: 495), RBRRBRRFQILYHB (SEQ ID NO: 496), RBRRBRRFQILYHB (SEQ ID NO: 497), RBRRBRRFQILYHB (SEQ ID NO: 498), RBRRBRRFQILYHB (SEQ ID NO: 49 ... 88), RBRRBRRFQILYB (SEQ ID NO: 489), RBRRBRRFQILYE (SEQ ID NO: 416), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBHB (SEQ ID NO: 490), RRBRRFQILYRBHBHB (SEQ ID NO: 491), BRRFQILYRBHBHB (SEQ ID NO: 492), RRFQILYRBHBHB (SEQ ID NO: 493), RRFQILYRBHBHE (SEQ ID NO: 417), RBRRBRRFQILYRBHBB (SEQ ID NO: 494), RBRRBRRF QILYRBHB (SEQ ID NO: 461), RBRRBRRFQILYRBB (SEQ ID NO: 495), RBRRBRRFQILYRB (SEQ ID NO: 463), HRBHRBHRBFQILYHRBRHB (SEQ ID NO: 496), HRBHRBHRBFQILYHRBHRBHRB (SEQ ID NO: 497), HRBHRBFQILYHRBHRB (SEQ ID NO: 498), HRBFQILYHRB (SEQ ID NO: 499), RBRBRBFQILYRBRBRB (SEQ ID NO: 500), RBRBFQILYRBRB (SEQ ID NO: 501), RB Provided is a peptide selected from the group consisting of FQILYRB (SEQ ID NO: 502), HRHRHRFQILYHRHRHRB (SEQ ID NO: 503), HRHRFQILYHRHRB (SEQ ID NO: 504), HRFQILYHRB (SEQ ID NO: 505), RRRRRRFQILYB (SEQ ID NO: 506), FQILYRRRRRRB (SEQ ID NO: 507), RRRRRRFQILYRRRRRRB (SEQ ID NO: 508), RBRRBRFQILYBRE (SEQ ID NO: 415), and RBRRBRFQILYE (SEQ ID NO: 509). In some embodiments, the peptide is Dpep1.9b-del2(RBRRBRFQILYBRE) (SEQ ID NO: 415).In some embodiments, the peptide is Dpep1.9b-del4(RBRRBRFQILYE) (SEQ ID NO:509). In some embodiments, the peptide is peptide E5-E(RBRRBRRFQILYE) (SEQ ID NO:416). In some embodiments, the peptide is peptide G5-E(RRFQILYRBHBHE) (SEQ ID NO:417). In another aspect, the invention provides a peptide that is rBrrBrfqilyBrBre (SEQ ID NO:405), where lowercase letters indicate D-amino acids. In another aspect, the peptide is as described above in this paragraph, but with the C-terminal amino acid removed or removed and replaced with, for example, a linker described herein (e.g., a B linker or an E linker).

[0093] In another aspect, the invention provides a peptide comprising or consisting of the sequence of SEQ ID NO: 415, 509, 416, 417, 418, 402, 512, 403, 404, 405, 478, 479, 456, or 460. In a further aspect, the invention provides a conjugate comprising such a peptide. In some embodiments, the conjugate comprises a therapeutic or diagnostic molecule (e.g., an oligonucleotide such as a morpholino). In some embodiments, the sequence of the oligonucleotide is selected from Tables 1, 2, 3, 4, 5, or 8.

[0094] definition Throughout this specification, "X" refers to any form of the amino acid aminohexanoic acid, such as 6-aminohexanoic acid.

[0095] Throughout this specification, "B" refers to the amino acid beta-alanine.

[0096] Throughout this specification, "[Hyp]" refers to the amino acid hydroxyproline.

[0097] Throughout this specification, "Ac" refers to an acetyl group (CH3-C(O)-).

[0098] Throughout this specification, other capital letters indicate the related amino acid residue according to the generally accepted alphabetic amino acid code.

[0099] The term "alkyl," as used herein, unless otherwise specified, refers to a straight- or branched-chain hydrocarbon group containing from 1 to 20 total carbon atoms (e.g., (1-6C)alkyl, (1-4C)alkyl, (1-3C)alkyl, or (1-2C)alkyl). Non-limiting examples of alkyl include methyl, ethyl, 1-methylethyl, propyl, 1-methylbutyl, 1-ethylbutyl, and the like. References to individual alkyl groups, such as "propyl," refer only to the straight-chain group, and references to individual branched-chain alkyl groups, such as "isopropyl," refer only to the branched-chain group.

[0100] The term "alkenyl," as used herein, unless otherwise specified, refers to an aliphatic group having one, two, or three carbon-carbon double bonds and containing a total of two to twenty carbon atoms (e.g., (2-6C)alkenyl, (2-4C)alkenyl, or (2-3C)alkenyl). Non-limiting examples of alkenyl include vinyl, allyl, homoallyl, isoprenyl, and the like. Unless otherwise specified, alkenyl is optionally substituted with one, two, three, four, or five groups selected from the group consisting of carbocyclyl, aryl, heterocyclyl, heteroaryl, oxo, halogen, and hydroxyl.

[0101] The term "alkynyl," as used herein, unless otherwise specified, refers to an aliphatic group containing 1, 2, or 3 carbon-carbon triple bonds and containing a total of 2 to 20 carbon atoms (e.g., (2-6C)alkynyl, (2-4C)alkynyl, or (2-3C)alkynyl). Non-limiting examples of alkynyl include ethynyl, propargyl, homopropargyl, but-2-yn-1-yl, 2-methyl-prop-2-yn-1-yl, and the like. Unless otherwise specified, alkynyl is optionally substituted with 1, 2, 3, 4, or 5 groups selected from the group consisting of carbocyclyl, aryl, heterocyclyl, heteroaryl, oxo, halogen, and hydroxyl.

[0102] By "arginine-rich" is meant that at least 40% of the cationic domain is formed by arginine residues.

[0103] The term "artificial amino acid" as used herein refers to non-biological (e.g., non-proteinogenic) amino acids. For example, artificial amino acids may include synthetic amino acids, modified amino acids (e.g., sugar-modified amino acids), unnatural amino acids, man-made amino acids, spacers, and non-peptide bond spacers. 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 are therefore natural amino acids, not artificial amino acids, in the context of the present invention. Artificial amino acids may include, for example, 6-aminohexanoic acid (X), tetrahydroisoquinoline-3-carboxylic acid (TIC), 1-(amino)cyclohexanecarboxylic acid (Cy), 3-azetidinecarboxylic acid (Az), and 11-aminoundecanoic acid. For the avoidance of doubt, the D enantiomers of naturally occurring L-amino acids are not considered artificial amino acids.

[0104] The term "aryl," as used herein, refers to a carbocyclic ring system containing one, two, or three rings, at least one of which is aromatic. Unsubstituted aryls contain a total of 6 to 14 carbon atoms. The term aryl includes both monovalent and divalent species. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, indanyl, and the like. In certain embodiments, the optionally substituted aryl is an optionally substituted phenyl.

[0105] As used herein, the term "bridged ring system" refers to a ring system in which two rings share more than two atoms, see, for example, Advanced Organic Chemistry, Jerry March, 4th Edition, Wiley Interscience, pp. 131-133, 1992. Examples of bridged heterocyclyl ring systems include aza-bicyclo[2.2.1]heptane, 2-oxa-5-azabicyclo[2.2.1]heptane, aza-bicyclo[2.2.2]octane, aza-bicyclo[3.2.1]octane, quinuclidine, and the like.

[0106] As used herein, the term "carbonyl" refers to the group of the following structure: -C(O)-. Non-limiting examples of carbonyl groups include those found in, for example, acetone, ethyl acetate, proteinogenic amino acids, acetamide, etc.

[0107] As used herein, "cationic" refers to an amino acid or amino acid domain that has an overall positive charge at neutral pH, although positively charged and neutral forms may coexist at this pH. For example, at neutral pH, histidine is believed to be in equilibrium between its neutral and cationic forms, with the pKa of the protonated histidine side chain being 6.3. Non-limiting examples of cationic amino acids include histidine, lysine, and arginine.

[0108] The term "(m-nC)" or "(m-nC) group" used alone or as a prefix, refers to a group that, when unsubstituted, has a total of m to n carbon atoms.

[0109] The term "complementary," as used herein with respect to a nucleobase sequence, refers to a nucleobase sequence having a pattern of consecutive nucleobases that allows an oligonucleotide having the nucleobase sequence to hybridize to another oligonucleotide or nucleic acid under physiological conditions to form a duplex structure. Complementary sequences include Watson-Crick base pairs formed from natural and / or modified nucleobases. Complementary sequences may also include non-Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, hypoxanthine-cytosine) and Hoogsteen base pairs.

[0110] The term "cycloalkyl," as used herein, unless otherwise specified, refers to a saturated carbocyclic ring system containing one or two rings and a total of 3 to 10 carbon atoms. Bicyclic cycloalkyls can be arranged as fused ring systems (two bridgehead carbon atoms are directly bonded to each other), bridged ring systems (two bridgehead carbon atoms are bonded to each other via a covalent linker containing at least one carbon atom), and spirocyclic ring systems (two rings are fused at the same carbon atom). Non-limiting examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, and the like.

[0111] The term "cycloalkenyl," as used herein, unless otherwise specified, refers to a non-aromatic, unsaturated, carbocyclic ring system containing one or two rings, one, two, or three endocyclic double bonds, and a total of three to ten carbon atoms. Bicyclic cycloalkenyls can be arranged as fused ring systems (two bridgehead carbon atoms are directly bonded to each other), bridged ring systems (two bridgehead carbon atoms are bonded to each other via a covalent linker containing at least one carbon atom), and spirocyclic ring systems (two rings are fused at the same carbon atom). Non-limiting examples of cycloalkenyls include cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 3-cyclohexen-1-yl, cyclooctenyl, and the like.

[0112] "Dystrophin" is a rod-shaped cytoskeletal protein and an integral part of the dystrophin-associated glycoprotein complex, which connects the cytoskeleton of muscle fibers through the cell membrane to the surrounding extracellular matrix. Dystrophin contains multiple functional domains. For example, dystrophin contains an actin-binding domain (approximately 14-240 amino acids) and a central rod domain (approximately 253-3040 amino acids). This large central domain is formed by 24 spectrin-like triple-helical elements (approximately 109 amino acids) and shares homology with α-actinin and spectrin. The repeats are typically separated by four proline-rich non-repeat segments, also known as hinge domains. Repeats 15 and 16 are separated by an 18-amino acid stretch that appears to provide the primary site for proteolytic cleavage of dystrophin. Sequence identity between most repeats ranges from 10 to 25%. Each repeat contains three α-helices: 1, 2, and 3. α-Helices 1 and 3 are each formed by seven helical turns, which interact as a coiled coil, presumably via a hydrophobic interface. α-Helix 2 has a more complex structure, formed by a sequence of four and three turns separated by glycine or proline residues. Each repeat is encoded by two exons, usually separated by an intron between amino acids 47 and 48 in the first part of α-helix 2. The second intron is located at a different position within the repeat, usually interspersed on helix 3. Dystrophin also contains a cysteine-rich domain at approximately amino acids 3080–3360, which contains a cysteine-rich segment (i.e., 15 cysteines out of 280 amino acids) that shows homology to the C-terminal domain of Dictyostelium discoideum α-actinin. The carboxy-terminal domain is located at approximately amino acids 3361–3685.

[0113] The amino terminus of dystrophin binds to F-actin, and the carboxy terminus binds to the dystrophin-associated protein complex (DAPC) in the muscle cell membrane. The DAPC contains dystroglycan, sarcoglycan, integrins, and caveolin, and mutations in any of these components cause autosomal recessive muscular dystrophies. Dystrophin deficiency destabilizes the DAPC, resulting in reduced levels of its constituent proteins and progressive fiber damage and membrane leakage. In various forms of muscular dystrophies, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), muscle cells produce either no dystrophin or a modified, functionally defective form of dystrophin, primarily due to incorrect splicing caused by mutations in the gene sequence. Predominant expression of defective dystrophin protein or the complete absence of dystrophin or dystrophin-like proteins leads to the rapid progression of muscle degeneration, as described above. In this regard, a "deficient" dystrophin protein may be characterized by the form of dystrophin produced in a particular subject with DMD or BMD, or by the absence of detectable dystrophin, as known in the art.

[0114] "Exon" refers to a defined portion of a nucleic acid that encodes a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after a portion of preprocessed (or precursor) RNA has been removed by splicing. A mature RNA molecule may be a functional form of messenger RNA (mRNA) or non-coding RNA such as rRNA or tRNA. The human dystrophin gene has approximately 79 exons.

[0115] "Exon skipping" generally refers to the process by which an entire exon or a portion thereof is removed from a given precursor RNA, thereby eliminating its presence in a mature RNA, such as a mature mRNA, that is translated into a protein. Thus, the portion of the protein encoded by the skipped exon is absent from the expressed form of the protein, typically creating a still-functional, but altered, form of the protein. In certain embodiments, the skipped exon is an aberrant exon of the human dystrophin gene, which may contain a mutation or other alteration in its sequence that causes aberrant splicing. In certain embodiments, the skipped exon is exon 44, 45, 51, and / or 53 of the human dystrophin gene.

[0116] The term "halo" or "halogen" as used herein refers to fluoro, chloro, bromo, and iodo.

[0117] The term "heteroalkyl," as used herein, unless otherwise specified, refers to a straight or branched chain hydrocarbon group containing a total of 1 to 30 carbon atoms and at least one heteroatom that is oxygen, nitrogen, or sulfur. For example, a heteroalkyl can be a (2-20C)heteroalkyl, a (2-12C)heteroalkyl, or a (2-10C)heteroalkyl. Non-limiting examples of alkyls include, for example, PEG3.

[0118] By "histidine-rich" is meant that at least 40% of the cationic domains are formed by histidine residues.

[0119] The terms "heteroaryl" and "heteroaromatic," used interchangeably herein, refer to a ring system containing one, two, or three rings, at least one of which is aromatic and contains from one to four (e.g., one, two, or three) heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unsubstituted heteroaryl groups contain a total of one to nine carbon atoms. The term heteroaryl includes both monovalent and divalent species. Examples of heteroaryl groups include monocyclic and bicyclic groups containing from five to twelve ring members, more typically from five to ten ring members. Heteroaryl groups may be, for example, 5- or 6-membered monocyclic rings or 9- or 10-membered bicyclic rings, e.g., bicyclic structures formed from fused 5- and 6-membered rings or two fused 6-membered rings. Each ring may contain up to about four heteroatoms, typically selected from nitrogen, sulfur, and oxygen. Typically, a heteroaryl ring contains up to three heteroatoms, more commonly up to two, e.g., a single heteroatom. In some embodiments, a heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atom of a heteroaryl ring may be basic, as in the case of imidazole or pyridine, or essentially non-basic, as in the case of an indole or pyrrole nitrogen. Generally, the number of basic nitrogen atoms present in a heteroaryl group will be less than five, including all ring amino group substituents.

[0120] Examples of heteroaryl include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazenyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, and isoaryl. Examples include quinolyl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthyridinyl, carbazolyl, phenazinyl, benzisoquinolinyl, pyridopyrazinyl, thieno[2,3-b]furanyl, 2H-furo[3,2-b]-pyranyl, 5H-pyrido[2,3-d]-oxazinyl, 1H-pyrazolo[4,3-d]-oxazolyl, 4H-imidazo[4,5-d]thiazolyl, pyrazino[2,3-d]pyridazinyl, imidazo[2,1-b]thiazolyl, and imidazo[1,2-b][1,2,4]triazinyl. "Heteroaryl" also includes partially aromatic bicyclic or polycyclic ring systems in which at least one ring is aromatic and one or more of the other rings is non-aromatic, saturated, or partially saturated, provided that at least one ring contains one or more heteroatoms selected from nitrogen, oxygen, or sulfur. Examples of partially aromatic heteroaryl groups include, for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo1,2,3,4-tetrahydroquinolinyl, dihydrobenzthienyl, dihydrobenzfuranyl, 2,3-dihydro-benzo[1,4]dioxinyl, benzo[1,3]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 5-membered heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, and tetrazolyl groups. Examples of 6-membered heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and triazinyl. Bicyclic heteroaryl groups include, for example, a benzene ring fused to a 5- or 6-membered ring containing one, two, or three ring heteroatoms; a pyridine ring fused to a 5- or 6-membered ring containing one, two, or three ring heteroatoms; a pyrimidine ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; a pyrrole ring fused to a 5- or 6-membered ring containing one, two, or three ring heteroatoms; a pyrazole ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; a pyrazine ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; an imidazole ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; the oxazole ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; an isoxazole ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; a thiazole ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; an isothiazole ring fused to a 5- or 6-membered ring containing one or two ring heteroatoms; a thiophene ring fused to a 5- or 6-membered ring containing one, two, or three ring heteroatoms; a furan ring fused to a 5- or 6-membered ring containing one, two, or three ring heteroatoms; a cyclohexyl ring fused to a 5- or 6-membered heteroaromatic ring containing one, two, or three ring heteroatoms; and a cyclopentyl ring fused to a 5- or 6-membered heteroaromatic ring containing one, two, or three ring heteroatoms.Illustrative examples of bicyclic heteroaryl groups containing a 6-membered ring fused to a 5-membered ring include, but are not limited to, benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzthiazolyl, benzisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, indolinyl, isoindolinyl, purinyl (e.g., adeninyl, guaninyl), indazolyl, benzodioxolyl, and pyrazolopyridinyl groups. Illustrative examples of bicyclic heteroaryl groups containing two fused 6-membered rings include, but are not limited to, quinolinyl, isoquinolinyl, chromanyl, thiochromanyl, chromenyl, isochromenyl, chromanyl, isochromanyl, benzodioxanyl, quinolidinyl, benzoxazinyl, benzodiazinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, and pteridinyl groups.

[0121] The term "heterocyclyl," as used herein, refers to a ring system containing one, two, or three rings, at least one of which contains one to four (e.g., one, two, or three) heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, with the proviso that the ring system does not contain an aromatic ring further containing an intracyclic heteroatom. Unsubstituted heterocyclyl groups contain a total of two to nine carbon atoms. The term heterocyclyl includes both monovalent and divalent species. Examples of heterocyclyl groups include monocyclic and bicyclic groups containing five to twelve ring members, more usually five to ten ring members. Heterocyclyl groups may be, for example, a five- or six-membered monocyclic ring, or a nine- or ten-membered bicyclic ring, e.g., a bicyclic structure formed from fused five- and six-membered rings or two fused six-membered rings. Each ring may contain up to about four heteroatoms, typically selected from nitrogen, sulfur, and oxygen. Non-limiting examples of heterocyclyl groups include, for example, pyrrolidine, piperazine, piperidine, azepane, 1,4-diazepane, tetrahydrofuran, tetrahydropyran, oxepane, 1,4-dioxepane, tetrahydrothiophene, tetrahydrothiopyran, indoline, benzopyrrolidine, 2,3-dihydrobenzofuran, phthalan, isochroman, and 2,3-dihydrobenzothiophene.

[0122] The terms "increase" or "ameliorate" or "ameliorate" may generally refer to the ability of one or more compounds of the invention to "increase" a relevant physiological or cellular response that may be decreased in a disease state as described herein and that is measured by routine methods in the diagnostic arts. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to one of skill in the art and may include, for example, an improvement in the symptoms or pathology of a neuromuscular or neurological disorder. An "increase" in response may be statistically significant compared to the response produced by a compound or control composition without the antisense compound and may include an increase of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all integers in between.

