HK40060220A - Cell-penetrating peptides - Google Patents

Description

Cell penetrating peptides

Technical Field

The present invention relates to peptides, in particular cell-penetrating peptides (cell-penetrating peptides), and to conjugates of such cell-penetrating peptides with therapeutic molecules. The invention further relates to the use of such peptides or conjugates in a method of treatment or as a medicament, in particular in the treatment of genetic disorders and especially muscular dystrophy (e.g. duchenne muscular dystrophy).

Background

Nucleic acid drugs are genomic drugs with the potential to alter human health care. Studies have shown that such therapies may find application in a wide range of disease areas, including neuromuscular diseases. The use of antisense oligonucleotide-based approaches to modulate pre-mRNA splicing in the neuromuscular disease Duchenne Muscular Dystrophy (DMD) has placed this monogenic disease at the forefront of accurate medical development.

However, insufficient cell penetration and poor distribution characteristics have hampered the therapeutic development of these promising antisense therapies-the large volume and dispersive nature of the muscle tissue matrix in DMD further highlights this challenge.

DMD affects one in every 3500 newborn boys. This severe X-linked recessive genetic disease is caused by a mutation in the DMD gene encoding the dystrophin protein. The disease is characterized by progressive muscle degeneration and wasting, with the appearance of respiratory failure and cardiac complications, ultimately leading to premature death. Most of the mutations behind DMD are out-of-frame (out-of-frame) deletions that result in truncation of the immature open reading frame, resulting in the deletion of dystrophin.

Exon skipping therapy uses splice switching antisense oligonucleotides (SSOs) to target specific regions of DMD transcripts, inducing exclusion of individual exons, leading to the restoration of an abnormal reading frame, and resulting in the production of an internally deleted but partially functional dystrophin protein. Although antisense oligonucleotide exon skipping therapy against DMD has undoubtedly potential, successful application of this approach is currently limited by relatively low targeting efficiency of skeletal muscle, and the inability of single-stranded oligonucleotides to adequately target other affected tissues (e.g., the heart).

In 2016, month 9, the united states Food and Drug Administration (FDA) approved an accelerated approval for "eteplirsen," a single-stranded oligonucleotide used to regulate exon 51 splicing. Although this predicts the first approved oligonucleotide that can modulate splicing in the united states, the level of recovery of dystrophin is disappointing, with only about 1% of normal dystrophin levels. Comparison with the allelic disorder becker muscular dystrophy and experiments in mdx mice indicate that at least-15% wild-type expression of the homeomorphic myodystrophin protein is required to protect muscles from exercise-induced damage.

Therefore, there is an urgent need to improve the delivery of antisense oligonucleotides in order to provide more effective therapies for devastating genetic diseases such as DMD.

The use of viruses as delivery vehicles has been proposed, but its use has been limited due to the immunotoxicity and potential carcinogenic effects of viral coat proteins. Alternatively, a variety of non-viral delivery vectors have been developed, among which it has been shown that peptides are most promising due to their small size, targeting specificity, and the ability of large biological cargo to be delivered across the capillaries. The ability of several peptides to penetrate cells, either alone or with biological cargo, has been reported.

For many years, Cell Penetrating Peptides (CPPs) have been conjugated with SSOs, especially charge neutral Phosphorodiamidate Morpholino Oligomers (PMOs) and Peptide Nucleic Acids (PNAs), to enhance cellular delivery of such oligonucleotide analogs by effectively carrying such oligonucleotide analogs across the cell membrane to their pre-mRNA target in the nucleus. It has been shown that PMO therapeutic agents conjugated to certain arginine-rich CPPs (known as P-PMO or peptide-PMO) can enhance dystrophin production in skeletal muscle following systemic administration in an mdx mouse model of DMD.