[0123] The term "internucleoside linkage," as used herein, refers to a group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. An internucleoside linkage may be an unmodified internucleoside linkage or a modified internucleoside linkage. An "unmodified internucleoside linkage" is a phosphate (-O-P(O)(OH)-O-) internucleoside linkage ("phosphodiester phosphate"). A "modified internucleoside linkage" is an internucleoside linkage other than a phosphodiester phosphate. Two major types of modified internucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, morpholino internucleoside linkages, methylphosphonates, and phosphoramidates. Non-limiting examples of non-phosphorus internucleoside linkages include methylenemethylimino (-CH2-N(CH3)-O-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Phosphorothioate linkages are phosphodiester and phosphotriester linkages in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. In some embodiments, the internucleoside linkage is a group of the following structure: [ka] (In the formula, Z is O, S, or Se; Y-XLR 1 and Each X is independently -O-, -S-, -N(-LR 1 )- or L, Each L is independently a covalent bond or a linker (e.g., an optionally substituted C 1-60 an aliphatic linker or optionally substituted C 2-60 heteroaliphatic linker), Each R 1 are independently hydrogen, -SSR 2 , -O-CO-R2 , -S-CO-R 2 , optionally substituted C 1-9 a heterocyclyl or hydrophobic moiety, and Each R 2 C may be independently substituted 1-10 Alkyl, optionally substituted C 2-10 Heteroalkyl, optionally substituted C 6-10 Aryl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 1-9 heterocyclyl or optionally substituted C 1-9 Heterocyclyl C 1-6 It is alkyl. L is a covalent bond, R 1 When L is a covalent bond, R is a hydrogen, Z is oxygen, and all X groups are -O-, the internucleoside group is known as a phosphodiester phosphate. 1 When R is hydrogen, Z is sulfur, and all X groups are -O-, the internucleoside group is known as a phosphorothioate diester. When Z is oxygen, all X groups are -O-, and (1) L is a linker, or (2) R 1 If is not hydrogen, the internucleoside group is known as a phosphotriester. Z is sulfur, all X groups are -O-, and (1) L is a linker, or (2) R 1 When is not hydrogen, the internucleoside group is known as a phosphorothioate triester. Non-limiting examples of phosphorothioate triester and phosphotriester linkages are described in US 2017 / 0037399, the disclosure of which is incorporated herein by reference.

[0124] "Intron" refers to a nucleic acid region (within a gene) that is not translated into protein. Introns are non-coding segments that are transcribed into precursor mRNA (pre-mRNA) and then removed by splicing during formation of the mature RNA.

[0125] The term "morpholino," as used herein with respect to types of oligonucleotides, refers to an oligomer of at least 10 morpholino monomer units interconnected by morpholino internucleoside linkages. A morpholino includes a 5' group and a 3' group. For example, a morpholino may have the following structure: [ka] (In the formula, n is an integer of at least 10 (e.g., 12 to 30) representing the number of morpholino subunits and associated groups L; each B is independently a nucleobase; R 1 is the 5' group (herein R 1 is sometimes referred to as the 5' end), R 2 is the 3' group (herein R 2 is sometimes referred to as the 3' end), and L is (i) a morpholino internucleoside linkage, or (ii) L is R 2 , it is a covalent bond.)

[0126] The 5' group of the morpholino can be, for example, a hydroxyl, a hydrophobic moiety, a phosphate, a diphosphate, a triphosphate, a phosphorothioate, a diphosphorothioate, a triphosphorothioate, a phosphorodithioate, a diphosphorodithioate, a triphosphorodithioate, a phosphonate, a phosphoramidate, a phosphorodiamidate, a bond to a peptide, a bond to a peptide / linker combination, an endosomal escape site, a neutral organic polymer, or the following structures: [ka] , (In the formula, Each R 1 are independently =O, alkyl optionally substituted with -OH and / or -NH, heteroalkyl optionally substituted with -O, -OH and / or -NH, cycloalkyl, heterocyclyl, heteroaryl, or -LR3 where L is a linker that is alkyl, cycloalkyl, heteroalkyl, heterocyclyl, or heteroaryl, and R 3 is the solid support and R 1 Ga-LR 3 There is no more than one R. 1 The groups, together with the nitrogen atom to which they are attached, 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. 2 are independently alkyl optionally substituted with =O, -OH and / or -NH, heteroalkyl optionally substituted with =O, -OH and / or -NH, cycloalkyl, heterocyclyl, or heteroaryl.

[0127] Preferably, the 5' group is a hydroxyl or a group of the following structure: [ka] or [ka]

[0128] A more preferred 5' group is the following structure: [ka]

[0129] The 3' group of the morpholino can be, for example, hydrogen, a hydrophobic moiety, a phosphate, a diphosphate, a triphosphate, a phosphorothioate, a diphosphorothioate, a triphosphorothioate, a phosphorodithioate, a diphosphorothioate, a triphosphorodithioate, a phosphonate, a phosphoramidate, a bond to a peptide, a bond to a peptide / linker combination, a group for conjugation (e.g., maleimide, thiol-substituted alkyl or aryl, alkynyl, or azide-substituted alkyl), an endosomal escape site, or a neutral organic polymer.

[0130] In a conjugate of an oligonucleotide that is a morpholino and a peptide covalently bonded or linked to the oligonucleotide, the preferred 3' group is the bond to the peptide or the bond to the peptide / linker combination.

[0131] The term "morpholino internucleoside linkage," as used herein, refers to a divalent group of the following structure: [ka] , (In the formula, Z is O or S; X 1 is a bond, —CH—, or —O—; X 2 is a bond, —CH—O—, or —O—, and Y is -NR2, where each R is independently H or C 1-6 alkyl (e.g., methyl), or both R together with the nitrogen atom to which they are attached are C 2-9 Forming a heterocyclyl (eg, N-piperazinyl). However, X 1 and X 2 where only one of the morpholino internucleoside linkages is a bond. Preferably, the morpholino internucleoside linkage is -P(O)(Nme2)O-. The morpholino internucleoside linkage is attached to the nitrogen atom of the morpholino ring of the morpholino subunit through its phosphorus atom.

[0132] The term "morpholino subunit" as used herein refers to the following structure: [ka] , (wherein B is a nucleobase).

[0133] The term "nucleobase" as used herein refers to the nitrogen-containing heterocyclic ring found at the 1' position of the ribofuranose / 2'-deoxyribofuranose of a nucleoside. Nucleobases may be unmodified or modified. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, alkyl- or alkynyl-substituted pyrimidines, alkyl-substituted purines, and N-2, N-6, and O-6 substituted purines, as well as synthetic and natural nucleobases such as 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine. These include adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 5-trifluoromethyluracil, 5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids. For example, 5-substituted pyrimidines, 6-azapyrimidines, and N2-, N6-, and / or O6-substituted purines. The stability of nucleic acid duplexes can be enhanced using, for example, 5-methylcytosine.Non-limiting examples of nucleobases include 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (—C≡C—CH3) uracil, 5-propynylcytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted propionates. phosphorus, 5-halo, especially 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp). Modified nucleobases can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deazaadenine, 7-deazaguanine, 2-aminopyridine, or 2-pyridone.Additional nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, The Concise Encyclopedia Of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288; and Chapters 6 and 15, Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, 163-166 and 442-443.

[0134] The term "nucleoside" as used herein refers to sugar-nucleobase compounds and groups known in the art, as well as modified or unmodified 2'-deoxyribofuranose-nucleobase compounds and groups known in the art. The sugar may be ribofuranose. The sugar may be modified or unmodified. An unmodified ribofuranose-nucleobase is a ribofuranose having an anomeric carbon bond to the unmodified nucleobase. Unmodified ribofuranose-nucleobases are adenosine, cytidine, guanosine, and uridine. Unmodified 2'-deoxyribofuranose-nucleobase compounds are 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, and thymidine. 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 the replacement of an unmodified nucleobase with a modified nucleobase. The sugar modification may be, for example, 2'-substituted, locked, carbocyclized, or non-locked. The 2'-substitution may replace the 2'-hydroxyl of the ribofuranose with, for example, 2'-fluoro, 2'-methoxy, or 2'-(2-methoxy)ethoxy. Alternatively, the 2'-substitution may be a 2'-(ara) substitution, corresponding to the following structure: [ka] where B is a nucleobase and R is a 2'-(ara) substitution (e.g., fluoro). The 2'-(ara) substituents are known in the art and can be identical to other 2'-substituents described herein. In some embodiments, the 2'-(ara) substitution is a 2'-(ara)-F substitution (R is fluoro). A locking modification is the incorporation of a bridge between the 4'-carbon atom and the 2'-carbon atom of a ribofuranose. Nucleosides with locking modifications are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. Bridged nucleic acids are typically used as affinity-enhancing nucleosides. "Nucleoside" can also refer to a morpholino subunit.

[0135] The term "nucleotide" as used herein refers to an internucleoside linkage or to a nucleotide having the following structure: -X 1 -P(X 2 )(R 1 ) 2, represents a nucleoside bonded to a monovalent group of the formula 1 is O, S, or NH, and X 2 is absent, ═O, or ═S, and each R 1 are independently -OH, -N(R 2 )2, or —O—CH2CH2CN, where each R 2 are independently optionally substituted alkyl, or both R 2 The groups, together with the nitrogen atom to which they are attached, are joined to form an optionally substituted heterocyclyl.

[0136] The term "oligonucleotide" as used herein refers to a structure containing 10 or more consecutive nucleosides covalently linked by internucleoside linkages, a morpholino containing 10 or more morpholino subunits, or a peptide nucleic acid containing 10 or more peptide nucleic acid subunits. Preferably, the oligonucleotide is a morpholino. In some embodiments, the oligonucleotide is a phosphorothioate (PS) oligonucleotide. In some embodiments, the oligonucleotide is a 2'-O-alkyl oligoribonucleotide, e.g., a 2'-O-methyl oligoribonucleotide. In some embodiments, the oligonucleotide is a 2'-O-alkyl phosphorothioate, e.g., a 2'-O-methyl phosphorothioate.

[0137] The term "optionally substituted" refers to a group, structure, or molecule that may be substituted or unsubstituted as described for each group. 1 The term "any CH, CH, CH group or heteroatom (i.e., NH) within the group may be substituted" is used to refer to 1 It means that (any) one of the hydrogen radicals of the group is replaced by the associated defined group.

[0138] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with individual (e.g., human) tissue and that are free from excessive toxicity, irritation, allergic response, or other problem complications, commensurate with a reasonable benefit / risk ratio.

[0139] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of the conjugate, oligonucleotide, or peptide disclosed herein. Pharmaceutically acceptable salts of any compound described herein may include those that are within the scope of sound medical judgment, suitable for use in contact with human and animal tissues, and lack undue toxicity, irritation, or allergic reaction, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977, and Pharmaceutical Salts: Properties, Selection, and Use (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free base with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, These include lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like.Representative alkali and alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations (including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine).

[0140] As used herein, the term "pharmaceutical composition" refers to a composition containing an oligonucleotide as described herein, formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease of interest.

[0141] The terms "reduce" or "inhibit" may generally refer to the ability of one or more compounds of the invention to "reduce" a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured by routine techniques in the diagnostic arts. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to one of skill in the art and may include, for example, a reduction in the symptoms or pathology of a neuromuscular or neurological disorder. A "reduction" in response may be statistically significant compared to the response produced by a compound or control composition without the antisense compound and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction, including all integers in between.

[0142] "Steric inhibition" refers to a therapeutic approach in which a complementary antisense oligonucleotide binds to a target sequence present in an mRNA transcript and prevents the transcript from binding to other molecules, which may include proteins or RNA species. The steric inhibition mechanism may be used to treat DM1 or DM2 or other genetic disorders, potentially correcting downstream mis-splicing pathology and corresponding phenotypic manifestations, or providing therapeutic benefit through other mechanisms of action.

[0143] As used herein, the term "subject" refers to a human or non-human animal (e.g., a mammal) that has been determined by a qualified professional (e.g., a doctor or nurse) to be suffering from or at risk for a disease, disorder, or condition, with or without art-known clinical testing of a sample from the subject. Non-limiting examples of diseases, disorders, and conditions include DMD, BMD, FSHD, DM1, DM2, CMT1a, CMT2a, FCD, FA, and SMA.

[0144] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring or structures that can replace the furanose ring of a nucleoside. The sugars contained in the nucleosides of the invention can also be non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures include simple variations on the naturally occurring furanose ring (e.g., a six-membered ring). Sugar substitutes can also include sugar surrogates in which the furanose ring is replaced with another ring system, such as, for example, a morpholino ring system or a hexitol ring system. Non-limiting examples of useful sugar moieties that can be included in the oligonucleotides of the invention include β-D-ribose, β-D-2′-deoxyribose, substituted sugars (e.g., 2′, 5′, and bis-substituted sugars), 4′-S-sugars (e.g., 4′-S-ribose, 4′-S-2′-deoxyribose, and 4′-S-2′-substituted riboses), bicyclic sugar moieties (e.g., bicyclic sugars derived from 2′-O-CH2-4′ or 2′-O-(CH2)2-4′ bridged ribose), and sugar surrogates (where the ribose ring is replaced with a morpholino or hexitol ring system).

[0145] As used herein, the terms "treatment" and "treating" refer to the medical management of a subject with the goal of improving, ameliorating, or stabilizing a disease, disorder, or condition (e.g., DMD, BMD, FSHD, DM1, DM2, CMT1a, FCD, FA, or SMA). This term includes active treatment (treatment for the improvement of DMD, BMD, FSHD, DM1, DM2, CMT1a, FCD, FA, and SMA), palliative treatment (treatment designed to relieve the symptoms of DMD, BMD, FSHD, DM1, DM2, CMT1a, FCD, FA, and SMA), and supportive treatment (treatment to supplement other treatments).

[0146] Throughout this description and the claims, the words "comprise" and "contain" and variations thereof mean "including, but not limited to," and are not intended to (and do not) exclude other moieties, additives, components, elements, or steps. Throughout this description and the claims, the singular encompasses the plural unless the context dictates otherwise. In particular, where the indefinite article is used, the specification is understood to contemplate the plural as well as the singular, unless the context requires otherwise.

[0147] All references to "complexes" also refer to salts and solvates thereof. As will be understood by those skilled in the art, the complexes disclosed herein may contain multiple ionizable and / or protonizable groups. Thus, the complexes of the present invention may be used in salt form, such as an acid addition salt, or in a substantially neutral form.

[0148] All references to "oligonucleotides" also refer to salts and solvates thereof.

[0149] Unless otherwise specified, all peptides are shown herein in N-terminal to C-terminal (left to right) orientation. Unless otherwise specified, all oligonucleotides are shown herein in 5' to 3' orientation (left to right).

[0150] It is understood that any feature, integer, property, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention is also applicable to other aspects, embodiments, or examples described herein, except where inconsistent therewith. All of the features disclosed in this specification (including the accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. [Brief explanation of the drawings]

[0151] [Figure 1] Figure 1 is a graph showing the level of Dmd exon 23 skipping in the quadriceps muscle of wild-type mice treated with PPMO. Results are mean ± standard deviation, n = 3–5. [Figure 2] Figure 2 is a graph showing the level of Dmd exon 23 skipping in the myocardium of wild-type mice administered PPMO. Results are mean ± standard deviation, n = 3 to 5. [Figure 3] Figure 3 is a schematic diagram of PMO synthesis. DETAILED DESCRIPTION OF THE INVENTION

[0152] Typically, the present invention provides novel peptides that can be advantageously used to deliver therapeutic or diagnostic molecules, such as oligonucleotides. In some embodiments, the peptide is present in a complex with the peptide and a therapeutic or diagnostic molecule (e.g., an oligonucleotide), where the peptide is covalently linked to the therapeutic or diagnostic molecule via a covalent bond or a linker. As further described below, the complex can be used to treat genetic disorders, such as neuromuscular and neurological disorders, including DMD, BMD, FSHD, DM1, DM2, CMT1a, CMT2a, FCD, FA, and SMA.

[0153] In some embodiments, the complex comprises a peptide and an oligonucleotide linked to the peptide by a covalent bond (i.e., a direct bond) or a non-cationic linker (e.g., a linker comprising a total of one amino acid), wherein the peptide comprises a total of one hydrophobic domain and a total of one cationic domain, wherein the cationic domain comprises at least one cationic amino acid residue, and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues.

[0154] In some embodiments, the complex comprises a peptide and an oligonucleotide linked to the peptide by a covalent bond (i.e., a direct bond) or a non-cationic linker (e.g., a linker comprising a total of one amino acid or an aliphatic dicarboxylic acid linker), wherein the peptide comprises a total of one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, the at least one cationic domain comprising a total of one cationic amino acid residue, and the hydrophobic domain comprising at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues.

[0155] In some embodiments, the complex comprises a peptide and an oligonucleotide covalently (i.e., directly) or via a non-cationic linker (e.g., via a linker comprising a total of one amino acid) linked to the peptide, wherein the peptide comprises a total of one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, all cationic domains comprising a total of five or fewer cationic amino acid residues, and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues.

[0156] In some embodiments, the complex comprises a peptide and an oligonucleotide covalently or via a non-cationic linker (e.g., via a linker comprising a total of one amino acid) linked to the peptide, wherein the peptide comprises a total of one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, at least one-third of the amino acid residues in the N-terminal cationic domain are histidine, and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues.

[0157] In some embodiments, the conjugate comprises a peptide and an oligonucleotide linked to the peptide by a covalent bond (i.e., a direct bond) or a non-cationic linker (e.g., a linker comprising a total of one amino acid), wherein the peptide is selected from the group consisting of RBRRFQILYRBHBH (SEQ ID NO: 447), RBRFQILYRBHBH (SEQ ID NO: 448), RBFQILYRBHBH (SEQ ID NO: 449), RFQILYRBHBH (SEQ ID NO: 450), FQILYRBHBH (SEQ ID NO: 451), R BRRBRRFQILYBHBHB (SEQ ID NO: 452), RBRRBRRFQILYHBH (SEQ ID NO: 453), RBRRBRRFQILYBH (SEQ ID NO: 454), RBRRBRRFQILYH (SEQ ID NO: 455), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBH (SEQ ID NO: 457), RRBRRFQILYRBHBH (SEQ ID NO: 458), BRRFQILYRBHBH (SEQ ID NO: 459), RRFQILYRBHBH (SEQ ID NO: 460), RBRRBRRFQILY RBHB (SEQ ID NO: 461), RBRRBRRFQILYRBH (SEQ ID NO: 462), RBRRBRRFQILYRB (SEQ ID NO: 463), RBRRBRRFQILYR (SEQ ID NO: 464), HRBHRBHRBFQILYHRBRH (SEQ ID NO: 465), HRBHRBHRBFQILYHRBHRBHR (SEQ ID NO: 466), HRBHRBFQILYHRBHR (SEQ ID NO: 467), HRBFQILYHR (SEQ ID NO: 468), RBRBRBFQILYRBRBR (SEQ ID NO: 469), RBRBFQIL Selected from the group consisting of YRBR (SEQ ID NO: 470), RBFQILYR (SEQ ID NO: 471), HRHRHRFQILYHRHRHR (SEQ ID NO: 472), HRHRFQILYHRHR (SEQ ID NO: 473), HRFQILYHR (SEQ ID NO: 474), RRRRRRFQILY (SEQ ID NO: 475), FQILYRRRRRR (SEQ ID NO: 476), RRRRRRFQILYRRRRRR (SEQ ID NO: 477), RBRRBRFQILYBR (SEQ ID NO: 478), and RBRRBRFQILY (SEQ ID NO: 479).

[0158] In some embodiments, the peptide of the complex is (RBRRBRFQILYBR (SEQ ID NO:478)). In some embodiments, the peptide of the complex is (RBRRBRFQILY (SEQ ID NO:479)). In some embodiments, the peptide of the complex is (RBRRBRRFQILY (SEQ ID NO:456)). In some embodiments, the peptide of the complex is (RRFQILYRBHBH (SEQ ID NO:460)). In some embodiments, the peptide of the complex is rBrrBrfqilyBrBr (SEQ ID NO:510), where lowercase letters indicate D-amino acids.

[0159] In some embodiments, the conjugate comprises a peptide and an oligonucleotide linked to the peptide by a covalent bond (i.e., a direct bond) or a non-cationic linker (e.g., a linker comprising a total of one amino acid), the peptide being selected from the group consisting of RBRRFQILYRBHBHB (SEQ ID NO: 481), RBRFQILYRBHBHB (SEQ ID NO: 482), RBFQILYRBHBHB (SEQ ID NO: 483), RFQILYRBHBHB (SEQ ID NO: 484), FQILYRBHBHB, RBRRBRRFQILYBHBHB (SEQ ID NO: 452), R BRRBRRFQILYHBHB (SEQ ID NO: 486), RBRRBRRFQILYBHB (SEQ ID NO: 487), RBRRBRRFQILYHB (SEQ ID NO: 488), RBRRBRRFQILYB (SEQ ID NO: 489), RBRRBRRFQILYE (SEQ ID NO: 416), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBHB (SEQ ID NO: 490), RRBRRFQILYRBHBHB (SEQ ID NO: 491), BRRFQILYRBHBHB (SEQ ID NO: 492), RRFQILYRBHBHB (SEQ ID NO: 493), RRFQILYR BHBHE (SEQ ID NO: 417), RBRRBRRFQILYRBHBB (SEQ ID NO: 494), RBRRBRRFQILYRBHB (SEQ ID NO: 461), RBRRBRRFQILYRBB (SEQ ID NO: 495), RBRRBRRFQILYRB (SEQ ID NO: 463), HRBHRBHRBFQILYHRBRHB (SEQ ID NO: 496), HRBHRBHRBFQILYHRBHRBHRB (SEQ ID NO: 497), HRBHRBFQILYHRBHRB (SEQ ID NO: 498), HRBFQILYHRB (SEQ ID NO: 499), RBRBRBFQILYRBRBRB (SEQ ID NO: 500), RBRBFQILYRBRB (SEQ ID NO: 501), RBFQILYRB (SEQ ID NO: 502), HRHRHRFQILYHRHRHRB (SEQ ID NO: 503), HRHRFQILYHRHRB (SEQ ID NO: 504), HRFQILYHRB (SEQ ID NO: 505), RRRRRRFQILYB (SEQ ID NO: 506), FQILYRRRRRRB (SEQ ID NO: 507), RRRRRRFQILYRRRRRRB (SEQ ID NO: 508), RBRRBRFQILYBRE (SEQ ID NO: 415), and RBRRBRFQILYE (SEQ ID NO: 509).In another embodiment, the conjugate comprises a peptide as described above in this paragraph, but with the C-terminal amino acid removed or removed and replaced with, for example, a linker described herein (e.g., a B linker or an E linker).

[0160] In some embodiments, the peptide of the complex is Dpep1.9b-del2(RBRRBRFQILYBRE) (SEQ ID NO: 415). In some embodiments, the peptide of the complex is Dpep1.9b-del4(RBRRBRFQILYE) (SEQ ID NO: 509). In some embodiments, the peptide of the complex is peptide E5-E(RBRRBRRFQILYE) (SEQ ID NO: 416). In some embodiments, the peptide of the complex is peptide G5-E(RRFQILYRBHBHE) (SEQ ID NO: 417). In some embodiments, the peptide of the complex is rBrrBrfqilyBrBre (SEQ ID NO: 405), where lowercase letters indicate D-amino acids.

[0161] The peptide may comprise at least one positively charged domain and at least one hydrophobic domain. Without being bound by theory, the peptide may act as a cell-penetrating peptide, enhancing the activity of the conjugated oligonucleotide, for example, by improving intracellular delivery of the conjugated oligonucleotide. Advantageously, as illustrated in the Examples below, the conjugates of the present disclosure exhibit reduced toxicity compared to certain alternative peptide structures.

[0162] In the case of therapeutic molecules that are oligonucleotides, the oligonucleotides may be, for example, antisense oligonucleotides complementary to a target sequence within or near any one of exons 8 to 55 (e.g., exon 8, exon 23, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53, or exon 55) of the human dystrophin gene. Alternatively, the oligonucleotides may be complementary to a target sequence within or near intron 7 of the transcript of the human SMN2 gene. Alternatively, the oligonucleotides may be complementary to a CUG repeat sequence (e.g., at least 9 consecutive nucleobases are complementary to a CUG repeat sequence). Alternatively, the oligonucleotides may be complementary to a CCUG repeat sequence.

[0163] In some embodiments, the antisense oligonucleotide sequence is for inducing exon skipping of a single exon of the dystrophin gene (e.g., having a target sequence in the dystrophin transcript) for use in treating DMD. In some embodiments, the single exon is selected from any exon involved in DMD, and may be any exon of the dystrophin gene (e.g., the human dystrophin gene), such as exons 8, 23, 44, 45, 50, 51, 52, 53, or 55. In some embodiments, the antisense oligonucleotide sequence is for promoting the inclusion of exon 7 in the splicing of SMN2 pre-mRNA and may therefore be used in the treatment of spinal muscular atrophy (SMA). In some embodiments, the antisense oligonucleotide sequence binds to SMN2 pre-mRNA intron 7 and promotes the inclusion of exon 7.

[0164] In some embodiments, the antisense oligonucleotide sequence is intended to reduce the deleterious effects associated with the presence of a CTG repeat expansion in the DMPK gene. expBy targeting the CUG repeat sequence (e.g., the oligonucleotide may have at least 9 consecutive nucleobases complementary to the CUG repeat sequence), the oligonucleotide can ameliorate the pathology associated with this gain-of-function repeat expansion in DM1.

[0165] In some embodiments, the antisense oligonucleotide sequence is intended to reduce the deleterious effects associated with the presence of a CCTG repeat expansion in the CNBP gene. exp By targeting the CCUG repeat sequence (e.g., the oligonucleotide may have at least 8 consecutive nucleobases complementary to the CCUG repeat sequence), the oligonucleotide can ameliorate pathology associated with this gain-of-function repeat expansion in DM2.

[0166] In some embodiments, the oligonucleotide of the conjugate is an oligonucleotide complementary to the pre-mRNA of the target gene. Thus, as used herein, targeting a gene also includes targeting the RNA transcript (e.g., pre-mRNA transcript) of that gene.

[0167] In some embodiments, an oligonucleotide complementary to the pre-mRNA of a gene target causes a steric inhibition event that alters the pre-mRNA, resulting in an altered mRNA and, ultimately, a protein of altered sequence. In some embodiments, the gene target is the dystrophin gene. In some embodiments, the steric inhibition event may be exon inclusion or exon skipping. In some embodiments, the steric inhibition event is exon skipping, for example, exon skipping of a single exon of the dystrophin gene. Optionally, lysine residues may be added to one or both ends of the oligonucleotide (such as a PMO or PNA) before conjugation to the peptide to improve water solubility.

[0168] In some embodiments, the oligonucleotide has a molecular weight of less than 15,000 Da, such as less than 10,000 Da, such as less than 5,000 Da, or such as less than 3,000 Da.

[0169] In some embodiments, the peptide is covalently attached to the oligonucleotide at the C-terminus.

[0170] In some embodiments, the peptide is covalently attached to the oligonucleotide via a linker, which may function as a spacer to separate the peptide sequence from the oligonucleotide.

[0171] The linker may be selected from any suitable sequence.

[0172] In some embodiments, a linker is present between the peptide and the oligonucleotide. In some embodiments, the linker is a separate group from the peptide and the oligonucleotide. Thus, the linker may include an artificial amino acid.

[0173] In some embodiments, the conjugate comprises a peptide covalently linked to an oligonucleotide via a linker. In some embodiments, the conjugate consists of the following structure: [Peptide]-[Linker]-[Oligonucleotide]

[0174] In some embodiments, the complex consists of the following structure: [Peptide]-[Linker]-[Oligonucleotide]

[0175] In some embodiments, any of the peptides described herein may be used in a conjugate according to the present invention.

[0176] Preferably, the oligonucleotide is a morpholino (more preferably a morpholino in which all morpholino internucleoside linkages are -P(O)(Nme2)O-).

[0177] In some embodiments, the oligonucleotide is a phosphorothioate, e.g., as described herein. In some embodiments, the oligonucleotide is a 2'-O-alkyl oligonucleotide, e.g., a 2'-O-methyl oligonucleotide, and / or a 2'-O-alkyl phosphorothioate, e.g., a 2'-O-methyl phosphorothioate.

[0178] In some embodiments, the oligonucleotide is a PNA.

[0179] Additional information regarding peptides, conjugates comprising peptides, linkers, molecules to which peptides are linked to form conjugates, pharmaceutical compositions, and methods is provided below.

[0180] peptide The present invention relates to cell-penetrating peptides used to deliver therapeutic cargo molecules in the treatment of medical diseases.

[0181] A peptide has a continuous sequence of a single molecule, and therefore the domains of the peptide are continuous. In some embodiments, the peptide comprises multiple domains linearly arranged between the N- and C-termini. In some embodiments, the domains are selected from the cationic and hydrophobic domains described above. In some embodiments, the peptide consists of a cationic domain and a hydrophobic domain, as defined herein.

[0182] While each domain shares common sequence characteristics as described herein, the exact sequence of each domain can vary and be modified. Thus, various sequences are possible for each domain. Each possible domain sequence combination results in a variety of peptide structures, each of which constitutes part of the present invention. The characteristics of the peptide structures are described herein.

[0183] In some embodiments, the peptide comprises a total of one hydrophobic domain and a total of one cationic domain, the cationic domain comprising at least one cationic amino acid residue, and the hydrophobic domain comprising at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40 or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues.

[0184] In some embodiments, the peptide comprises one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, at least one cationic domain comprising one cationic amino acid residue, and the hydrophobic domain comprising at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues.

[0185] In some embodiments, the peptide comprises one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, all cationic domains comprising five or fewer cationic amino acid residues, and the hydrophobic domain comprising at least three amino acid residues, provided that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues.

[0186] In some embodiments, the peptide comprises a total of one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, at least one-third of the amino acid residues in the N-terminal cationic domain are histidines, and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues.

[0187] In some embodiments, the peptide is RBRRFQILYRBHBH (SEQ ID NO: 447), RBRFQILYRBHBH (SEQ ID NO: 448), RBFQILYRBHBH (SEQ ID NO: 449), RFQILYRBHBH (SEQ ID NO: 450), FQILYRBHBH (SEQ ID NO: 451), RBRRBRRFQILYBHBHB (SEQ ID NO: 452), RBRRBRRFQILYHBH (SEQ ID NO: 453), RBRRBRRFQILYBH (SEQ ID NO: 4 54), RBRRBRRFQILYH (SEQ ID NO: 455), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBH (SEQ ID NO: 457), RRBRRFQILYRBHBH (SEQ ID NO: 458), BRRFQILYRBHBH (SEQ ID NO: 459), RRFQILYRBHBH (SEQ ID NO: 460), RBRRBRRFQILYRBHB (SEQ ID NO: 461), RBRRBRRFQILYRBH (SEQ ID NO: 462), RBRRBRRF QILYRB (SEQ ID NO: 463), RBRRBRRFQILYR (SEQ ID NO: 464), HRBHRBHRBFQILYHRBRH (SEQ ID NO: 465), HRBHRBHRBFQILYHRBHRBHR (SEQ ID NO: 466), HRBHRBFQILYHRBHR (SEQ ID NO: 467), HRBFQILYHR (SEQ ID NO: 468), RBRBRBFQILYRBRBR (SEQ ID NO: 469), RBRBFQILYRBR (SEQ ID NO: 470), RBFQILYR (SEQ ID NO: 471) SEQ ID NO: 471), HRHRHRFQILYHRHRHR (SEQ ID NO: 472), HRHRFQILYHRHR (SEQ ID NO: 473), HRFQILYHR (SEQ ID NO: 474), RRRRRRFQILY (SEQ ID NO: 475), FQILYRRRRRR (SEQ ID NO: 476), RRRRRRFQILYRRRRRR (SEQ ID NO: 477), RBRRBRFQILYBR (SEQ ID NO: 478), and RBRRBRFQILY (SEQ ID NO: 479). In some embodiments, the peptide is rBrrBrfqilyBrBr (SEQ ID NO: 510), where lowercase letters indicate D-amino acids.

[0188] In some embodiments, the peptide is RBRRFQILYRBHBHB (SEQ ID NO: 481), RBRFQILYRBHBHB (SEQ ID NO: 482), RBFQILYRBHBHB (SEQ ID NO: 483), RFQILYRBHBHB (SEQ ID NO: 484), FQILYRBHBHB (SEQ ID NO: 485), RBRRBRRFQILYBHBHB (SEQ ID NO: 452), RBRRBRRFQILYHBHB (SEQ ID NO: 486), RBRRBRRFQILYBHB (SEQ ID NO: 487), RBRRBRRFQILYHB (SEQ ID NO: 488), RBRRBRRFQILYB (SEQ ID NO: 489), RBRRBRRFQILYE (SEQ ID NO: 416), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBHB (SEQ ID NO: 490), RRBRRFQILYRBHBHB (SEQ ID NO: 491), BRRFQILYRBHBHB (SEQ ID NO: 492), RRFQILYRBHBHB (SEQ ID NO: 493), RRFQILYRBHBHE (SEQ ID NO: 417), RBRRBRRFQILYRBHBB (SEQ ID NO: 494), R BRRBRRFQILYRBHB (SEQ ID NO: 461), RBRRBRRFQILYRBB (SEQ ID NO: 495), RBRRBRRFQILYRB (SEQ ID NO: 463), HRBHRBHRBFQILYHRBRHB (SEQ ID NO: 496), HRBHRBHRBFQILYHRBHRBHRB (SEQ ID NO: 497), HRBHRBFQILYHRBHRB (SEQ ID NO: 498), HRBFQILYHRB (SEQ ID NO: 499), RBRBRBFQILYRBRBRB (SEQ ID NO: 500), RBRBFQILYRBRB (SEQ ID NO: No. 501), RBFQILYRB (SEQ ID NO: 502), HRHRHRFQILYHRHRHRB (SEQ ID NO: 503), HRHRFQILYHRHRB (SEQ ID NO: 504), HRFQILYHRB (SEQ ID NO: 505), RRRRRRFQILYB (SEQ ID NO: 506), FQILYRRRRRRB (SEQ ID NO: 507), RRRRRRFQILYRRRRRRB (SEQ ID NO: 508), RBRRBRFQILYBRE (SEQ ID NO: 415), and RBRRBRFQILYE (SEQ ID NO: 509). In some embodiments, the peptide of the complex is Dpep1.9b-del2(RBRRBRFQILYBRE) (SEQ ID NO: 415).In some embodiments, the peptide of the complex is Dpep1.9b-del4(RBRRBRFQILYE) (SEQ ID NO: 509). In some embodiments, the peptide of the complex is peptide E5-E(RBRRBRRFQILYE) (SEQ ID NO: 416). In some embodiments, the peptide of the complex is peptide G5-E(RRFQILYRBHBHE) (SEQ ID NO: 417). In some embodiments, the peptide of the complex is rBrrBrfqilyBrBre (SEQ ID NO: 405), where lowercase letters indicate D-amino acids. In another aspect, the peptide is as described above in this paragraph, but with the C-terminal amino acid removed or removed and replaced with, for example, a linker described herein (e.g., a B linker or an E linker).

[0189] 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.

[0190] In some embodiments, the peptide contains only, and thus consists of, naturally occurring amino acid residues.

[0191] In some embodiments, an artificial amino acid, such as 6-aminohexanoic acid, typically used in cell-penetrating peptides, is replaced with a naturally occurring amino acid, hi some embodiments, an artificial amino acid, such as 6-aminohexanoic acid, typically used in cell-penetrating peptides, is replaced with an amino acid selected from beta-alanine, serine, proline, arginine, and histidine or hydroxyproline.

[0192] In some embodiments, the aminohexanoic acid is replaced by beta-alanine. In some embodiments, the 6-aminohexanoic acid is replaced by beta-alanine.

[0193] In some embodiments, aminohexanoic acid is substituted with histidine. In some embodiments, 6-aminohexanoic acid is substituted with histidine.

[0194] In some embodiments, the aminohexanoic acid is substituted with hydroxyproline. In some embodiments, the 6-aminohexanoic acid is substituted with hydroxyproline.

[0195] In some embodiments, artificial amino acids such as 6-aminohexanoic acid, which are typically used in cell-penetrating peptides, can be replaced with any combination of beta-alanine, serine, proline, arginine, and histidine or hydroxyproline, for example, any combination of beta-alanine, histidine, and hydroxyproline.

[0196] In some embodiments, peptides having a total length of 40 amino acid residues or less are provided, the peptides comprising two or more cationic domains, each comprising at least four amino acid residues, and one or more hydrophobic domains, each comprising at least three amino acid residues, wherein at least one cationic domain comprises histidine residues. In some embodiments, at least one cationic domain is histidine-rich.

[0197] In some embodiments, histidine-rich is defined herein with respect to the cationic domain.

[0198] In some embodiments, the peptide comprises L-amino acids, e.g., all chiral amino acids are L-amino acids. In some embodiments, the peptide comprises D-amino acids, e.g., all chiral amino acids are D-amino acids. In some embodiments, the peptide comprises L-amino acids and D-amino acids.

[0199] In some embodiments, the peptide comprises 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 acids. In some embodiments, in peptides comprising D-amino acids, the remaining amino acids in the peptide, if any, are L-amino acids or non-chiral amino acids.

[0200] In some embodiments, additional groups such as linkers, terminal modifications and / or oligonucleotides may be present.

[0201] In some embodiments, the peptide is N-terminally modified.

[0202] In some embodiments, the peptide is N-acetylated, N-methylated, N-trifluoroacetylated, N-trifluoromethylsulfonylated, or N-methylsulfonylated, hi some embodiments, the peptide is N-acetylated.

[0203] Optionally, the N-terminus of the peptide may be unmodified.

[0204] In some embodiments, the peptide is C-terminally modified.

[0205] In some embodiments, the peptide comprises a C-terminal modification selected from a carboxy group, a thioacid group, an aminooxy group, a hydrazino group, a thioester group, an azide group, a strained alkyne, a strained alkene, an aldehyde group, a thiol group, or a haloacetyl group.

[0206] Advantageously, the C-terminal modification provides a means for attaching the peptide to the oligonucleotide.

[0207] Thus, the C-terminal modification may comprise a linker, or vice versa. In some embodiments, the C-terminal modification may consist of a linker, or vice versa. Suitable linkers are described herein.

[0208] In some embodiments, the peptide comprises a C-terminal carboxyl group.

[0209] In some embodiments, the C-terminal carboxyl group is provided by a glycine residue or a beta-alanine residue.

[0210] In some embodiments, the C-terminal carboxyl group is provided by a beta-alanine residue. In some embodiments, the C-terminal beta-alanine residue is a linker.

[0211] The peptides of the present invention may have a total length of 40 amino acid residues or less, and therefore may be considered oligopeptides.

[0212] In some embodiments, the peptide has a total length of 3 to 30 amino acid residues, for example, 5 to 25 amino acid residues, 10 to 25 amino acid residues, 13 to 23 amino acid residues, or 15 to 20 amino acid residues.

[0213] In some embodiments, the peptide has an overall length of at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 amino acid residues.

[0214] In some embodiments, the peptide is capable of penetrating cells, and therefore can be considered a cell-penetrating peptide.

[0215] In some embodiments, the peptide is for attaching to an oligonucleotide. In some embodiments, the peptide is for transporting the oligonucleotide to a target cell. In some embodiments, the peptide is for delivering the oligonucleotide to a target cell. Thus, the peptide can be considered a carrier peptide.

[0216] In some embodiments, the peptides are capable of penetrating into cells and tissues, for example, into the nucleus of cells, and in some embodiments, into muscle tissue.

[0217] Cationic Domain In the peptides described herein, each cationic domain comprises at least one cationic amino acid residue. In some embodiments, each cationic domain comprises at least one cationic amino acid residue, and at least one of the cationic domains comprises a total of one cationic amino acid residue. In some embodiments, all of the cationic domains comprise a total of seven or fewer (e.g., five or fewer) cationic amino acid residues.

[0218] In some embodiments, the peptide comprises a total of one or two cationic domains flanking a hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue.

[0219] In some embodiments, the peptide comprises up to four cationic domains, for example, up to three cationic domains.

[0220] In some embodiments, the peptide comprises two cationic domains, eg, a total of two cationic domains.

[0221] In some embodiments, the peptide comprises two or more cationic domains, each having a length of at least four amino acid residues.

[0222] In some embodiments, each cationic domain has a length of 4 to 12 amino acid residues, for example, a length of 4 to 7 amino acid residues.

[0223] In some embodiments, each cationic domain has a length of 4, 5, 6, or 7 amino acid residues.

[0224] In some embodiments, each cationic domain is of a similar length, eg, each cationic domain is the same length.

[0225] In some embodiments, each cationic domain contains cationic amino acids and may contain polar and / or nonpolar amino acids.

[0226] The non-polar amino acids may be selected from alanine, beta-alanine, proline, glycine, cysteine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine.

[0227] Polar amino acids may be selected from serine, asparagine, hydroxyproline, histidine, arginine, threonine, tyrosine, and glutamine. In some embodiments, the selected polar amino acids do not carry a negative charge.

[0228] The cationic amino acid may be selected from arginine, histidine, and lysine.

[0229] In some embodiments, each cationic domain does not contain anionic or negatively charged amino acid residues, hi some embodiments, each cationic domain contains arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues.

[0230] In some embodiments, each cationic domain consists of arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues.

[0231] In some embodiments, each cationic domain comprises at least 40%, at least 45%, at least 50% cationic amino acids.