In particular, PNA/PMO internalizing peptides (pips) have been developed, which are arginine-rich CPPs consisting of two arginine-rich sequences separated by a central short hydrophobic sequence. These "Pip" peptides were designed to improve serum stability while maintaining high levels of exon skipping, initially by attaching them to PNA cargo. Other derivatives of these peptides were designed as conjugates of PMO, which were shown to lead to the production of systemic skeletal muscle dystrophin and, importantly, also the heart, following systemic administration in mice. Although these peptides are effective, their associated toxicity precludes their therapeutic use.

Alternative cell penetrating peptides, such as R, having a single arginine-rich domain have also been generated₆Gly. These CPPs have been used to produce peptide conjugates with reduced toxicity, but these conjugates show lower efficacy compared to Pip peptides.

Thus, the currently available CPPs have not proven suitable for use in humans to treat diseases such as DMD.

A challenge in the field of cell penetrating peptide technology is to isolate efficacy and toxicity. Now, the present inventors have identified, synthesized and tested a number of improved CPPs having a particular structure according to the present invention, which at least solves this problem.

These peptides maintain a good level of efficacy in skeletal muscle when tested in vivo and in vitro with cargo therapeutic molecules. Furthermore, these peptides show an improvement in efficacy when used in the same conjugate compared to previously available CPPs. At the same time, these peptides are effective in vivo with reduced clinical symptoms after systemic injection and lower toxicity is observed by measuring biochemical markers. Critically, the peptides of the invention proved to exhibit surprisingly reduced toxicity following similar systemic injection into mice, as compared to previous CPPs. Thus, the peptides of the invention provide improved applicability as a therapy for humans compared to previously available peptides, and can be used in therapeutic conjugates to safely and effectively treat human subjects.

Disclosure of Invention

According to a first aspect of the present invention there is provided a peptide having a total length of 40 amino acid residues or less, the peptide comprising:

two or more cationic domains, each cationic domain comprising at least 4 amino acid residues;

and

one or more hydrophobic domains, each hydrophobic domain comprising at least 3 amino acid residues; wherein the peptide does not comprise artificial amino acid residues.

According to a second aspect of the invention there is provided a conjugate comprising a peptide of the first aspect covalently linked to a therapeutic molecule.

According to a third aspect of the invention there is provided a conjugate comprising a peptide of the first aspect covalently linked to an imaging molecule.

According to a fourth aspect of the invention, there is provided a pharmaceutical composition comprising the conjugate of the second aspect.

According to a fifth aspect of the invention there is provided a conjugate according to the second aspect for use as a medicament.

In an embodiment of the fifth aspect, there is provided a pharmaceutical composition according to the fourth aspect for use as a medicament.

According to a sixth aspect of the invention there is provided a method of treating a disease in a subject, the method comprising administering to the subject the conjugate of the second aspect in a therapeutically effective amount.

In an embodiment of the sixth aspect, there is provided a method of treating a disease in a subject comprising administering to the subject a pharmaceutical composition according to the fourth aspect in a therapeutically effective amount.

According to a seventh aspect of the invention there is provided an isolated nucleic acid encoding a peptide of the first aspect or a conjugate of the second aspect or a conjugate of the third aspect.

According to an eighth aspect of the present invention there is provided an expression vector comprising the nucleic acid sequence of the seventh aspect.

According to a ninth aspect of the present invention there is provided a host cell comprising the expression vector of the eighth aspect.

Detailed Description

The present inventors have prepared a series of peptides suitable for use as cell penetrating peptides to deliver therapeutic molecules into cells.

Surprisingly, the present inventors have found a set of peptides having at least two cationic domains and at least one hydrophobic domain of defined length without any artificial amino acids, which peptides provide enhanced cell penetration into muscle compared to currently available cell penetrating peptides. This effect is observed when delivered into cells as a conjugate with an antisense oligonucleotide therapeutic agent, or when administered in vivo.

In the case of the disease DMD, the enhanced cell penetration by the peptides of the invention linked to a suitable therapeutic molecule can be shown by specific exon exclusion (exon exclusion) within the transcript. Directing antisense oligonucleotides to appropriate sequences results in forced skipping of exons, correction of open reading frames, and restoration of internal deletions of dystrophin but isoforms with partial function.