[0232] In some embodiments, each cationic domain comprises a majority of cationic amino acids, ie, 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.

[0233] In some embodiments, each cationic domain has an isoelectric point (pI) of at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 10.5, at least 11.0, at least 11.5, or at least 12.0.

[0234] In some embodiments, each cationic domain has an isoelectric point (pI) of at least 10.0.

[0235] In some embodiments, each cationic domain has an isoelectric point (pI) between 10.0 and 13.0.

[0236] In some embodiments, each cationic domain has an isoelectric point (pI) between 10.4 and 12.5.

[0237] In some embodiments, the isoelectric point of the cationic domain is calculated at physiological pH by any suitable means available in the art, in some embodiments, by using IPC (www.isoelectric.org), a web-based algorithm developed by Lukasz Kozlowski (Biol. Direct. 2016, 11:55. DOI:10.1186 / s13062-016-0159-9).

[0238] In some embodiments, each cationic domain comprises at least one cationic amino acid, e.g., 1 to 5 cationic amino acids, hi some embodiments, each cationic domain comprises at least two cationic amino acids, e.g., 2 to 5 cationic amino acids.

[0239] In some embodiments, each cationic domain is arginine-rich and / or histidine-rich. In some embodiments, a cationic domain can contain both histidine and arginine.

[0240] In some embodiments, each cationic domain comprises a majority of arginine and / or histidine residues.

[0241] In some embodiments, each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% arginine and / or histidine residues. In some embodiments, a cationic domain may comprise at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% arginine residues.

[0242] In some embodiments, the cationic domain may comprise at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, at least 70% histidine residues.

[0243] In some embodiments, the cationic domain may comprise a total of 1 to 5 histidine and 1 to 5 arginine residues. In some embodiments, the cationic domain may comprise 1 to 5 arginine residues. In some embodiments, the cationic domain may comprise 1 to 5 histidine residues. In some embodiments, the cationic domain may comprise a total of 2 to 5 histidine and 3 to 5 arginine residues. In some embodiments, the cationic domain may comprise 3 to 5 arginine residues. In some embodiments, the cationic domain may comprise 2 to 5 histidine residues.

[0244] In some embodiments, each cationic domain contains no beta-alanine residues. In some embodiments, each cationic domain contains one or more beta-alanine residues. In some embodiments, each cationic domain may contain a total of 2 to 5 beta-alanine residues, for example, a total of 2 or 3 beta-alanine residues.

[0245] In some embodiments, the cationic domain may include one or more hydroxyproline or serine residues.

[0246] In some embodiments, the cationic domain may comprise one to two hydroxyproline residues. In some embodiments, the cationic domain may comprise one to two serine residues.

[0247] In some embodiments, all of the cationic amino acids in a given cationic domain can be histidine, or, for example, all of the cationic amino acids in a given cationic domain can be arginine.

[0248] In some embodiments, the peptide may comprise at least one histidine-rich cationic domain. In some embodiments, the peptide may comprise at least one arginine-rich cationic domain.

[0249] In some embodiments, the peptide may comprise at least one arginine-rich cationic domain and may comprise at least one histidine-rich cationic domain.

[0250] In some embodiments, the peptide comprises two arginine-rich cationic domains.

[0251] In some embodiments, the peptide comprises two histidine-rich cationic domains.

[0252] In some embodiments, the peptide comprises two arginine- and histidine-rich cationic domains.

[0253] In some embodiments, the peptide comprises one arginine-rich cationic domain and one histidine-rich cationic domain, hi some embodiments, each cationic domain comprises no more than three consecutive arginine residues, e.g., no more than two consecutive arginine residues.

[0254] In some embodiments, each cationic domain does not comprise consecutive histidine residues.

[0255] In some embodiments, each cationic domain comprises arginine, histidine, and / or beta-alanine residues. In some embodiments, each cationic domain comprises 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%, or 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.

[0256] In some embodiments, the peptide comprises a first cationic domain comprising arginine and beta-alanine residues and a second cationic domain comprising arginine and beta-alanine residues.

[0257] In some embodiments, the peptide comprises a first cationic domain comprising arginine and beta-alanine residues, and a second cationic domain comprising histidine, beta-alanine, and optionally arginine residues.

[0258] In some embodiments, the peptide comprises a first cationic domain comprising arginine and beta-alanine residues, and a second cationic domain comprising histidine and beta-alanine residues.

[0259] In some embodiments, the peptide comprises a first cationic domain consisting of arginine and beta-alanine residues and a second cationic domain consisting of arginine and beta-alanine residues.

[0260] In some embodiments, the peptide comprises a first cationic domain consisting of arginine and beta-alanine residues, and a second cationic domain consisting of arginine, histidine, and beta-alanine residues.

[0261] In some embodiments, the peptide comprises at least two cationic domains, e.g., these cationic domains form arms of the peptide. In some embodiments, the cationic domains are located at the N- and C-termini of the peptide. Thus, in some embodiments, the cationic domains may be known as cationic arm domains.

[0262] In some embodiments, the peptide comprises two cationic domains, one located at the N-terminus of the peptide and one located at the C-terminus of the peptide. In some embodiments, no additional amino acids or domains are present at the N-terminus or C-terminus of the peptide, except for other groups such as terminal modifications, linkers, and / or oligonucleotides. For the avoidance of doubt, such other groups may be present in addition to the "peptide" described and claimed herein. Thus, in some embodiments, each cationic domain forms a terminus of the peptide. In some embodiments, this does not preclude the presence of additional linker groups as described herein.

[0263] In some embodiments, the peptide may comprise up to four cationic domains, hi some embodiments, the peptide comprises two cationic domains.

[0264] In some embodiments, the peptide comprises two cationic domains that are both arginine-rich.

[0265] In some embodiments, the peptide comprises one cationic domain that is arginine-rich.

[0266] In some embodiments, the peptide comprises two cationic domains that are both arginine-rich and histidine-rich.

[0267] In some embodiments, the peptide comprises one cationic domain that is arginine-rich and one cationic domain that is histidine-rich.

[0268] In some embodiments, at least one cationic domain (e.g., all cationic domains) is selected from the group consisting of RBRR (SEQ ID NO: 419), RBR, RB, R, RBRRBRR (SEQ ID NO: 420), RRBRR (SEQ ID NO: 425), BRR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRB, RBRBRB (SEQ ID NO: 424), RBRB (SEQ ID NO: 425), RB, HRHRHR (SEQ ID NO: 426), HRHR (SEQ ID NO: 427), H R, RRRRRR (SEQ ID NO: 428), RBRRBR (SEQ ID NO: 429), RBHBHB (SEQ ID NO: 430), RBHBH (SEQ ID NO: 431), BHBHB (SEQ ID NO: 432), BHBH (SEQ ID NO: 433), HBHB (SEQ ID NO: 434), HBH, BHB, BH, HB, H, RBHBHE (SEQ ID NO: 435), RBHBB (SEQ ID NO: 436), RBHB (SEQ ID NO: 437), RBB, HRBRHB (SEQ ID NO: 438), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440). 40), HRB, RBRBR (SEQ ID NO: 441), HRHRHRB (SEQ ID NO: 442), HRHRB (SEQ ID NO: 443), HRBRH (SEQ ID NO: 444), HRB, RRRRRRB (SEQ ID NO: 445), BRE, and BR, such as RBRR (SEQ ID NO: 419), RBR, RB, R, RBRRBRR (SEQ ID NO: 420), RRBRR (SEQ ID NO: 421), BRR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRB, RBRBRB (SEQ ID NO: 4 24), RBRB (SEQ ID NO: 425), RB, HRHRHR (SEQ ID NO: 426), HRHR (SEQ ID NO: 427), HR, RRRRRR (SEQ ID NO: 428), RBRRBR (SEQ ID NO: 429), RBHBH (SEQ ID NO: 431), BHBH (SEQ ID NO: 433), HBH, BH, H, RBHB (SEQ ID NO: 437), HRBRH (SEQ ID NO: 444), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), RBRBR (SEQ ID NO: 441), and BR.In some embodiments, at least one cationic domain (e.g., all cationic domains) is selected from the group consisting of RBR, RBRB (SEQ ID NO: 425), RB, R, RBRRBRR (SEQ ID NO: 420), BRR, BR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRB, HRHRHR (SEQ ID NO: 426), HR, RRRRRR (SEQ ID NO: 428). , RRRRRRB (SEQ ID NO: 445), RBHBH (SEQ ID NO: 431), BH, BHB, H, HB, RBH, and RBHB (SEQ ID NO: 437), e.g., RBR, R, RBRRBRR (SEQ ID NO: 420), BRR, BR, RR, HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRHRHR (SEQ ID NO: 426), HR, RRRRRR (SEQ ID NO: 428), RBHBH (SEQ ID NO: 431), BH, H, and RBH.

[0269] In some embodiments, the cationic domain comprises an amino acid unit selected from R, H, B, RR, HH, BB, RH, HR, RB, BR, HB, BH, RBR, RBB, BRR, BBR, BRB, RBH, RHB, HRB, BRH, HRR, RRH, HRH, HBB, BBH, RHR, BHB, HBH, or any combination thereof.

[0270] In some embodiments, the cationic domain may also comprise serine, proline, and / or hydroxyproline residues. In some embodiments, the cationic domain may further comprise an amino acid unit selected from RP, PR, RPR, RRP, PRR, PRP, Hyp, R[Hyp]R, RR[Hyp], [Hyp]RR, [Hyp]R[Hyp], [Hyp][Hyp]R, R[Hyp][Hyp], SB, BS, or any combination thereof, or any combination with the above amino acid units.

[0271] In some embodiments, each cationic domain in the peptide can be the same or different. In some embodiments, each cationic domain in the peptide is different.

[0272] In some embodiments, each cationic domain may further comprise an N- or C-terminal modification. In some embodiments, the C-terminal cationic domain comprises a C-terminal modification. In some embodiments, the N-terminal cationic domain comprises an N-terminal modification. In some embodiments, the C-terminal cationic domain comprises a linker group. In some embodiments, the C-terminal cationic domain comprises a C-terminal beta-alanine. In some embodiments, the N-terminal cationic domain is N-acetylated.

[0273] Hydrophobic domain In some embodiments, the peptide comprises up to 3 hydrophobic domains, or up to 2 hydrophobic domains, hi some embodiments, the peptide comprises 1 hydrophobic domain, e.g., a total of 1 hydrophobic domain.

[0274] The peptide may include one or more hydrophobic domains, each having a length of at least three amino acid residues.

[0275] In some embodiments, each hydrophobic domain has a length of 3 to 6 amino acid residues, hi some embodiments, each hydrophobic domain has a length of 5 amino acid residues.

[0276] In some embodiments, each hydrophobic domain may comprise apolar, polar, and hydrophobic amino acid residues.

[0277] The hydrophobic amino acid residue may be selected from alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, methionine, tryptophan.

[0278] The non-polar amino acid residue may be selected from proline, glycine, cysteine, alanine, valine, leucine, isoleucine, tryptophan, phenylalanine, and methionine.

[0279] Polar amino acid residues may be selected from serine, asparagine, hydroxyproline, histidine, arginine, threonine, tyrosine, and glutamine.

[0280] In some embodiments, the hydrophobic domain does not include hydrophilic amino acid residues.

[0281] In some embodiments, each hydrophobic domain comprises a majority of hydrophobic amino acid residues. In some embodiments, each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids. In some embodiments, each hydrophobic domain consists of hydrophobic amino acid residues.

[0282] 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, at least 1.3.

[0283] In some embodiments, each hydrophobic domain has a hydrophobicity index of at least 0.3, at least 0.35, at least 0.4, or at least 0.45.

[0284] In some embodiments, each hydrophobic domain has a hydrophobicity index of at least 1.2, at least 1.25, at least 1.3, or at least 1.35.

[0285] In some embodiments, each hydrophobic domain has a hydrophobicity index between 0.4 and 1.4.

[0286] In some embodiments, each hydrophobic domain has a hydrophobicity index between 0.45 and 0.48.

[0287] In some embodiments, each hydrophobic domain has a hydrophobicity index between 1.27 and 1.39.

[0288] In some embodiments, hydrophobicity is measured as described by White and Wimley: WC Wimley and SH White, "Experimentally determined hydrophobicity scale for proteins at membrane interfaces," Nature Struct Biol 3:842 (1996).

[0289] In some embodiments, each hydrophobic domain comprises at least 3 or at least 4 hydrophobic amino acid residues.

[0290] In some embodiments, each hydrophobic domain comprises phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and glutamine residues, hi some embodiments, each hydrophobic domain consists of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues.

[0291] In some embodiments, each hydrophobic domain consists of phenylalanine, leucine, isoleucine, tyrosine and / or glutamine residues.

[0292] In some embodiments, each hydrophobic domain consists of tryptophan and / or proline residues.

[0293] In some embodiments, the peptide comprises one hydrophobic domain. In some embodiments, the hydrophobic domain is located at the center of the peptide. Thus, in some embodiments, the hydrophobic domain may be known as the core hydrophobic domain. In some embodiments, the hydrophobic core domain is flanked on both sides by arm domains. In some embodiments, the arm domains may comprise one or more cationic domains and one or more additional hydrophobic domains. In some embodiments, each arm domain comprises a cationic domain.

[0294] In some embodiments, the peptide comprises two arm domains flanking a hydrophobic core domain, wherein each arm domain comprises a cationic domain.

[0295] In some embodiments, the peptide consists of two cationic arm domains flanking a hydrophobic core domain.

[0296] In some embodiments, the or each hydrophobic domain comprises FQILY (SEQ ID NO: 446).

[0297] In some embodiments, the or each hydrophobic domain consists of FQILY (SEQ ID NO: 446).

[0298] In some embodiments, the hydrophobic domain comprises or consists of YQFLI (SEQ ID NO: 513), IQFLI (SEQ ID NO: 514), YRFLI (SEQ ID NO: 515), or ILFRY (SEQ ID NO: 516).

[0299] In some embodiments, each hydrophobic domain in the peptide may be the same sequence or a different sequence.

[0300] In some embodiments, a hydrophobic domain separates any two cationic domains, hi some embodiments, each hydrophobic domain is flanked on either side by a cationic domain.

[0301] In some embodiments, a cationic domain is not contiguous with other cationic domains.

[0302] In some embodiments, the peptide comprises one hydrophobic domain flanked by two cationic domains in the following arrangement: [cationic domain]-[hydrophobic domain]-[cationic domain]

[0303] In some embodiments, the peptide consists of two cationic domains and one hydrophobic domain.

[0304] In some embodiments, the peptide consists of a hydrophobic core domain flanked by two cationic arm domains.

[0305] In some embodiments, the peptide consists of one hydrophobic core domain comprising FQILY (SEQ ID NO: 446).

[0306] In some embodiments, the peptide consists of one hydrophobic core domain that is FQILY (SEQ ID NO: 446).

[0307] Complex The peptides of the invention may be covalently linked to therapeutic or diagnostic molecules to provide conjugates.

[0308] The therapeutic molecule can be any molecule for treating a disease. Thus, the therapeutic molecule can optionally be selected from nucleic acids, peptide nucleic acids, antisense oligonucleotides (PNAs, PMOs, etc.), mRNA, gRNA (e.g., using CRISPR / Cas9 technology), short interfering RNA, microRNA, antagomiR, peptides, cyclic peptides, proteins, pharmaceuticals, drugs, or nanoparticles.

[0309] In some embodiments, the therapeutic molecule is an antisense oligonucleotide (see, e.g., below and elsewhere herein). Preferably, the antisense oligonucleotide comprises a phosphorodiamide amorpholino oligonucleotide (PMO). Alternatively, the oligonucleotide is a modified PMO or peptide nucleic acid (PNA), a chemically modified PNA such as gamma PNA (Bahal, Nat. Comm. 2016 13304), an oligonucleotide phosphoramidate (in which the non-bridging oxygen of the phosphate is replaced with an amine or alkylamine, as described in WO 2016 / 028187 A1), or other partially or fully charge-neutralized oligonucleotide. In other embodiments, the antisense oligonucleotide is a phosphorothioate, e.g., as described herein. In some embodiments, the oligonucleotide is a 2'-O-alkyl oligonucleotide, e.g., a 2'-O-methyl oligonucleotide, and / or a 2'-O-alkyl phosphorothioate, e.g., a 2'-O-methyl phosphorothioate. In some embodiments, the oligonucleotide is a PNA.

[0310] Therapeutic antisense oligonucleotide sequences can be selected, for example, from those known in the art. Examples of therapeutic antisense oligonucleotides that can be included in the conjugates of the invention are described herein.

[0311] In some embodiments, the peptide of the conjugate is directly covalently linked to a diagnostic or therapeutic molecule (eg, an oligonucleotide).

[0312] In some embodiments, the peptide of the conjugate is linked to the diagnostic or therapeutic molecule via a linker.

[0313] The peptides of the invention may be covalently linked to an imaging molecule to provide a conjugate.

[0314] In some embodiments, the imaging molecule can be any molecule that allows visualization of the complex. In some embodiments, the imaging molecule can indicate the location of the complex. In some embodiments, the location of the complex is in vitro or in vivo. In some embodiments, a method of monitoring the location of a complex comprising an imaging molecule is provided, the method comprising administering the complex to a subject and imaging the subject to identify the location of the complex.

[0315] Examples of imaging molecules include detection molecules, contrast molecules, or enhancement molecules. Suitable imaging molecules may be selected from radionuclides, fluorophores, nanoparticles (such as nanoshells), nanocages, chromogenic agents (e.g., enzymes), radioisotopes, dyes, radiopaque materials, fluorescent compounds, and combinations thereof.

[0316] In some embodiments, the imaging molecule is visualized using an imaging technique, which may be a cellular imaging technique or a medical imaging technique. Suitable cellular imaging techniques include, for example, image cytometry, fluorescence microscopy, phase contrast microscopy, SEM, TEM, etc. Suitable medical imaging techniques include, for example, X-ray, fluoroscopy, MRI, scintigraphy, SPECT, PET, CT, CAT, FNRI, etc.

[0317] In some cases, imaging molecules may be considered diagnostic molecules. In some embodiments, the diagnostic molecule allows for the diagnosis of a disease using a conjugate. In some embodiments, the imaging molecule can be used to determine the location of the conjugate, thereby diagnosing the disease. In some embodiments, a method for diagnosing a disease is provided, comprising administering to a subject an effective amount of a conjugate comprising an imaging molecule and monitoring the location of the conjugate.

[0318] In some embodiments, further details, such as binding, of the conjugates comprising the imaging molecule are similar to those described above for the conjugates comprising the oligonucleotides.

[0319] In some embodiments, the peptides of the present invention may be covalently linked to an oligonucleotide and an imaging molecule to provide a conjugate.

[0320] In some embodiments, the complex is capable of penetrating into cells and tissues, eg, into the nucleus of cells, eg, into muscle tissue.

[0321] Additional information regarding linkers that may be included in the conjugates of the invention is provided below.

[0322] Linker In some embodiments, the peptide of the conjugate is linked to the diagnostic or therapeutic molecule via a linker.

[0323] Suitable linkers include, for example, a C-terminal cysteine ​​residue that allows for the formation of a disulfide, thioether, or thiol-maleimide bond, a C-terminal aldehyde to form an oxime, a click reaction, or the formation of a morpholino bond with a basic amino acid on the peptide, or a carboxylic acid moiety on the peptide covalently linked to an amino group to form a carboxamide bond.

[0324] In some embodiments, the linker is 1 to 5 amino acids in length. In some embodiments, the linker may include any linker known in the art. In some embodiments, the linker is selected from any of the following sequences: G, BC, XC, C, GGC, BBC, BXC, XBC, X, XX, B, BB, BX, and XB, where X is 6-aminohexanoic acid.

[0325] In some embodiments, the linker may be a polymer such as, for example, PEG.

[0326] In some embodiments, the linker is beta-alanine.

[0327] In some embodiments, the peptide is attached to a therapeutic or diagnostic molecule, such as an oligonucleotide, via a carboxamide bond.

[0328] The linker of the conjugate may form part of the therapeutic or diagnostic molecule (e.g., an oligonucleotide) to which the peptide is attached. Alternatively, the therapeutic or diagnostic molecule (e.g., an oligonucleotide) may be attached directly to the C-terminus of the peptide (e.g., an amide bond). In some embodiments, no linker is required.

[0329] Alternatively, the peptide can be chemically linked to the therapeutic or diagnostic molecule (e.g., an oligonucleotide) via, for example, a disulfide, alkenyl, alkynyl, aryl, ether, thioether, triazole, amide, carboxamide, urea, thiourea, semicarbazide, carbazide, hydrazine, oxime, phosphate, phosphoramidate, thiophosphate, borane phosphate, iminophosphate, or thiolmaleimide bond.

[0330] Optionally, a cysteine ​​may be added to the terminus of the therapeutic or diagnostic molecule (e.g., an oligonucleotide) to form a disulfide bond to the peptide, or the terminus may be bromoacetylated to attach a thioether to the peptide.

[0331] The linker may be a non-cationic linker, for example, a linker containing a total of 1 to 5 amino acids (e.g., a linker containing a total of 1 amino acid) or an aliphatic dicarboxylic acid linker. The peptide may be linked to the linker at its side chain (e.g., the side chain of an amino acid having a covalently bondable functional group, such as a glutamic acid side chain, a lysine side chain, or an aspartic acid side chain).

[0332] In some embodiments, the linker is glutamic acid (eg, via the gamma-carboxyl group), beta-alanine, glycine, delta-aminovaleric acid, or gamma-aminobutyric acid.

[0333] The linker may be at the N-terminus or C-terminus of the peptide.

[0334] Examples of linkers that can be used in the conjugates of the invention are described above. Additional examples of linkers that can be used are described in WO 2020 / 115494 and WO 2020 / 030927, the contents of which are incorporated herein by reference.

[0335] In some embodiments, the linker has the following structure: [ka] , [ka] , [ka] , [ka] , or [ka]

[0336] In some embodiments, the linker has the following structure: [ka]

[0337] In some embodiments, the linker has the following structure: [ka]

[0338] In some embodiments, the linker has the following structure: [ka]

[0339] In some embodiments, the linker has the following structure: [ka]

[0340] In some embodiments, the linker has the following structure: [ka]

[0341] In some embodiments, the complex is of the following structure: [ka]

[0342] In some embodiments, the complex is of the following structure: [ka]

[0343] In some embodiments, the complex is of the following structure: [ka]

[0344] In some embodiments, the complex is of the following structure: [ka]

[0345] In some embodiments, the complex is of the following structure: [ka]

[0346] Oligonucleotides The oligonucleotides in the conjugates described herein include those that can be used in therapeutic approaches involving, for example, promoting exon skipping, restoring cryptic splicing, altering the level of alternatively spliced ​​mRNA, blocking intermolecular interactions, or suppressing RNA toxicity. In some examples, the oligonucleotides promote exon skipping in DMD pre-mRNA or SMN2 pre-mRNA and can therefore be used in therapeutic approaches for muscular dystrophy (e.g., DMD or BMD) or SMA. In other examples, the oligonucleotides form heterodimers with the 3'-UTR of the DMPK transcript and can therefore be used in therapeutic approaches for myotonic dystrophy type 1 (e.g., DM1). Examples of oligonucleotides targeting DMD are listed in Table 1 (exon 45), Table 2 (exon 51), Table 3 (exon 53), and Table 4 (exon 44). Examples of oligonucleotides targeting the 3'-UTR of the DMPK transcript are also listed. Additionally, examples of oligonucleotides targeting the first intron of the CNBP transcript are also listed. Further examples of oligonucleotides targeting the SMN2 transcript are shown in Table 5.

[0347] In some embodiments, the oligonucleotides used in the conjugates disclosed herein may be complementary to a target site within the dystrophin transcript. Without being bound by theory, oligonucleotides that hybridize to specific target regions within the human dystrophin transcript are complementary to exons 8-55 during dystrophin pre-mRNA splicing. (e.g., exon 8, exon 23, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53, or exon 55), thereby improving Duchenne muscular dystrophy. Non-limiting examples of nucleobase sequences that can be used in the oligonucleotides of the present invention are described in US 8,084,601, US 8,324,371, US 8,461,325, US 8,552,172, US 9,018,368, US 9,079,934, US 9,243,251, US 9,243,252, US 9,447,417, US 9,650,632, US 9,970,010, US 10,385,092, and US 10,781,450.

[0348] The oligonucleotide may comprise a nucleobase sequence complementary to a human dystrophin transcript, e.g., capable of inducing exon 45 skipping. Non-limiting examples of such sequences are listed in Table 1. For example, the oligonucleotide may comprise at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) contiguous nucleobases selected from any one of the sequences listed in Table 1. In certain preferred embodiments, the oligonucleotide comprises at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) contiguous nucleobases selected from 5'-CAAUGCCAUCCUGGAGUUCCUG-3' (SEQ ID NO: 265) or its thymine-substituted analog, 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 275). In certain preferred embodiments, the oligonucleotide comprises a nucleobase sequence selected from the group consisting of 5'-CAAUGCCAUCCUGGAGUUCCUG-3' (SEQ ID NO: 265) or its thymine substituted analog, 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 275). Table 1 # Sequence number 1 253 5′-CCAAUGCCAUCCUGGAGUUCCUGUAAGAUA-3′ 2 254 5′-GCUGCCCAAUGCCAUCCUGGAGUUCCUGUA-3′ 3 255 5′-CAAUGCCAUCCUGGAGUUCCUGUAAGA-3′ 4 256 5′-GCUGCCCAAUGCCAUCCUGGAGUUCCUGUAAGAUACCAA-3′ 5 257 5′-GCCCAAUGCCAUCCUGGAGUUCCUGUAAGA-3′ 6 258 5′-UGCCAUCCUGGAGUUCCUGUAAGAUACC-3′ 7 259 5′-UGCCAUCCUGGAGUUCCUGUAAGAU-3′ 8 260 5′-CAAUGCCAUCCUGGAGUUCCUGUAAGAU-3′ 9 261 5′-GCCCAAUGCCAUCCUGGAGUUCCUGUAAGAU-3′ 10 262 5′-UUGCCGCUGCCCAAUGCCAUCCUGGAGUUC-3′ 11 263 5′-GCCCAAUGCCAUCCUGGAGUUCCUGAAA-3′ 12 264 5′-GCCGCUGCCCAAUGCCAUCCUGGAGUUCCU-3′ 13 265 5′-CAAUGCCAUCCUGGAGUUCCUG-3′ 14 266 5′-GCCCAAUGCCAUCCUGGAGUUCCUG-3′ 15 267 5′-GCUGCCCAAUGCCAUCCUGGAGUUCCUG-3′ 16 268 5′-GCUGCCCAAUGCCAUCCUGGAGUUCCUGAAA-3′ 17 269 5′-CAAUGCCAUCCUGGAGUUCCUGUAAGAUACC-3′ 18 270 5'-TTGCCGCTGCCCAATGCCATCCTGGAGTTC-3' 19 271 5'-CAGTTTGCCGCTGCCCAATGCCATCCTGGA-3' 20 272 5'-CCAAUGCCAUCCUGGAGUUCCUGUAA-3' 21 273 5'-CCAATGCCATCCTGGAGTTCCTGTA-3' 22 274 5'-CTGACAACAGTTTGCCGCTGCCCCAA-3' 23 275 5'-CAATGCCATCCTGGAGTTCCTG-3' 24 276 5'-GCTGCCCAATGCCATCCTGGAGTTCCTGTAA-3'

[0349] In some embodiments, one or more uracils (e.g., all uracils) in the oligonucleotide sequences shown in Table 1 are substituted with thymine. For example, the oligonucleotide sequence may be 5'-CAAUGCCAUCCUGGAGUUCCUG-3' (SEQ ID NO: 265). Alternatively, the oligonucleotide sequence may be, for example, 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 275). In some embodiments, one or more thymines (e.g., all thymines) in the oligonucleotide sequences shown in Table 1 are substituted with uracil.

[0350] The oligonucleotide may comprise a nucleobase sequence complementary to a human dystrophin transcript, e.g., capable of inducing skipping of exon 51. Non-limiting examples of such sequences are set forth in Table 2. For example, the oligonucleotide may comprise at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) contiguous nucleobases from, e.g., any one of the sequences set forth in Table 2. In certain preferred embodiments, the oligonucleotide may comprise at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) contiguous nucleobases from 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 282) or its thymine-substituted analog, 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 287). In certain preferred embodiments, the oligonucleotide comprises a nucleobase sequence selected from the group consisting of 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 282) or its thymine substituted analog, 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 287). Table 2 # Sequence number 1 277 5′-ACCAGAGUAACAGUCUGAGUAGGAGC-3′ 2 278 5′-CUCAUACCUUCUGCUUGAUGAUC-3′ 3 279 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ 4 280 5′-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3′ 5 281 5′-ACAUCAAGGAAGAUGGCAUUUCUAG-3′ 6 282 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ 7 283 5′-AUCAUUUUUUCUCAUACCUUCUGCUAG-3′ 8 284 5′-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3′ 9 285 5′-CACCCACCAUCACCCUCUGUG-3′ 10 286 5′-AUCAUCUCGUUGAAUUCCUCAA-3′ 11 287 5'-CTCCAACATCAAGGAAGATGGCATTTCT AG-3'

[0351] In some embodiments, one or more uracils (e.g., all uracils) in the oligonucleotide sequences shown in Table 2 are substituted with thymine. For example, the oligonucleotide sequence may be, for example, 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (SEQ ID NO: 282). Alternatively, the oligonucleotide sequence may be, for example, 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 287). In some embodiments, the sequence may be 5'-GGCCAAACCTCGGCTTACCTGAAAT-3' (SEQ ID NO: 288). In some embodiments, one or more thymines (e.g., all thymines) in the oligonucleotide sequences shown in Table 2 are substituted with uracil.

[0352] The oligonucleotide may comprise a nucleobase sequence complementary to a human dystrophin transcript, e.g., capable of inducing skipping of exon 53. Non-limiting examples of such sequences are shown in Table 3. For example, the oligonucleotide may comprise at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) contiguous nucleobases from, e.g., any one of the sequences set forth in Table 3. In certain preferred embodiments, the oligonucleotide may comprise at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) contiguous nucleobases from 5'-CCTCCGGTTCTGAAGGTGTTCT-3' (SEQ ID NO: 316) or 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 325). In certain preferred embodiments, the oligonucleotide comprises a nucleobase sequence selected from the group consisting of 5'-CCTCCGGTTCTGAAGGTGTTCT-3' (SEQ ID NO: 316) and 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 325). Table 3 # Sequence number 1 289 5′-CCGGTTCTGAAGGTGTTCTTGTA-3′ 2 290 5′-TCCGGTTCTGAAGGTGTTCTTGTA-3′ 3 291 5′-CTCCGGTTCTGAAGGTGTTCTTGTA-3′ 4 292 5′-CCTCCGGTTCTGAAGGTGTTCTTGTA-3′ 5 293 5′-GCCTCCGGTTCTGAAGGTGTTCTTGTA-3′ 6 294 5′-TGCCTCCGGTTCTGAAGGTGTTCTTGTA-3′ 7 295 5′-CCGGTTCTGAAGGTGTTCTTGT-3′ 8 296 5′-TCCGGTTCTGAAGGTGTTCTTGT-3′ 9 297 5′-CTCCGGTTCTGAAGGTGTTCTTGT-3′ 10 298 5′-CCTCCGGTTCTGAAGGTGTTCTTGT-3′ 11 299 5′-GCCTCCGGTTCTGAAGGTGTTCTTGT-3′ 12,300 5′-TGCCTCCGGTTCTGAAGGTGTTCTGT-3′ 13 301 5′-CCGGTTCTGAAGGTGTTCTTG-3′ 14 302 5′-TCCGGTTCTGAAGGTGTTCTTG-3′ 15 303 5′-CTCCGGTTCTGAAGGTGTTCTTG-3′ 16 304 5′-CCTCCGGTTCTGAAGGTGTTCTTG-3′ 17 305 5′-GCCTCCGGTTCTGAAGGTGTTTCTTG-3′ 18,306 5′-TGCCTCCGGTTCTGAAGGTGTTCTTG-3′ 19 307 5′-CCGGTTCTGAAGGTGTTCTT-3′ 20 308 5′-TCCGGTTCTGAAGGTGTTCTT-3′ 21 309 5′-CTCCGGTTCTGAAGGTGTTCTT-3′ 22 310 5′-CCTCCGGTTCTGAAGGTGTTCTTT-3′ 23 311 5′-GCCTCCGGTTCTGAAGGTGTTCTT-3′ 24 312 5′-TGCCTCCGGTTCTGAAGGTGTTCCTT-3′ 25 313 ​​5′-CCGGTTCTGAAGGTGTTCT-3′ 26 314 5′-TCCGGTTCTGAAGGTGTTCT-3′ 27 315 ​​5′-CTCCGGTTCTGAAGGTGTTCT-3′ 28 316 5′-CCTCCGGTTCTGAAGGTGTTCT-3′ 29 317 5′-GCCTCCGGTTCTGAAGGTGTTCT-3′ 30 318 5′-TGCCTCCGGTTCTGAAGGTGTTCT-3′ 31 319 5′-CCGGTTCTGAAGGTGTTC-3′ 32 320 5′-TCCGGTTCTGAAGGTGTTC-3′ 33 321 5′-CTCCGGTTCTGAAGGTGTTC-3′ 34 322 5′-CCTCCGGTTCTGAAGGTGTTC-3′ 35 323 5′-GCCTCCGGTTCTGAAGGTGTTC-3′ 36 324 5′-TGCCTCCGGTTCTGAAGGTGTTC-3′ 37 325 5′-GTTGCCTCCGGTTCTGAAGGTGTTC-3′ 38 326 5'-CAUUCAACUGUUGCCUCCGGUUCUGAAGGUG-3' 39 327 5'-CTGTTGCCTCCGGTTCTGAAGGTGTTCTTG-3' 40 328 5'-CAACTGTTGCCTCCGGTTCTGAAGGTGTTC-3' 41 329 5'-TTGCCTCCGGTTCTGAAGGTGTTCTTGTAC-3' 42 330 5'-CTGAAGGTGTTCTTGTACTTCATCC-3' 43 331 5'-CATTCAACTGTTGCCTCCGGTTCTGAAGGTG-3'

[0353] In some embodiments, one or more thymines (e.g., all thymines) in the oligonucleotide sequences shown in Table 3 are substituted with uracil. In some embodiments, one or more uracils (e.g., all uracils) in the oligonucleotide sequences shown in Table 3 are substituted with thymine.

[0354] The oligonucleotide may comprise a nucleobase sequence complementary to a human dystrophin transcript, e.g., capable of inducing skipping of exon 44. Non-limiting examples of such sequences are set forth in Table 4. For example, the oligonucleotide may comprise at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) contiguous nucleobases from, e.g., any one of the sequences set forth in Table 4. Table 4 # Sequence number 1 332 5'-UUUGUGUCUUUCUGAGAAAC-3' 2 333 5'-AAAGACUUACCUUAAGAUAC-3' 3 334 5'-AUCUGUCAAAUCGCCUGCAG-3' 4 335 5'-TGAAAACGCCGCCATTTCTCAACAGATCTG-3' 5 336 5'-CATAATGAAAACGCCGCCATTTCTCAAACAG-3' 6 337 5'-TGTTCAGCTTCTGTTAGCCACTGATTAAAT-3' 7 338 5'-CGCCGCCATTTCTCAACAG-3' 8 339 5'-ATCTGTCAAATCGCCTGCAG-3'

[0355] In some embodiments, one or more uracils (e.g., all uracils) in the oligonucleotide sequences shown in Table 4 are substituted with thymine. In some embodiments, one or more thymines (e.g., all thymines) in the oligonucleotide sequences shown in Table 4 are substituted with uracil.

[0356] Alternatively, the oligonucleotide may be r(CUG) exp (e.g., the oligonucleotide may have at least 9 consecutive nucleobases complementary to the CUG repeat sequence). Thus, the oligonucleotide may have a 5'-[CAG] n -3',5'-[AGC]n -3' and 5'-[GCA] n -3', where n is an integer from 5 to 8, inclusive. Non-limiting examples of nucleobase sequences that may be included in the oligonucleotides described herein are 5'-[CAG]5-3' (SEQ ID NO: 340), 5'-[CAG]6-3' (SEQ ID NO: 341), 5'-[CAG]7-3' (SEQ ID NO: 342), 5'-[CAG]8-3' (SEQ ID NO: 343), 5'-[AGC]5-3' (SEQ ID NO: 344), 5'-[AGC]6-3' (SEQ ID NO: 345), 5'-[AGC]7-3' (SEQ ID NO: 346), 5'-[AGC]8-3' (SEQ ID NO: 347), 5'-[GCA]5-3' (SEQ ID NO: 348), 5'-[GCA]6-3' (SEQ ID NO: 349), 5'-[GCA]7-3' (SEQ ID NO: 350), and 5'-[GCA]8-3' (SEQ ID NO: 351).

[0357] Alternatively, the oligonucleotide may be r(CCUG) exp (e.g., the oligonucleotide may have at least 8 consecutive nucleobases complementary to the CCUG repeat sequence). Thus, the oligonucleotide may have a 5'-[CAGG] n -3',5'-[AGGC] n -3',5'-[GGCA] n -3',5'-[GCAG] n-3', where n is an integer from 4 to 8. Non-limiting examples of nucleobase sequences that may be included in the oligonucleotides described herein are 5'-[CAGG]4-3' (SEQ ID NO: 352), 5'-[CAGG]5-3' (SEQ ID NO: 353), 5'-[CAGG]6-3' (SEQ ID NO: 354), 5'-[CAGG]7-3' (SEQ ID NO: 355), 5'-[CAGG]8-3' (SEQ ID NO: 356), 5'-[AGGC]4-3' (SEQ ID NO: 357), 5'-[AGGC]5-3' (SEQ ID NO: 358), 5'-[AGGC]6-3' (SEQ ID NO: 359), 5'-[AGGC]7-3' (SEQ ID NO: 360), 5'-[A GGC]8-3' (SEQ ID NO: 361), 5'-[GGCA]4-3' (SEQ ID NO: 362), 5'-[GGCA]5-3' (SEQ ID NO: 363), 5'-[GGCA]6-3' (SEQ ID NO: 364), 5'-[GGCA]7-3' (SEQ ID NO: 365), 5'-[GGCA]8-3' (SEQ ID NO: 366), 5'-[GCAG]4-3' (SEQ ID NO: 367), 5'-[GCAG]5-3' (SEQ ID NO: 368), 5'-[GCAG]6-3' (SEQ ID NO: 369), 5'-[GCAG]7-3' (SEQ ID NO: 370), and 5'-[GCAG]8-3' (SEQ ID NO: 371).

[0358] Alternatively, the oligonucleotide may be complementary to a target sequence within or near intron 7 of the human SMN2 gene. Thus, the oligonucleotide comprises a nucleobase sequence complementary to the human SMN2 gene, e.g., to induce inclusion of exon 7. Non-limiting examples of such sequences include sequences that are at least 80% (e.g., at least 85%, at least 90%, or at least 95%) complementary to a sequence set forth in Table 5. In certain embodiments, the oligonucleotide comprises a nucleobase sequence that is 5'-UCACUUUCAUAAUGCUGG-3' (SEQ ID NO: 401) or 5'-ATTCACTTTCATAATGCTGG-3' (SEQ ID NO: 400). Table 5 # Sequence number 1 372 5'-CCAGCAUUAUGAAAG-3' 2 373 5′-C U AGCA AC AUGAAAAG-3' 3,374 5'- A CAG GCCG AUGAAAAG-3' 4,375 5'- UG AG A A CC AUGAAAAG-3' 5 376 5'-C G AG UUAG AUGAAAAG-3' 6 377 5′-CCAG GGGA AUGAAAAG-3' 7 378 5′-CCAG A A GG AUGAAAAG-3' 8 379 5'-C G AG UC U C AUGAAAAG-3' 9 380 5'-C G AGC GG UAUGAAAAG-3' 10,381 5'- GG AGC GG UAUGAAAAG-3' 11 382 5′-CCAG AGG UAUGAAAAG-3' 12 383 5′-CCAGC GG UAUGAAAAG-3' 13 384 5′-CCAGCA G UAUGAAAAG-3' 14 378 5′-CCAG A A G GAAUGAAG-3' 15,385 5'- UA AGC CC UAUGAAAAG-3' 16 386 5′-C U AG UU UUAUGAAAAG-3' 17 387 5′-CC UUA AUU UA GAAAG-3′ 18,388 5'- AA AGCAUUAUGAAAG-3' 19 389 5'- UU AGCAUUAUGAAAG-3' 20 390 5'-C GU GCAUUAUGAAAG-3' 21 391 5'-C UGU CAUUAUGAAAG-3' 22 392 5'-C UUU CAUUAUGAAAG-3' 23 393 5'-C AUU CAUUAUGAAAG-3' 24 394 5'-CCAGCAUUAUGA UUA -3' 25 395 5'-CCAGCAUUAU CU AAG-3' 26 396 5'-CCAGCAUUAU CCC AG-3' 27 397 5'-CCAGCAUUAU UUU AG-3' 28 398 5′-ATTCACTTTCATAATGCTGG-3 29 399 5'-UCACUUUCAUAAUGCUGG-3'

[0359] In the above sequences, underlined nucleotides indicate mutations in the ISS-N1 wild-type sequence. All of the above mutant forms retain the inhibitory function of ISS-N1. In some embodiments, one or more uracils (e.g., all uracils) in the oligonucleotide sequences shown in Table 5 are substituted with thymine. In some embodiments, one or more thymines (e.g., all thymines) in the oligonucleotide sequences shown in Table 5 are substituted with uracil.