It is shown herein that the peptides of the invention, when used as conjugates with antisense oligonucleotide therapeutics intended to target the dystrophin gene, have high levels of exon exclusion and dystrophin recovery.

In particular, conjugates comprising the peptides of the invention show significantly increased cell penetration compared to currently available peptides conjugated to the same antisense oligonucleotide therapeutic agent. In the present invention, this is demonstrated by the increased exon skipping of the dystrophin gene in various muscle groups.

In vivo, the results described herein show that the exon skipping levels and the expression levels of functional dystrophin protein when using the peptide conjugates of the present invention are close to twice the levels produced by using the same antisense oligonucleotide therapeutic conjugated to a previously available cell penetrating peptide.

This is a significant improvement in the efficacy of this peptide vector to penetrate muscle cells in the development of neuromuscular diseases.

Without wishing to be bound by theory, the inventors believe that the removal of artificial amino acids, such as 6-aminocaproic acid residues, commonly used for cell penetrating peptides, and replacement with, for example, naturally occurring beta-alanine residues, has the beneficial effect of reducing the overall toxicity of the peptide and increasing its cell penetration.

However, it was completely unexpected that such a peptide structure that did not contain any artificial amino acid residues would improve the previously reported delivery properties of cell penetrating peptides to transport therapeutic molecular cargo (e.g., oligonucleotides) into muscle. The effectiveness of a peptide depends in large part on its ability to have serum stability for the length of time it takes to enter the cell. It is expected that the peptides formed in the absence of artificial amino acids are too unstable in vivo and are susceptible to degradation by proteases to the extent that they do not penetrate muscle cells and tissues in sufficient quantities and do not produce enhanced therapeutic effects. Contrary to this expectation, the inventors found that the claimed peptides with specific structures are sufficiently stable to enter cells and maintain good or even improved efficacy, but also have the advantage of reduced toxicity due to the lack of artificial amino acids.

Unexpectedly, as demonstrated herein, such transport would be increased, resulting in therapeutic molecules (e.g., antisense oligonucleotides) that successfully increased exon skipping and production of functional dystrophin in a variety of different muscles.

Additionally, unexpectedly, this peptide structure significantly reduces the toxicity of cell penetrating peptides when the therapeutic cargo is transported in vivo to the extent that treatment of humans with such conjugates can be performed. In vivo, the results described herein show a reduction in renal toxicity as measured by biochemical markers.

For the avoidance of doubt, and in order to clarify the manner in which the present disclosure is explained, certain terms used in accordance with the present invention will now be further defined.

The invention includes any combination of the described aspects and features except where such combination is explicitly excluded or explicitly avoided.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

All the times "X" denotes any form of the artificial, synthetically prepared amino acid aminocaproic acid.

The natural but not genetically encoded amino acid beta-alanine is always denoted by "B".

The acetylation of the relevant peptide is always denoted by "Ac".

The natural but non-genetically encoded amino acid hydroxyproline is always denoted by "Hyp".

Amino acid residues genetically encoded in accordance with the relevant art recognized alphabetic amino acid code are always indicated in capital letters.

Artificial amino acids

The present invention relates to short cell penetrating peptides with a specific structure, wherein artificial amino acid residues are absent.

Reference herein to "artificial" amino acids or residues denotes any amino acid that is not naturally occurring and includes synthetic amino acids, modified amino acids (e.g., amino acids modified with sugars), unnatural amino acids, artificial amino acids, spacers, and non-peptide bonded spacers.

Synthetic amino acids may be those chemically synthesized by man.

For the avoidance of doubt, in the context of the present invention, aminocaproic acid (X) is an artificial amino acid. For the avoidance of doubt, β -alanine (B) and hydroxyproline (Hyp) are naturally occurring and therefore in the context of the present invention are not artificial amino acids but natural amino acids.