[0360] In some embodiments, the oligonucleotide comprises a nucleobase sequence disclosed in Table 2 of WO 2020 / 028832, which is incorporated by reference in its entirety. In some embodiments, one or more uracils (e.g., all uracils) in the oligonucleotide sequences set forth in Table 2 of WO 2020 / 028832 are substituted with thymine.

[0361] In some embodiments, the oligonucleotides comprise a nucleobase sequence disclosed in Table 8 (see below). In some embodiments, one or more uracils (e.g., all uracils) in the oligonucleotide sequences shown in Table 8 are substituted with thymine. In some embodiments, all Us in all oligonucleotides shown in Table 8 are substituted with Ts.

[0362] [Table 8] JPEG2025538552000039.jpg239166JPEG2025538552000040.jpg149166

[0363] In some embodiments, the oligonucleotide is a morpholino, e.g., as described herein. In some embodiments, the oligonucleotide is a phosphorothioate, e.g., as described herein. In some embodiments, the oligonucleotide is a 2'-O-alkyl oligonucleotide, e.g., a 2'-O-methyl oligonucleotide, and / or a 2'-O-alkyl phosphorothioate, e.g., a 2'-O-methyl phosphorothioate. In some embodiments, the oligonucleotide is a PNA.

[0364] Pharmaceutical Composition The conjugates of the present invention may be formulated into pharmaceutical compositions.

[0365] In some embodiments, the pharmaceutical composition comprises a conjugate of the invention or a pharmaceutically acceptable salt thereof.

[0366] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable diluent, adjuvant, or carrier.

[0367] Suitable pharmaceutically acceptable diluents, adjuvants or carriers are well known in the art.

[0368] It is to be understood that the pharmaceutical compositions of the present disclosure may further comprise additional known therapeutic agents, drugs, modifications of compounds into prodrugs, etc. for medical use to alleviate, ameliorate, prevent, and treat the diseases, disorders, and conditions described herein.

[0369] In some embodiments, the pharmaceutical composition is for use as a medicament, e.g., for use as a medicament in the same manner as described herein for the conjugate, and all features described herein in relation to medical treatment using the conjugate apply to the pharmaceutical composition.

[0370] Thus, in a further aspect of the present invention there is provided a pharmaceutical composition according to the fourth aspect for use as a medicament. In a further aspect there is provided a method of treating a subject for a disease state comprising administering an effective amount of a pharmaceutical composition as disclosed herein.

[0371] medical use The conjugates of the present invention can be used as pharmaceuticals for the treatment of diseases.

[0372] The medicament may be in the form of a pharmaceutical composition as defined above.

[0373] Also provided are methods of treating a patient or subject in need of treatment for a disease condition, the methods comprising administering to the patient or subject a therapeutically effective amount of the conjugate. In some embodiments, the medical treatment requires delivery of a therapeutic molecule (e.g., an oligonucleotide) into a cell, e.g., into the nucleus of a cell.

[0374] The disease to be treated may include any disease in which improved penetration of the cell membrane and / or nuclear membrane by a therapeutic molecule (e.g., an oligonucleotide) may result in improved therapeutic efficacy.

[0375] In some embodiments, the disease is a genetic disease.

[0376] In some embodiments, the conjugate is used to treat a disease of the neuromuscular or nervous system.

[0377] A conjugate comprising the peptide of the present invention (e.g., one that targets the dystrophin gene) is suitable for the treatment of Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). A conjugate comprising the peptide of the present invention (e.g., r(CUG) exp The conjugates containing the peptides of the present invention (e.g., r(CCUG)) are suitable for the treatment of myotonic dystrophy type 1 (DM1). exp Complexes containing the peptides of the present invention (e.g., those targeting SMN2, e.g., SMN2 intron 7) are suitable for the treatment of myotonic dystrophy type 2 (DM2). Complexes containing the peptides of the present invention (e.g., those targeting SMN2, e.g., SMN2 intron 7) are suitable for the treatment of spinal muscular atrophy. Complexes containing the peptides of the present invention (e.g., those targeting DUX4) are suitable for the treatment of facioscapulohumeral muscular dystrophy (FSHD). Complexes containing the peptides of the present invention (e.g., those targeting PMP22) are suitable for the treatment of Charcot-Marie-Tooth disease type 1a (CMT1a). Complexes containing the peptides of the present invention (e.g., those targeting MFN2) are suitable for the treatment of Charcot-Marie-Tooth disease type 2a (CMT2a). Complexes containing the peptides of the present invention (e.g., those targeting TCF4) are suitable for the treatment of Fuchs' corneal dystrophy (FCD). Complexes containing the peptides of the present invention (e.g., those targeting FXN) are suitable for the treatment of Friedreich's ataxia (FA).

[0378] In some embodiments, the complex is used to treat diseases in which protein production can be restored or prevented by modulating the splicing process (e.g., promoting exon skipping). In such embodiments, the complex may comprise an oligonucleotide that can induce splicing to restore the open reading frame of a mutated transcript and / or prevent or correct a splicing defect and / or increase production of correctly spliced ​​mRNA molecules. In some embodiments, the complex may comprise an oligonucleotide that reduces or ameliorate harmful effects associated with the presence of toxic RNA. The efficacy of treatment may be evaluated using any suitable method. In some embodiments, the efficacy of treatment may be evaluated by assessment of observed symptoms associated with a genetic disease, such as a neuromuscular or neurological disease (e.g., DMD, BMD, FSHD, DM1, DM2, CMT1a, CMT2a, FCD, FA, or SMA). In some embodiments, evaluation is by assessment of muscle atrophy or weakness, e.g., by measuring subject self-reported outcomes (e.g., motor capacity, self-care, usual activities, pain / discomfort, anxiety / depression), or by assessing quality of life indicators (e.g., lifespan). In some embodiments, evaluation is by assessment of functional outcomes. In some embodiments, evaluation is by assessment of molecular markers of disease. In some embodiments, evaluation is by assessment of relevant RNA transcript and / or protein levels. In some embodiments, evaluation is by assessment of imaging parameters.

[0379] In some embodiments, the conjugate is used to treat a neuromuscular disease or a genetic disease such as a neuromuscular disease (e.g., DMD, BMD, FSHD, DM1, DM2, CMT1a, CMT2a, FCD, FA, or SMA).

[0380] In some embodiments, there is provided a conjugate according to the second aspect for use in treating a genetic disease, such as a neuromuscular disease (e.g., DMD, BMD, FSHD, DM1, DM2, CMT1a, CMT2a, FCD, FA, or SMA). In some embodiments, in the case of DMD or BMD, the oligonucleotide of the conjugate acts to increase expression of a dystrophin protein. In some such embodiments, the oligonucleotide of the conjugate acts to increase expression of a functional dystrophin protein.

[0381] In some embodiments, the complex increases dystrophin expression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the complex increases dystrophin expression by up to 50%. In some embodiments, the complex restores dystrophin expression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the complex restores dystrophin expression by up to 50%.

[0382] In some embodiments, the complex restores 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the function of the dystrophin protein, hi some embodiments, the complex restores up to 50% of the function of the dystrophin protein.

[0383] In some embodiments, the oligonucleotides of the complex can act to do so by causing the skipping of one or more exons during transcription of dystrophin.

[0384] In some embodiments, the oligonucleotides of the conjugate cause skipping of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of one or more exons of the dystrophin gene, hi some embodiments, the oligonucleotides of the conjugate cause skipping of up to 50% of one or more exons of the dystrophin gene.

[0385] 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.

[0386] In some embodiments, the patient or subject to be treated as described above may be of any age, hi some embodiments, the patient or subject to be treated is 0 to 40 years old, e.g., 0 to 30 years old, e.g., 0 to 25 years old, e.g., 0 to 20 years old.

[0387] In some embodiments, the conjugate is for administration to a subject systemically or locally, for example, by intramedullary, intrathecal, intracerebroventricular, intravitreal, enteral, parenteral, intravenous, intraarterial, intramuscular, intratumoral, subcutaneous, oral, or nasal routes.

[0388] In some embodiments, the conjugate is for intravenous administration to a subject.

[0389] In some embodiments, the conjugate is for administration to a subject by intravenous injection.

[0390] In some embodiments, the conjugate is administered to a subject in a "therapeutically effective amount." As used herein, "therapeutically effective amount" means an amount sufficient to provide benefit to the individual. The actual amount administered, the rate and duration of administration will depend on the nature and severity of the condition being treated. Dosage determination is the responsibility of general practitioners and other medical professionals. Exemplary techniques and protocols are described in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins. Exemplary doses may be 1-50 mg / kg, 10-40 mg / kg, 10-35 mg / kg, 10-30 mg / kg, 10-25 mg / kg, 10-20 mg / kg, 15-40 mg / kg, 15-35 mg / kg, 15-30 mg / kg, 15-25 mg / kg, 15-20 mg / kg, 20-40 mg / kg, 20-35 mg / kg, 20-30 mg / kg, 20-25 mg / kg, 25-40 mg / kg, 25-35 mg / kg, and 25-30 mg / kg. In some embodiments, the dose is about 20 mg / kg, 25 mg / kg, or 30 mg / kg.

[0391] Advantageously, the dosage of the conjugates of the invention may be lower, eg, an order of magnitude or more lower, than the dosage required to see any effect from the therapeutic molecule (eg, oligonucleotide) alone.

[0392] In some embodiments, treatment according to the present invention results in one or more improvements compared to conventional conjugates using currently available peptide carriers: clinical observation, at least one clinical chemistry marker (e.g., one or more of urea, creatinine, alanine transferase, aspartate transferase, alkaline phosphatase, TRIG, magnesium, potassium, calcium, sodium, chloride, KIM-1), hematology, coagulation, urinalysis (e.g., one or more of magnesium, potassium, creaine, KIM-1), nephrotoxicity, complement activation, hepatotoxicity, histopathology, and other established toxicity markers used in the field.

[0393] In some embodiments, after administration of the conjugates of the present invention, one or more toxicity markers are significantly improved compared to conventional conjugates using currently available peptide carriers.

[0394] In some embodiments, after administration of the conjugates of the present invention, the level of at least one clinical chemistry marker (e.g., see above) is improved compared to conventional conjugates using currently available peptide carriers.

[0395] In some embodiments, the levels of each of the above clinical chemistry markers are improved after administration of the conjugates of the present invention compared to conventional conjugates using currently available peptide carriers.

[0396] In some embodiments, the levels of each marker are significantly improved compared to conventional conjugates using currently available peptide carriers.

[0397] In some embodiments, the levels of each marker are improved by up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% after administration of the conjugates of the present invention compared to conventional conjugates using currently available peptide carriers.

[0398] Advantageously, the toxicity of the peptides and resulting conjugates is significantly reduced compared to conventional cell-penetrating peptides and conjugates.

[0399] Preparation of peptides The peptides of the present invention can be produced by any standard protein synthesis method (e.g., solid-phase peptide synthesis), for example, chemical or semi-chemical synthesis. Thus, the present invention also relates to nucleotide sequences comprising or consisting of DNA encoding the peptides, expression systems, for example, vectors comprising such sequences with sequences necessary for expression and expression control, and host cells and host organisms transformed with the expression systems.

[0400] Thus, nucleic acids encoding the peptides according to the invention are also provided.

[0401] In some embodiments, the nucleic acid may be provided in an isolated or purified form.

[0402] Typically, techniques for preparing peptides, oligonucleotides, and their conjugates are well known in the art, see, for example, US 9,302,014, US 2021 / 0299263, US 2021 / 0299264, and WO 2020 / 115494.

[0403] The following examples are intended to illustrate the present invention, but are not intended to limit the invention in any way.

[0404] Example Example 1. Synthesis Peptide synthesis The peptides in Table 6 were prepared according to the following general procedure. Table 6. Peptides synthesized on Rink Amide resin [Table 6] E and e refer to the gamma Glu linker. The C-terminal NH2 refers to the amidation of the alpha carboxyl group of E or e. *All chiral amino acids are D-amino acids

[0405] Solid-phase synthesis of resin-bound protected peptides on rink amide resin. All steps were carried out at 20°C under an inert atmosphere. 1. Preparation of resin: Fmoc-Rink Amide MBHA resin (1.00 eq., substitution: 0.53 mmol / g) in DMF was stirred at 20° C. for 2 hours. 2. Deprotection: The resin was treated with 20% piperidine for 30 minutes and washed five times with DMF. 3. Coupling: The C-terminal residue was added as a solution of Fmoc-Glu(OtBu)-OH or Fmoc-D-Glu(OtBu)-OH (3.00 eq.), and a solution of HBTU (2.85 eq.) and DIEA (6.00 eq.) in DMF (0 eq.) was added to the resin and stirred for 50 min. [Fmoc-AA-OH] initial ≒0.2-0.3 M. The resin was then washed five times with DMF. 4. Steps 2 and 3 above were repeated to couple the remaining amino acids. 5. An N-terminal acetyl (Ac) group was added by treatment with a solution (Ac2O / DIEA / DMF = 10 / 5 / 85) for 30 minutes.

[0406] The peptides in Table 7 were prepared according to the following general procedure. Table 7.2-Peptides synthesized on chlorotrityl resin [Table 7]

[0407] Solid-phase synthesis of resin-bound protected peptides on 2-chlorotrityl resin All steps were carried out at 20°C under an inert atmosphere. 1. Preparation of resin: To chloro-2-chlorotrityl resin (1.00 eq., Sub: 1.08 mmol / g) in DCM was added Fmoc-β-Ala-OH or Fmoc-Gly-OH (0.71 eq.) and DIEA (2.84 eq.). The resin was stirred for 3 h. Then, MeOH (1 mL / mmol of resin) was added and stirred for 30 min. The resin was washed with DMF (5 times) and filtered. 2. Deprotection: The resin was treated with 20% piperidine for 30 minutes. The resin was washed with DMF (5 times). 3. Coupling: The second residue [Fmoc-His(Trt)-OH or Fmoc-Arg(Pbf)-OH] was added as a solution (3.00 eq.), and a solution of HBTU (2.85 eq.) and DIEA (6.00 eq.) in DMF was added to the resin and stirred for 50 min ([Fmoc-AA-OH] initial(≒0.2-0.3 M). The resin was then washed five times with DMF. 4. Repeat steps 2 and 3 above to couple the remaining amino acids. 5. The N-terminal acetyl (Ac) group was added by treatment with a solution of Ac2O / DIEA / DMF = 10 / 5 / 85 for 30 minutes. Peptide cleavage and purification: 1. Cleavage solution (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% 3-mercaptopropionic acid) was added to the flask containing the side-chain protected peptide and stirred for 2.0 hours. 2. The precipitated peptide was washed with cold isopropyl ether. 3. The crude peptide was collected as a cake-like mass by filtration. 4. The filter cake was washed two more times with isopropyl alcohol. 5. The crude peptide was dried under vacuum for 2 hours. 6. The crude peptide was purified by prep-HPLC (conditions: A: 0.075% TFA aqueous solution, B: ACN) and lyophilized to obtain a solid. It was then converted to the HCl salt by prep-HPLC (conditions: A: 0.05% HCl aqueous solution, B: ACN) to obtain a white solid. The yield of the final product was over 30%, and the final purity was over 95±2%.

[0408] Synthesis of PMO23 PMO23 was synthesized according to a published protocol (Ghosh ChemRxiv, 2020, 1-1, schematic diagram) using commercially available phosphate chloride monomer (Figure 3). To reduce side reactions, O-methylation of guanosine was used. 64-Nitrophenylethyl (NPE) protection was used for the PMO oligomer (Pon 1986). NPE was removed according to published methods (Garcia et al., Antisense and Nucleic Acid Drug Development 11(6):369-378, 2001; Avino et al., Nucleosides, Nucleotides & Nucleic Acids 13(10):2059-2069, 1994). Briefly, the synthesis begins with N-Trt-sarcosine attached to polystyrene via an acid-stable ester bond. The N-Trt group is removed with mild acid, and the resin is neutralized. 5'-Phosphonochloramidate coupling is performed using NEM in DMI solvent. Unreacted amino groups are capped with benzoic anhydride. The deprotection, coupling, and capping cycle is repeated until the full-length PMO oligomer is obtained. The oligonucleotide and C and A protecting groups are cleaved with aqueous ammonia. Crude PMO oligomers containing NPE protecting groups were deblocked using DBU in the presence of thymine to prevent reverse alkylation of the bases by NPE (Avino 1994). A final cleavage with ammonia was performed to remove the guanine-N2 isobutyryl group. The crude PMO oligomers were then purified by ionic reverse-phase chromatography and lyophilized to yield a white powder. The final purity was 93%, with an overall yield of 39%.

[0409] Conjugation of peptide PMO23 JPEG2025538552000043.jpg78170 Conjugation of the peptide to the 3'-morpholine ring of PMO23 was achieved by in situ activation of the free gamma carboxylic acid of the peptide.

[0410] Example PMO23 (1.0 eq.) and Dpep1.9b (2.0 eq.) were dissolved in DMSO, and EDCl (2.0 eq.), HOBt (4.0 eq.), and DIPEA (1.0 eq.) were added to carry out the coupling reaction. After 4 h, the reaction was quenched with purified water, and the crude solution was purified by cation exchange chromatography and desalted by ultrafiltration / diafiltration. The final product was recovered as a white powder with a purity of 99% and a yield of 66% after lyophilization.

[0411] The same procedure was used for the polyarginine-PMO23 complex, and after freeze-drying, a white powder with a purity of 99% and a yield of 53% was obtained.

[0412] Example 2. In vitro efficacy overview This method describes the in vitro evaluation of exon skipping and cell viability in C2C12 myoblasts after incubation with a peptide conjugated to a mouse Dmd-specific PMO. The peptide is conjugated to the mouse Dmd-specific PMO, PMO23, with the sequence 5′-GGCCAAACCTCGGCTTACCTGAAAT-3′ (SEQ ID NO: 288). In this protocol, the efficacy of the PPMO conjugate is evaluated in vitro using C2C12 myoblasts, an immortalized cell line isolated from Mus musculus. The C2C12 cell line was developed as a subclone of the mouse myoblast cell line originally isolated by Yaffe and Saxel (Nature, 270:725-727, 1977). C2C12 myoblasts rapidly differentiate to form contractile myotubes and produce characteristic muscle proteins. C2C12 myoblast cells have been shown to express high levels of dystrophin (DMD) and have been widely used in studies evaluating the restoration of dystrophin levels by ASOs (Xu et al., Mol. Ther., 24:564-569, 2016; Lehto et al., Nuc. Acids Res., 42:3207-3217, 2014).

[0413] Exon skipping was assessed by qPCR analysis amplifying exons 22 and 23-24, which amplify skipped and non-skipped transcripts, respectively. By quantitatively measuring the levels of skipped and non-skipped transcripts, the percentage of exon skipping in each sample could be calculated. This mDMDex23 qPCR assay can be used in in vitro experiments with C2C12 myoblasts, enabling high-throughput screening of PPMO efficacy.

[0414] protocol Myoblast proliferation C2C12 myoblasts can be subcultured. To maintain the differentiation potential of C2C12 cells, under normal culture conditions, the cell confluence should be below 75%. The procedure for subculture of C2C12 cells is as follows. 1. Remove the medium and briefly wash the cell monolayer with warm PBS. 2. Detach cells using TrypLE. Neutralize TrypLE Express using DMEM. 3. Count the number of cells and adjust the cell concentration to 1 cm 2 5.0 x 10 per culture vessel surface area 3 Adjust to the cells. 4. Dispense sufficient medium into tissue culture vessels, dispense an appropriate amount of cell suspension into each vessel to complete seeding, and culture the cells under conditions of 37°C, 5% CO2.

[0415] Myoblast-myotube differentiation C2C12 myoblasts spontaneously differentiate to form myotubes when they reach over 90% confluence. To ensure a high level of differentiation, a specific differentiation medium is used instead of growth medium. This differentiation medium consists of DMEM supplemented with 2% horse serum and 1% antibiotic / antimycotic. 1. Cells for differentiation must reach a confluence of 80% or more, and should be seeded preferably 1 to 2 days in advance. 2. Remove growth medium and wash briefly with pre-warmed PBS. 3. Add 1 mL of pre-warmed differentiation medium and return the plate to the incubator. 4. Assess myotube formation daily and replace the medium with fresh differentiation medium daily. After 5.7 days, high levels of myotube formation can be observed.

[0416] In vitro PPMO treatment C2C12 myotubes are treated 6-8 days after the initiation of differentiation, when a high level of differentiation is observed. Myotubes are exposed to gymnotic delivery of PPMO at various concentrations (typically 0.1-20 μM) for a minimum of 48 hours.

[0417] Cell harvesting and RNA extraction After treatment with PPMO, C2C12 myotubes were harvested and RNA was extracted to quantify the level of DMD exon 23 skipping by qPCR using a Maxwell RSC 48 and Promega simplyRNA Tissue Kit according to the manufacturer's guidelines.

[0418] RNA normalization 1. Quantify the extracted RNA to determine the quality (260 / 280 ratio >1.7) and quantity (>50 ng / μL) of RNA in all samples.

[0419] cDNA synthesis cDNA synthesis from normalized RNA samples is performed using the High-Capacity cDNA Reverse Transcription Kit (4368813) from ThermoFisher Scientific. 1. Prepare a master mix (RT buffer, dNTP mix, RT random primers, Multiscribe reverse transcriptase, and nuclease-free water) for all samples. 3. Pipette 10 μL of master mix into each reaction tube and add 10 μL of normalized RNA (50 ng / μL). 4. RNA samples are reverse transcribed as follows: Thermocycler conditions required for reverse transcription using the High Capacity cDNA Reverse Transcription Kit. JPEG2025538552000044.jpg3656

[0420] qPCR mDMDex23 Gene expression can be measured by quantitation of cDNA relative to calibration standard samples (standards, ie, gBlocks). 1. Sequences correspond to WT cDNA (mouse Dmd gene Ref NM007868), with the non-skipped sequence covering exon 20 to exon 26 and the skipped sequence covering exon 20 to exon 26 but excluding exon 23 (Δ23). 2. Standard curves were prepared by performing four 10-fold dilution series in triplicate for each standard curve. The highest standard value without skipping was 797,100 copies, and with skipping was 965,300 copies. 3. Primer / probe preparation. Primers and probes are provided by IDT at a concentration of 100 μM in IDTE pH 8.0 solution. Primers are used at a final concentration of 0.5 μM and probes at 0.25 μM. Primer and probe sequences are listed below. JPEG2025538552000045.jpg49166JPEG2025538552000046.jpg491664. Prepare qPCR reaction mixes. Prepare reaction mixes for each standard and sample. Standards may be run in triplicate, and samples in duplicate. 5. qPCR mDMDex23 assays were performed using TaqMan Fast Advanced Master Mix (4444557). TaqMan® Fast Advanced Master Mix contains AmpliTaq® Fast DNA polymerase, uracil-N-glycosylase (UNG), dNTPs (including dUTP), ROX, and ribosomal RNA. TM It contains a dye (passive reference) and an optimized buffer. 6. Dilute the cDNA 1:5 (20 μL cDNA and 80 μL nuclease-free water) and add 5 μL of diluted cDNA to each well. 7. Prepare the TaqMan master mix (TaqMan Fast Advanced Master Mix, primers / probes, cDNA template, and nuclease-free water) and transfer the appropriate amount of each reaction mix to each well of the optical plate. Cover the plate with optical adhesive film. 8. Run qPCR using QuantStudio6 (Thermo) under the following conditions: Temperature cycling conditions for the qPCR mDMDex23 assay. JPEG2025538552000047.jpg37709. Post-run analysis: When using an Applied Biosystems real-time PCR instrument, the baseline and threshold of the amplification curve can be automatically calculated using Sequence Detection System (SDS) software, or can be set manually. The threshold is set above background and within the exponential growth phase of the amplification curve. Raw data is extracted and analyzed. The level of each transcript is determined by calibration with a standard curve prepared using known amounts of transcript, and the skip ratio is calculated as ([with skip] / [with skip + without skip]) x 100.

[0421] Example 2. In vivo efficacy Mouse studies may be performed to evaluate the efficacy of the conjugates described herein. Conjugates can be evaluated for efficacy (e.g., exon skipping in the heart and quadriceps) and / or toxicity (e.g., clinical signs, clinical chemistry, gross findings, histopathology (liver and kidney), and body weight profile). The peptides used in the conjugates evaluated in this example are listed in the table below. JPEG2025538552000048.jpg73163E and e refer to gamma Glu linkers. The alpha carboxyl group of E or e is amidated. *All chiral amino acid residues are D-amino acids.

[0422] This test may be performed as shown in the table below. JPEG2025538552000049.jpg149160

[0423] The conjugates may be administered intravenously to mice as a bolus (over approximately 20 seconds) into the tail vein, and 7 days after administration, the mice may be necropsied to obtain serum and tissue samples.

[0424] Safety and tolerability analyses may include clinical observations, clinical chemistry (e.g., markers of liver and kidney function), gross findings, histopathology (e.g., liver and kidney), and / or body / tissue weight profiles.

[0425] Activity analysis can be based on observations (e.g., exon skipping) in skeletal muscle (e.g., quadriceps) and cardiac tissue.

[0426] The results of exon 23 skipping in the quadriceps and heart of PPMO-treated mice are shown in Figures 1 and 2, respectively. Mice were administered candidate PPMOs at 30 or 60 mg / kg via bolus injection (over approximately 20 seconds) into the tail vein. Treatment was performed on day 1, and necropsy was performed as scheduled on day 8. Exon skipping was assessed by qPCR. Graphs are plotted as the percentage of exon skipping (mean ± SD, n = 3–5). The results showed that specific peptides improved exon skipping in both the quadriceps and heart compared to the polyarginine control.

[0427] Other embodiments Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the present invention.

[0428] Other embodiments are within the claims.

Claims

1. A conjugate comprising a peptide and a therapeutic or diagnostic molecule linked to the peptide via a covalent or non-cationic linker, The peptide comprises a total of one hydrophobic domain and a total of one cationic domain, the cationic domain comprising at least one cationic amino acid residue, and the hydrophobic domain comprising at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues.

2. A conjugate comprising a peptide and a therapeutic or diagnostic molecule linked to the peptide via a covalent or non-cationic linker, The peptide comprises one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, the at least one cationic domain comprising one cationic amino acid residue, and the hydrophobic domain comprising at least three amino acid residues, the peptide comprising a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and no artificial amino acid residues, and the amino acid residues of each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine.

3. A conjugate comprising a peptide and a therapeutic or diagnostic molecule linked to the peptide via a covalent or non-cationic linker, The peptide comprises one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, all cationic domains comprising five or fewer cationic amino acid residues, and each hydrophobic domain comprising at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues, and the amino acid residues in each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine.

4. A conjugate comprising a peptide and a therapeutic or diagnostic molecule linked to the peptide via a covalent or non-cationic linker, The peptide comprises a total of one hydrophobic domain and a total of one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, at least one-third of the amino acid residues in the N-terminal cationic domain being histidine, and the hydrophobic domain comprising at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 (e.g., 6 to 40, or 7 to 40) amino acid residues and does not comprise any artificial amino acid residues.

5. 10. The complex of any preceding claim, wherein each cationic domain comprises at least 40%, at least 45%, or at least 50% cationic amino acids.

6. 10. The complex of any preceding claim, wherein each cationic domain comprises a majority of cationic amino acids, preferably at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% cationic amino acids.

7. 7. The complex of claim 1 or 4 to 6, wherein each cationic domain comprises an arginine, histidine, beta-alanine, hydroxyproline, and / or serine residue, preferably each cationic domain consists of an arginine, histidine, beta-alanine, hydroxyproline, and / or serine residue, provided that at least one arginine or histidine is present.

8. 10. The complex of any preceding claim, wherein each cationic domain is arginine-rich and / or histidine-rich, preferably each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% arginine and / or histidine residues.

9. At least one cationic domain may be selected from the group consisting of RBRR (SEQ ID NO: 419), RBR, RB, R, RBRRBRR (SEQ ID NO: 420), RRBRR (SEQ ID NO: 426), BRR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRB, RBRBRB (SEQ ID NO: 424), RBRB (SEQ ID NO: 425), RB, HRHRHR (SEQ ID NO: 426), HRHR (SEQ ID NO: 427), HR, RRRRRR (SEQ ID NO: 428), RBRRBR (SEQ ID NO: 429), RBHBHB (SEQ ID NO: 430), RBHBH (SEQ ID NO: 431), BHBHB (SEQ ID NO: 432), BHB 5. The complex of any one of claims 1 to 4, wherein the complex is selected from the group consisting of H (SEQ ID NO: 433), HBHB (SEQ ID NO: 434), HBH, BHB, BH, HB, H, RBHBHE (SEQ ID NO: 435), RBHBB (SEQ ID NO: 436), RBHB (SEQ ID NO: 437), RBB, HRBRHB (SEQ ID NO: 438), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRB, RBRBR (SEQ ID NO: 441), HRHRHRB (SEQ ID NO: 442), HRHRB (SEQ ID NO: 443), HRBRH (SEQ ID NO: 444), HRB, RRRRRRB (SEQ ID NO: 445), BRE, and BR.

10. 5. The complex of any one of claims 1 to 4, wherein the at least one cationic domain is selected from the group consisting of RBR, RBRB (SEQ ID NO: 425), RB, R, RBRRBRR (SEQ ID NO: 420), BRR, BR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRB, HRHRHR (SEQ ID NO: 426), HR, RRRRRR (SEQ ID NO: 428), RRRRRRB (SEQ ID NO: 445), RBHBH (SEQ ID NO: 431), BH, BHB, H, HB, RBH, and RBHB (SEQ ID NO: 437).

11. 10. A conjugate according to any preceding claim, wherein each hydrophobic domain has a length of 3 to 6 amino acids, preferably each hydrophobic domain is 5 amino acids in length.

12. 10. The conjugate of any preceding claim, wherein each hydrophobic domain comprises a majority of hydrophobic amino acid residues, preferably wherein each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids.

13. 10. A conjugate according to any preceding claim, wherein each hydrophobic domain comprises phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues, preferably each hydrophobic domain consists of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues, with the proviso that at least one of phenylalanine, leucine, isoleucine, tyrosine, and tryptophan is present.

14. 10. The complex of any preceding claim, wherein the hydrophobic domain is FQILY (SEQ ID NO: 446).

15. A conjugate of a peptide and a therapeutic or diagnostic molecule linked to said peptide via a covalent or non-cationic linker, comprising: The peptides were RBRRFQILYRBHBH (SEQ ID NO: 447), RBRFQILYRBHBH (SEQ ID NO: 448), RBFQILYRBHBH (SEQ ID NO: 449), RFQILYRBHBH (SEQ ID NO: 450), FQILYRBHBH (SEQ ID NO: 451), RBRRBRRFQILYBHBHB (SEQ ID NO: 452), RBRRBRRFQILYHBH (SEQ ID NO: 453), RBRRBRRFQILYBH (SEQ ID NO: 454), RBRRBRRFQILYH (SEQ ID NO: 455), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBH (SEQ ID NO: 457), RRBRRFQILYRBHBH (SEQ ID NO: 458), BRRFQILYRBHBH (SEQ ID NO: 459), RRFQILYRBHBH (SEQ ID NO: 460), RBRRBRRFQILYRBHB (SEQ ID NO: 461), RBRRBRRFQILYRBH (SEQ ID NO: 462), RBRRBRRFQILYRB (SEQ ID NO: 463), RBRRBRRFQILYRB (SEQ ID NO: 464), RBRRBRRFQILYRB (SEQ ID NO: 465), RBRRBRRFQILYRB (SEQ ID NO: 466), RBRRBRRFQILYRB (SEQ ID NO: 467), RBRRBRRFQILYRB (SEQ ID NO: 468), RBRRBRRFQILYRB (SEQ ID NO: 469), RBRRBRRFQILYRB (SEQ ID NO: 470), RBRRBRRFQILYRBHB (SEQ ID NO: 471), RBRRBR SEQ ID NO: 463), RBRRBRRFQILYR (SEQ ID NO: 464), HRBHRBHRBFQILYHRBRH (SEQ ID NO: 465), HRBHRBHRBFQILYHRBHRBHR (SEQ ID NO: 466), HRBHRBFQILYHRBHR (SEQ ID NO: 467), HRBFQILYHR (SEQ ID NO: 468), RBRBRBFQILYRBRBR (SEQ ID NO: 469), RBRBFQILYRBR (SEQ ID NO: 470), RBFQILYR (SEQ ID NO: 471) ), HRHRHRFQILYHRHRHR (SEQ ID NO: 472), HRHRFQILYHRHR (SEQ ID NO: 473), HRFQILYHR (SEQ ID NO: 474), RRRRRRFQILY (SEQ ID NO: 475), FQILYRRRRRR (SEQ ID NO: 476), RRRRRRFQILYRRRRRR (SEQ ID NO: 477), RBRRBRFQILYBR (SEQ ID NO: 478), and RBRRBRFQILY (SEQ ID NO: 479).

16. A conjugate of a peptide and a therapeutic or diagnostic molecule linked to said peptide via a covalent or non-cationic linker, comprising: The peptides are RBRRFQILYRBHBHB (SEQ ID NO: 481), RBRFQILYRBHBHB (SEQ ID NO: 482), RBFQILYRBHBHB (SEQ ID NO: 483), RFQILYRBHBHB (SEQ ID NO: 484), FQILYRBHBHB (SEQ ID NO: 485), RBRRBRRFQILYBHBHB (SEQ ID NO: 452), RBRRBRRFQILYHBHB (SEQ ID NO: 486), RBRRBRRFQILYBHB (SEQ ID NO: 487), RBRRBRRFQILYHB (SEQ ID NO: 488), R BRRBRRFQILYB (SEQ ID NO: 489), RBRRBRRFQILYE (SEQ ID NO: 416), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBHB (SEQ ID NO: 490), RRBRRFQILYRBHBHB (SEQ ID NO: 491), BRRFQILYRBHBHB (SEQ ID NO: 492), RRFQILYRBHBHB (SEQ ID NO: 493), RRFQILYRBHBHE (SEQ ID NO: 417), RBRRBRRFQILYRBHBB (SEQ ID NO: 494), RBRRBRRFQI LYRBHB (SEQ ID NO: 461), RBRRBRRFQILYRBB (SEQ ID NO: 495), RBRRBRRFQILYRB (SEQ ID NO: 463), HRBHRBHRBFQILYHRBRHB (SEQ ID NO: 496), HRBHRBHRBFQILYHRBHRBHRB (SEQ ID NO: 497), HRBHRBFQILYHRBHRB (SEQ ID NO: 498), HRBFQILYHRB (SEQ ID NO: 499), RBRBRBFQILYRBRBRB (SEQ ID NO: 500), RBRBFQILYRBRB (SEQ ID NO: 501), A complex selected from the group consisting of RBFQILYRB (SEQ ID NO: 502), HRHRHRFQILYHRHRHRB (SEQ ID NO: 503), HRHRFQILYHRHRB (SEQ ID NO: 504), HRFQILYHRB (SEQ ID NO: 505), RRRRRRFQILYB (SEQ ID NO: 506), FQILYRRRRRRB (SEQ ID NO: 507), RRRRRRFQILYRRRRRRB (SEQ ID NO: 508), RBRRBRFQILYBRE (SEQ ID NO: 415), and RBRRBRFQILYE (SEQ ID NO: 509).

17. 10. A conjugate according to any preceding claim, wherein the peptide is attached to the remainder of the conjugate via its N-terminus.

18. 18. The conjugate of claim 17, wherein the C-terminus of the peptide is amidated.

19. 17. The conjugate of any one of claims 1 to 16, wherein the peptide is linked to the remainder of the conjugate via its C-terminus.

20. 20. The conjugate of claim 19, wherein the peptide is acylated at the N-terminus.

21. 10. A conjugate according to any preceding claim, wherein the peptide is covalently attached to a therapeutic or diagnostic molecule.

22. 21. The conjugate of any one of claims 1 to 20, wherein the peptide is covalently linked to the therapeutic or diagnostic molecule via a non-cationic linker, optionally comprising a total of one amino acid or aliphatic dicarboxylic acid linker.

23. 23. The conjugate of claim 22, wherein the linker is selected from the group consisting of glutamic acid, beta-alanine, glycine, delta-aminovaleric acid, and gamma-aminobutyric acid.

24. 21. The conjugate of any one of claims 1 to 20, wherein the linker has the following structure: 【Chemistry 1】

25. 23. The conjugate of claim 22, wherein the linker has the following structure: 【Chemistry 2】

26. 23. The conjugate of claim 22, wherein the linker has the following structure: 【Transformation 3】

27. 23. The conjugate of claim 22, wherein the linker has the following structure: 【Chemistry 4】

28. 23. The conjugate of claim 22, wherein the linker has the following structure: 【Transformation 5】

29. 23. The conjugate of claim 22, wherein the conjugate has the following structure: 【Transformation 6】

30. 23. The conjugate of claim 22, wherein the conjugate has the following structure: 【Transformation 7】

31. 23. The conjugate of claim 22, wherein the conjugate has the following structure: 【Transformation 8】

32. 23. The conjugate of claim 22, wherein the conjugate has the following structure: 【Chemistry 9】

33. 23. The conjugate of claim 22, wherein the conjugate has the following structure: 【Chemistry 10】

34. 10. The conjugate of any preceding claim, wherein the therapeutic or diagnostic molecule optionally comprises an oligonucleotide attached at its 3' end to the linker or the peptide.

35. 35. The conjugate of claim 34, wherein the oligonucleotide comprises a sequence complementary to a target sequence, and wherein targeting by the oligonucleotide alters the processing or recognition of the target sequence, and optionally, the targeting can be used to treat, ameliorate, or prevent one or more symptoms of a genetic disease.

36. 36. The conjugate of claim 35, wherein the genetic disease is optionally a neuromuscular disease selected from the group consisting of muscular dystrophy (e.g., DMD, BMD, or FSHD), DM1, DM2, and SMA, and optionally, the target sequence is present in a transcript of a DMD, DMPK, CNBP, SMN2, or DUX4 gene.

37. 36. The conjugate of claim 35, wherein the genetic disease is a neurological disease or a disease that targets another organ system of the body, optionally CMT1a, CMT2a, FCD, or FA, and optionally the target sequence is present in a transcript of a PMP22, MFN2, TF4, or FXN gene.

38. 37. The conjugate of any one of claims 34 to 36, wherein the oligonucleotide comprises at least 12 consecutive nucleobases complementary to a target sequence in the dystrophin gene.

39. 39. The conjugate of claim 38, wherein the target sequence is located within or near any one of exons 8 to 55 of the transcript of the DMD gene.

40. 40. The conjugate of claim 39, wherein the target sequence is located within or near any one of exons 8, 23, 44, 45, 50, 51, 52, 53, or 55 of the transcript of the DMD gene.

41. 41. The conjugate of any one of claims 35, 36, and 38 to 40, wherein the target sequence is present within a transcript of the human DMD sequence.

42. 42. The complex of any one of claims 38 to 41, wherein the target sequence includes the exon 45 splice site or is located within 150 nucleobases of the exon 45 splice site.

43. 43. The conjugate of Claim 42, wherein said oligonucleotide comprises at least 12 contiguous nucleobases from any one of the sequences in Table 1, and thymine or uracil substituted forms thereof.

44. 43. The conjugate of claim 42, wherein the oligonucleotide comprises any one of the sequences of Table 1, or a thymine or uracil substitution thereof.

45. The sequences in Table 1 are 45. The complex of claim 44, which is 5'-GCTGCCCAATGCCATCCTGGAGTTCCTGTAA-3' (sequence number 276).