The artificial amino acids may include, for example, 6-aminocaproic acid (X), tetrahydroisoquinoline-3-carboxylic acid (TIC), 1- (amino) cyclohexanecarboxylic acid (Cy), and 3-azetidine-carboxylic acid (Az), 11-aminoundecanoic acid.

Suitably, the peptide does not comprise an aminocaproic acid residue. Suitably, the peptide does not comprise any form of aminocaproic acid residues. Suitably, the peptide does not comprise a 6-aminocaproic acid residue.

Suitably, the peptide comprises only natural amino acid residues and thus consists of natural amino acid residues.

Suitably, artificial amino acids such as 6-aminocaproic acid, typically used in cell penetrating peptides, are replaced by natural amino acids. Suitably, the artificial amino acid typically used in cell penetrating peptides, such as 6-aminocaproic acid, is replaced by an amino acid selected from beta-alanine, serine, proline, arginine and histidine or hydroxyproline.

In one embodiment, aminocaproic acid is replaced by beta-alanine. Suitably, 6-aminocaproic acid is replaced by beta-alanine.

In one embodiment, aminocaproic acid is replaced with histidine. Suitably, 6-aminocaproic acid is replaced by histidine.

In one embodiment, the aminocaproic acid is replaced by hydroxyproline. Suitably, 6-aminocaproic acid is replaced by hydroxyproline.

Suitably, the artificial amino acids typically used in cell penetrating peptides, such as 6-aminocaproic acid, may be replaced with any combination of beta-alanine, serine, proline, arginine and histidine or hydroxyproline, suitably any combination of beta-alanine, histidine and hydroxyproline.

In one embodiment, there is provided a peptide having a total length of 40 amino acid residues or less, the peptide comprising:

two or more cationic domains, each cationic domain comprising at least 4 amino acid residues; and

one or more hydrophobic domains, each hydrophobic domain comprising at least 3 amino acid residues; wherein at least one cationic domain comprises a histidine residue.

Suitably, at least one of the cationic domains is histidine-rich.

Suitably, the meaning of histidine-rich is defined herein with respect to the cationic domain.

Cationic domain

The present invention relates to short cell penetrating peptides with a specific structure, wherein at least two cationic domains with a certain length are present.

Reference herein to a "cation" means an amino acid or domain of amino acids that has an overall positive charge at physiological pH.

Suitably, the peptide comprises up to 4 cationic domains, up to 3 cationic domains.

Suitably, the peptide comprises 2 cationic domains.

As defined above, the peptide comprises two or more cationic domains, each cationic domain having a length of at least 4 amino acid residues.

Suitably, each cationic domain is 4 to 12 amino acid residues in length, suitably 4 to 7 amino acid residues in length.

Suitably, each cationic domain has a length of 4, 5, 6 or 7 amino acid residues.

Suitably, each cationic domain has a similar length, suitably each cationic domain has the same length.

Suitably, each cationic domain comprises cationic amino acids, and may also comprise polar and/or non-polar amino acids.

The non-polar amino acid may be selected from: alanine, beta-alanine, proline, glycine, cysteine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine. Suitably, the non-polar amino acid has no charge.

The polar amino acids may be selected from: serine, asparagine, hydroxyproline, histidine, arginine, threonine, tyrosine, glutamine. Suitably, the selected polar amino acids do not have a negative charge.

The cationic amino acid may be selected from: arginine, histidine and lysine. Suitably, the cationic amino acid has a positive charge at physiological pH.

Suitably, each cationic domain does not comprise anionic or negatively charged amino acid residues.

Suitably, each cationic domain comprises arginine, histidine, beta-alanine, hydroxyproline and/or serine residues.

Suitably, each cationic domain consists of arginine, histidine, β -alanine, hydroxyproline and/or serine residues.

Suitably, each cationic domain comprises at least 40%, at least 45%, at least 50% cationic amino acids.