46. The sequences in Table 1 are 45. The complex of claim 44, wherein the complex is 5'-CAATGCCATCCTGGAGTTCCTG-3' (sequence number 275).

47. The sequences in Table 1 are 45. The complex of claim 44, wherein the complex is selected from the group consisting of 5'-TTGCCGCTGCCCAATGCCATCCTGGAGTTC-3' (SEQ ID NO: 270), 5'-CAGTTTGCCGCTGCCCAATGCCATCCTGGA-3' (SEQ ID NO: 271), 5'-CCAAUGCCAUCCUGGAGUUCCUGUAA-3' (SEQ ID NO: 272), 5'-CCAATGCCATCCTGGAGTTCCTGTA-3' (SEQ ID NO: 273), and 5'-CTGACAACAGTTTGCCGCTGCCCAA-3' (SEQ ID NO: 274).

48. 42. The complex of any one of claims 38 to 41, wherein the target sequence includes the exon 51 splice site or is located within 150 nucleobases of the exon 51 splice site.

49. 49. The conjugate of Claim 48, wherein said oligonucleotide comprises at least 12 contiguous nucleobases from any one of the sequences in Table 2, and thymine substituted forms thereof.

50. 49. The conjugate of claim 48, wherein the oligonucleotide comprises any one of the sequences in Table 2, or a thymine substituted version thereof.

51. The sequences in Table 2 are 51. The complex of claim 50, wherein the complex is 5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3' (sequence number 282).

52. The sequences in Table 2 are 51. The complex of claim 50, wherein the complex is 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (sequence number 287).

53. 42. The complex of any one of claims 38 to 41, wherein the target sequence comprises the exon 53 splice site or is located within 150 nucleobases of the exon 53 splice site.

54. 54. The conjugate of Claim 53, wherein said oligonucleotide comprises at least 12 contiguous nucleobases from any one of the sequences in Table 3, and thymine or uracil substituted forms thereof.

55. 54. The conjugate of claim 53, wherein the oligonucleotide comprises any one of the sequences in Table 3, or a thymine or uracil substituted version thereof.

56. The sequences in Table 3 are 56. The complex of claim 55, which is 5'-CCTCCGGTTCTGAAGGTGTTCT-3' (sequence number 316) or 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (sequence number 325).

57. The sequences in Table 3 are 56. The complex of claim 55, wherein the complex is selected from the group consisting of 5'-CTGTTGCCTCCGGTTCTGAAGGTGTTCTTG-3' (SEQ ID NO: 327), 5'-CAACTGTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 328), 5'-TTGCCTCCGGTTCTGAAGGTGTTCTTGTAC-3' (SEQ ID NO: 329), 5'-CTGAAGGTGTTCTTGTACTTCATCC-3' (SEQ ID NO: 330), and 5'-CATTCAACTGTTGCCTCCGGTTCTGAAGGTG-3' (SEQ ID NO: 331).

58. 42. The complex of any one of claims 38 to 41, wherein the target sequence comprises the exon 44 splice site or is located within 150 nucleobases of the exon 44 splice site.

59. 59. The conjugate of Claim 58, wherein said oligonucleotide comprises at least 12 contiguous nucleobases from any one of the sequences in Table 4, and thymine or uracil substituted forms thereof.

60. 59. The conjugate of claim 58, wherein the oligonucleotide comprises any one of the sequences in Table 4, or a thymine or uracil substituted version thereof.

61. The sequences in Table 4 are 61. The complex of claim 60, wherein the complex is selected from the group consisting of 5'-TGAAAACGCCGCCATTTCTCAACAGATCTG-3' (SEQ ID NO: 335), 5'-CATAATGAAAACGCCGCCATTTCTCAACAG-3' (SEQ ID NO: 336), 5'-TGTTCAGCTTCTGTTAGCCACTGATTAAAT-3' (SEQ ID NO: 337), 5'-CGCCGCCATTTCTCAACAG-3' (SEQ ID NO: 338), and 5'-ATCTGTCAAATCGCCTGCAG-3' (SEQ ID NO: 339).

62. 40. The conjugate of any one of claims 34 to 36, 38 or 39, wherein the oligonucleotide comprises the sequence 5'-GGCCAAACCTCGGCTTACCTGAAAT-3' (SEQ ID NO: 288).

63. 59. The complex of claim 42, 48, 53, or 58, wherein the splice site is an acceptor splice site.

64. 59. The complex of claim 42, 48, 53, or 58, wherein the splice site is a donor splice site.

65. 37. The conjugate of any one of claims 34 to 36, wherein the oligonucleotide comprises at least 9 consecutive nucleobases complementary to a CUG repeat sequence.

66. The oligonucleotide is 5'-[CAG] n -3',5'-[AGC] n -3' and 5'-[GCA] n -3', wherein n is an integer from 5 to 8.

67. The oligonucleotide is 5'-[CAG] 5 -3' (SEQ ID NO: 340), 5'-[CAG] 6 -3' (SEQ ID NO: 341), 5'-[CAG] 7 -3' (SEQ ID NO: 342), 5'-[CAG] 8 -3' (SEQ ID NO: 343), 5'-[AGC] 5 -3' (SEQ ID NO: 344), 5'-[AGC] 6 -3' (SEQ ID NO: 345), 5'-[AGC] 7 -3' (SEQ ID NO: 346), 5'-[AGC] 8 -3' (SEQ ID NO: 347), 5'-[GCA] 5 -3' (SEQ ID NO: 348), 5'-[GCA] 6 -3' (SEQ ID NO: 349), 5'-[GCA] 7 -3' (SEQ ID NO: 350), and 5'-[GCA] 8 66. The conjugate of claim 65, having a sequence selected from the group consisting of: -3' (SEQ ID NO: 351).

68. 37. The conjugate of any one of claims 34 to 36, wherein the oligonucleotide comprises at least 8 consecutive nucleobases complementary to a CCUG repeat sequence.

69. The oligonucleotide is 5'-[CAGG] n -3',5'-[AGGC] n -3',5'-[GGCA] n -3',5'-[GCAG] n -3', wherein n is an integer from 4 to 8.

70. The oligonucleotide is 5'-[CAGG] 4 -3' (SEQ ID NO: 352), 5'-[CAGG] 5 -3' (SEQ ID NO: 353), 5'-[CAGG] 6 -3' (SEQ ID NO: 354), 5'-[CAGG] 7 -3' (SEQ ID NO: 355), 5'-[CAGG] 8 -3' (SEQ ID NO: 356), 5'-[AGGC] 4 -3' (SEQ ID NO: 357), 5'-[AGGC] 5 -3' (SEQ ID NO: 358), 5'-[AGGC] 6 -3' (SEQ ID NO: 359), 5'-[AGGC] 7 -3' (SEQ ID NO: 360), 5'-[AGGC] 8 -3' (SEQ ID NO: 361), 5'-[GGCA] 4 -3' (SEQ ID NO: 362), 5'-[GGCA] 5 -3' (SEQ ID NO: 363), 5'-[GGCA] 6 -3' (SEQ ID NO: 364), 5'-[GGCA] 7 -3' (SEQ ID NO: 365), 5'-[GGCA] 8 -3' (SEQ ID NO: 366), 5'-[GCAG] 4 -3' (SEQ ID NO: 367), 5'-[GCAG] 5 -3' (SEQ ID NO: 368), 5'-[GCAG] 6 -3' (SEQ ID NO: 369), 5'-[GCAG] 7 -3' (SEQ ID NO: 370), and 5'-[GCAG] 8 69. The conjugate of claim 68, having a sequence selected from the group consisting of: -3' (SEQ ID NO: 371).

71. 37. The conjugate of any one of claims 34 to 36, wherein the oligonucleotide comprises at least 12 consecutive nucleobases complementary to a target sequence in a transcription product of the SMN2 gene.

72. 72. The conjugate of claim 71, wherein the target sequence is located in SMN2 intron 7.

73. 72. The conjugate of claim 71, wherein said oligonucleotide comprises any one of the sequences in Table 5 and thymine substituted versions thereof.

74. 74. The conjugate of any one of claims 34 to 73, wherein the oligonucleotide comprises or consists of a sequence of a table herein (Table 1, Table 2, Table 3, Table 4, Table 5, or Table 8), optionally wherein one or more (e.g., all) uracils are substituted with thymine, or one or more (e.g., all) thymines are substituted with uracil.

75. 75. The conjugate of any one of claims 34 to 74, wherein the oligonucleotide comprises at its 5' end a group: 【Chemistry 11】

76. 75. The conjugate of any one of claims 34 to 74, wherein the oligonucleotide comprises at its 5' end a group: 【Chemistry 12】

77. 77. The conjugate of any one of claims 34 to 76, wherein the oligonucleotide comprises a hydroxyl at its 5' end.

78. 78. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 77 and a pharmaceutically acceptable excipient.

79. 79. A method of treating a subject having a genetic disease, comprising administering to the subject a therapeutically effective amount of a conjugate of any one of claims 1 to 77, or a pharmaceutical composition of claim 78.

80. 80. The method of claim 79, wherein the genetic disease is optionally a neuromuscular disease selected from the group consisting of muscular dystrophy (e.g., DMD, BMD, or FSHD), DM1, DM2, and SMA, and optionally, the target sequence is present within a transcript of a DMD, DMPK, CNBP, SMN2, or DUX4 gene.

81. 80. The method of claim 79, wherein the genetic disease is optionally a neurological disease or a disease targeting another organ system selected from the group consisting of CMT1a, CMT2a, FCD, and FA, and optionally the target sequence is present within a transcript of a PMP22, MFN2, TF4, or RXN gene.

82. 80. The method of claim 79, comprising administering to the subject a therapeutically effective amount of a conjugate of any one of claims 1 to 77 or a pharmaceutical composition of claim 78, wherein in the treatment of DMD or BMD, the conjugate is of any one of claims 1 to 36, 38 to 64, or 74 to 77; in the treatment of DM1, the conjugate is of any one of claims 1 to 36, 65 to 67, or 74 to 77; in the treatment of DM2, the conjugate is of any one of claims 1 to 36, 68 to 70, or 74 to 77; and in the treatment of SMA, the conjugate is of any one of claims 1 to 36 or 71 to 77.

83. 82. The method of any one of claims 79, 80, or 81, wherein the subject has DMD or BMD.

84. 82. The method of any one of claims 79, 80, or 81, wherein the subject has DM1.

85. 82. The method of any one of claims 79, 80, or 81, wherein the subject has DM2.

86. 82. The method of any one of claims 79, 80, or 81, wherein the subject has SMA.

87. A peptide comprising a total of one hydrophobic domain and a total of one cationic domain, wherein the cationic domain comprises at least one cationic amino acid residue and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 amino acid residues and does not comprise any artificial amino acid residues.

88. 1. A peptide comprising one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, wherein each cationic domain comprises at least one cationic amino acid residue, the at least one cationic domain comprises one cationic amino acid residue, and the hydrophobic domain comprises at least three amino acid residues, the peptide comprising a total of 5 to 40 amino acid residues and no artificial amino acid residues, and wherein the amino acid residues in each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine.

89. 1. A peptide comprising one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, wherein each cationic domain comprises at least one cationic amino acid residue, all cationic domains comprise five or fewer cationic amino acid residues, and the hydrophobic domain comprises at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 amino acid residues and does not comprise any artificial amino acid residues, and the amino acid residues in each cationic domain are selected from the group consisting of arginine, histidine, and beta-alanine.

90. A peptide comprising one hydrophobic domain and one or two cationic domains flanking the hydrophobic domain, each cationic domain comprising at least one cationic amino acid residue, at least one-third of the amino acid residues in the N-terminal cationic domain being histidine, and the hydrophobic domain comprising at least three amino acid residues, with the proviso that the peptide comprises a total of 5 to 40 amino acid residues and does not comprise any artificial amino acid residues.

91. 91. The peptide of any one of claims 87 to 90, wherein each cationic domain comprises at least 40%, at least 45%, or at least 50% cationic amino acids.

92. 92. The peptide of any one of claims 87 to 91, wherein each cationic domain comprises a majority of cationic amino acids, preferably at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% cationic amino acids.

93. 93. The peptide of any one of claims 87 or 90-92, wherein each cationic domain comprises arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues, preferably each cationic domain consists of arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues, provided that at least one arginine or histidine is present.

94. 94. The peptide of any one of claims 87 to 93, wherein each cationic domain is arginine-rich and / or histidine-rich, preferably each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% arginine and / or histidine residues.

95. At least one cationic domain may be selected from the group consisting of RBRR (SEQ ID NO: 419), RBR, RB, R, RBRRBRR (SEQ ID NO: 420), RRBRR (SEQ ID NO: 421), BRR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRB, RBRBRB (SEQ ID NO: 424), RBRB (SEQ ID NO: 425), RB, HRHRHR (SEQ ID NO: 426), HRHR (SEQ ID NO: 427), HR, RRRRRR (SEQ ID NO: 428), RBRRBR (SEQ ID NO: 434), RBHBHB (SEQ ID NO: 430), RBHBH (SEQ ID NO: 431), BHBHB (SEQ ID NO: 432), BHBH ( 95. The peptide of any one of claims 87 to 94, wherein the peptide is selected from the group consisting of SEQ ID NO: 433), HBHB (SEQ ID NO: 434), HBH, BHB, BH, HB, H, RBHBHE (SEQ ID NO: 435), RBHBB (SEQ ID NO: 436), RBHB (SEQ ID NO: 437), RBB, HRBRHB (SEQ ID NO: 438), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRB, RBRBR (SEQ ID NO: 441), HRHRHRB (SEQ ID NO: 442), HRHRB (SEQ ID NO: 443), HRBRH (SEQ ID NO: 444), HRB, RRRRRRB (SEQ ID NO: 445), BRE, and BR.

96. 96. The peptide of any one of claims 87 to 95, wherein the at least one cationic domain is selected from the group consisting of RBR, RBRB (SEQ ID NO: 425), RB, R, RBRRBRR (SEQ ID NO: 420), BRR, BR, RR, HRBHRBHRB (SEQ ID NO: 422), HRBHRB (SEQ ID NO: 423), HRBHRBHR (SEQ ID NO: 439), HRBHR (SEQ ID NO: 440), HRB, HRHRHR (SEQ ID NO: 426), HR, RRRRRR (SEQ ID NO: 428), RRRRRRB (SEQ ID NO: 445), RBHBH (SEQ ID NO: 431), BH, BHB, H, HB, RBH, and RBHB (SEQ ID NO: 437).

97. 97. The peptide of any one of claims 87 to 96, wherein each hydrophobic domain has a length of 3 to 6 amino acids, preferably each hydrophobic domain has a length of 5 amino acids.

98. 98. The peptide of any of claims 87 to 97, wherein each hydrophobic domain comprises a majority of hydrophobic amino acid residues, preferably each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids.

99. 99. The peptide of any of claims 87 to 98, wherein each hydrophobic domain comprises phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and glutamine residues, preferably each hydrophobic domain consists of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues, with the proviso that at least one of phenylalanine, leucine, isoleucine, tyrosine, and tryptophan is present.

100. 100. The peptide of any one of claims 87 to 99, wherein the peptide comprises one hydrophobic domain.

101. 101. The peptide of any one of claims 87 to 100, wherein the hydrophobic domain is FQILY (SEQ ID NO: 446).

102. RBRRFQILYRBHBHB (SEQ ID NO: 481), RBRFQILYRBHBHB (SEQ ID NO: 482), RBFQILYRBHBHB (SEQ ID NO: 483), RFQILYRBHBHB (SEQ ID NO: 484), FQILYRBHBHB (SEQ ID NO: 485), RBRRBRRFQILYBHBHB (SEQ ID NO: 452), RBRRBRRFQILYHBHB (SEQ ID NO: 486), RBRRBRRFQILYBHB (SEQ ID NO: 487), RBRRBRRFQILYHB (SEQ ID NO: 488), RBRRBRR FQILYB (SEQ ID NO: 489), RBRRBRRFQILYE (SEQ ID NO: 416), RBRRBRRFQILY (SEQ ID NO: 456), BRRBRRFQILYRBHBHB (SEQ ID NO: 490), RRBRRFQILYRBHBHB (SEQ ID NO: 491), BRRFQILYRBHBHB (SEQ ID NO: 492), RRFQILYRBHBHB (SEQ ID NO: 493), RRFQILYRBHBHE (SEQ ID NO: 417), RBRRBRRFQILYRBHBB (SEQ ID NO: 494), RBRRBRRFQILYRB HB (SEQ ID NO: 461), RBRRBRRFQILYRBB (SEQ ID NO: 495), RBRRBRRFQILYRB (SEQ ID NO: 463), HRBHRBHRBFQILYHRBRHB (SEQ ID NO: 496), HRBHRBHRBFQILYHRBHRBHRB (SEQ ID NO: 497), HRBHRBFQILYHRBHRB (SEQ ID NO: 498), HRBFQILYHRB (SEQ ID NO: 499), RBRBRBFQILYRBRBRB (SEQ ID NO: 500), RBRBFQILYRBRB (SEQ ID NO: 501), RBF A peptide selected from the group consisting of QILYRB (SEQ ID NO: 502), HRHRHRFQILYHRHRHRB (SEQ ID NO: 503), HRHRFQILYHRHRB (SEQ ID NO: 504), HRFQILYHRB (SEQ ID NO: 505), RRRRRRFQILYB (SEQ ID NO: 506), FQILYRRRRRRB (SEQ ID NO: 507), RRRRRRFQILYRRRRRRB (SEQ ID NO: 508), RBRRBRFQILYBRE (SEQ ID NO: 415), and RBRRBRFQILYE (SEQ ID NO: 509).

103. A peptide comprising or consisting of rBrrBrfqilyBrBr (SEQ ID NO: 510) or rBrrBrfqilyBrBre (SEQ ID NO: 513), where lower case letters indicate D-amino acids.

104. A conjugate described in any one of claims 1 to 77, a pharmaceutical composition described in claim 78, a method described in any one of claims 79 to 86, or a peptide described in any one of claims 87 to 102, wherein the peptide or the peptide of the conjugate is or comprises DPep1.9b-del2 (RBRRBRFQILYBRE) (SEQ ID NO: 415), DPep1.9b-del4 (RBRRBRFQILYE) (SEQ ID NO: 509), E5-E (RBRRBRRFQILYE) (SEQ ID NO: 416), or G5-E (RRFQILYRBHBHE) (SEQ ID NO: 417).

105. 104, a pharmaceutical composition according to claim 78 or 104, or a method according to claim 79 to 86 or 103, wherein the therapeutic or diagnostic molecule is or comprises an oligonucleotide.

106. 106. The conjugate, pharmaceutical composition, or method of claim 105, wherein the oligonucleotide is a morpholino.

107. 107. The conjugate, pharmaceutical composition, or method of claim 105 or 106, wherein the oligonucleotide is morpholino having all morpholino internucleoside linkages.

108. The oligonucleotides have the following internucleoside linkages: -P(O)(NMe 2 108. The conjugate, pharmaceutical composition, or method of any one of claims 105 or 107, wherein the morpholino has O-.

109. 106. The conjugate, pharmaceutical composition, or method of claim 105, wherein the oligonucleotide is a phosphorothioate (PS) oligonucleotide.

110. 106. The complex, pharmaceutical composition, or method of claim 105, wherein the oligonucleotide is a 2'-O-alkyl oligoribonucleotide, such as a 2'-O-methyl oligoribonucleotide.

111. 106. The conjugate, pharmaceutical composition, or method of claim 105, wherein the oligonucleotide is a 2'-O-alkyl phosphorothioate, such as a 2'-O-methyl phosphorothioate.

112. 106. The conjugate, pharmaceutical composition, or method of claim 105, wherein the oligonucleotide is a peptide nucleic acid (PNA).

113. A peptide comprising or consisting of the sequence of SEQ ID NO: 415, 509, 416, 417, 418, 402, 512, 403, 404, 405, 478, 479, 456, or 460.

114. 114. A conjugate comprising the peptide of claim 113 and a therapeutic or diagnostic molecule.

115. 115. The conjugate of claim 114, wherein the therapeutic or diagnostic molecule comprises an oligonucleotide, such as a morpholino.

116. 116. The conjugate of claim 115, wherein the sequence of the oligonucleotide is selected from Table 1, 2, 3, 4, 5, or 8.

117. 103. A peptide comprising or consisting of the sequence of the peptide of claim 102, wherein the C-terminal amino acid of said peptide has been removed or removed and replaced with a linker as described herein.

Images