Suitably, each cationic domain comprises a majority of cationic amino acids. Suitably, each cationic domain comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% cationic amino acids.

Suitably, each cationic domain has an isoelectric point (pI) of at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 10.5, at least 11.0, at least 11.5, at least 12.0.

Suitably, the isoelectric point (pI) of each cationic domain is at least 10.0.

Suitably, each cationic domain has an isoelectric point (pI) of from 10.0 to 13.0.

In one embodiment, each cationic domain has an isoelectric point (pI) between 10.4 and 12.5.

Suitably, the isoelectric point of the cationic domain can be calculated at physiological pH by any suitable method available in the art. Suitably, by using IPC (www.isoelectric.org), the product of Lukasz Kozlowski, Biol direct.2016; 11:55.DOI:10.1186/s13062-016 and 0159-9.

Suitably, each cationic domain comprises at least 1 cationic amino acid, suitably 1-5 cationic amino acids. Suitably, each cationic domain comprises at least 2 cationic amino acids, suitably 2-5 cationic amino acids.

Suitably, each cationic domain is arginine-rich and/or histidine-rich. Suitably, the cationic domain may comprise both histidine and arginine.

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

Suitably, each cationic domain comprises a majority of arginine and/or histidine residues.

Suitably, each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% arginine and/or histidine residues.

Suitably, the cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% arginine residues.

Suitably, the cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% histidine residues.

Suitably, the cationic domain may comprise a total of 1-5 histidine and 1-5 arginine residues. Suitably, the cationic domain may comprise 1-5 arginine residues. Suitably, the cationic domain may comprise 1-5 histidine residues. Suitably, the cationic domain may comprise a total of 2-5 histidine and 3-5 arginine residues. Suitably, the cationic domain may comprise 3-5 arginine residues. Suitably, the cationic domain may comprise 2-5 histidine residues.

Suitably, each cationic domain comprises one or more β -alanine residues. Suitably, each cationic domain may comprise a total of 2-5 β -alanine residues, suitably a total of 2 or 3 β -alanine residues.

Suitably, the cationic domain may comprise one or more hydroxyproline residues or serine residues.

Suitably, the cationic domain may comprise 1-2 hydroxyproline residues. Suitably, the cationic domain may comprise 1-2 serine residues.

Suitably, all cationic amino acids in a given cationic domain may be histidine, or, suitably, all cationic amino acids in a given cationic domain may be arginine.

Suitably, the peptide may comprise at least one histidine-rich cationic domain. Suitably, the peptide may comprise at least one arginine-rich cationic domain.

Suitably, the peptide may comprise at least one arginine-rich cationic domain and at least one histidine-rich cationic domain.

In one embodiment, the peptide comprises two arginine-rich cationic domains.

In one embodiment, the peptide comprises two histidine-rich cationic domains.

In one embodiment, the peptide comprises two arginine-and histidine-rich cationic domains.

In one embodiment, the peptide comprises an arginine-rich cationic domain and a histidine-rich cationic domain.

Suitably, each cationic domain comprises no more than 3 consecutive arginine residues, suitably no more than 2 consecutive arginine residues.

Suitably, each cationic domain does not comprise consecutive histidine residues.

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

In one embodiment, the peptide comprises a first cationic domain comprising arginine and β -alanine residues and a second cationic domain comprising arginine and β -alanine residues.

In one embodiment, the peptide comprises a first cationic domain comprising arginine and β -alanine residues and a second cationic domain comprising histidine, β -alanine, and optionally arginine residues.

In one embodiment, the peptide comprises a first cationic domain comprising arginine and β -alanine residues and a second cationic domain comprising histidine and β -alanine residues.

In one embodiment, the peptide comprises a first cationic domain consisting of arginine and β -alanine residues and a second cationic domain consisting of arginine and β -alanine residues.

In one embodiment, the peptide comprises a first cationic domain consisting of arginine and β -alanine residues and a second cationic domain consisting of arginine, histidine and β -alanine residues.

Suitably, the peptide comprises at least two cationic domains, suitably these cationic domains form the arms of the peptide. Suitably, the cationic domains are located at the N-terminus and C-terminus of the peptide. Suitably, the cationic domain may therefore be referred to as a cationic arm domain.

In one embodiment, the peptide comprises two cationic domains, one at the N-terminus and one at the C-terminus of the peptide. Suitably at either terminus of the peptide. Suitably, no other amino acids or domains are present at the N-terminus and C-terminus of the peptide, other than other groups such as terminal modifications, linkers and/or therapeutic molecules. For the avoidance of doubt, such other groups may be present in addition to the "peptide" described and claimed herein. Suitably, each cationic domain thus forms a terminus of the peptide. Suitably, this does not preclude the presence of further linker groups as described herein.

Suitably, the peptide may comprise up to 4 cationic domains. Suitably, the peptide comprises two cationic domains.

In one embodiment, the peptide comprises two cationic domains, both rich in arginine.

In one embodiment, the peptide comprises an arginine-rich cationic domain.

In one embodiment, the peptide comprises two cationic domains each rich in both arginine and histidine.

In one embodiment, the peptide comprises an arginine-rich cationic domain and a histidine-rich cationic domain.

Suitably, 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.

Suitably, the cationic domain may also comprise serine, proline and/or hydroxyproline residues. Suitably, 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 having the amino acid units listed above.

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

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

Suitably, each cationic domain consists of one of the following sequences: RBRRBRR (SEQ ID NO:1), RBRBRBR (SEQ ID NO:2), RBRRBR (SEQ ID NO:4), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO: 9).

Suitably, each cationic domain in the peptide may be the same or different. Suitably, each cationic domain in the peptide is different.

Hydrophobic domains

The present invention relates to short cell penetrating peptides with a specific structure, wherein at least one hydrophobic domain with a certain length is present.

"hydrophobic" as referred to herein denotes an amino acid or a domain of an amino acid that has the ability to repel water or is not mixed with water.

Suitably, the peptide comprises up to 3 hydrophobic domains, up to 2 hydrophobic domains.

Suitably, the peptide comprises 1 hydrophobic domain.

As defined above, the peptide comprises two or more hydrophobic domains, each hydrophobic domain having a length of at least 3 amino acid residues.

Suitably, each hydrophobic domain has a length of 3-6 amino acids. Suitably, each hydrophobic domain has a length of 5 amino acids.

Suitably, each hydrophobic domain may comprise non-polar, polar and hydrophobic amino acid residues.

The hydrophobic amino acid residue may be selected from: alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, methionine, and tryptophan.

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

The polar amino acid residues may be selected from: serine, asparagine, hydroxyproline, histidine, arginine, threonine, tyrosine, glutamine.

Suitably, the hydrophobic domain does not comprise hydrophilic amino acid residues.

Suitably, each hydrophobic domain comprises a majority of hydrophobic amino acid residues. Suitably, each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100% hydrophobic amino acids. Suitably, each hydrophobic domain consists of hydrophobic amino acid residues.

Suitably, the hydrophobicity of each hydrophobic domain is 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 1.0, at least 1.1, at least 1.2, at least 1.3.

Suitably, each hydrophobic domain has a hydrophobicity of at least 0.3, at least 0.35, at least 0.4, at least 0.45.

Suitably, each hydrophobic domain has a hydrophobicity of at least 1.2, at least 1.25, at least 1.3, at least 1.35.

Suitably, the hydrophobicity of each hydrophobic domain is from 0.4 to 1.4.

In one embodiment, each hydrophobic domain has a hydrophobicity of 0.45 to 0.48.

In one embodiment, each hydrophobic domain has a hydrophobicity of 1.27 to 1.39.

Suitably, the hydrophobicity is as defined by White and Wimley: wimley and S.H.white, "Experimental defined hydropathic scale for proteins at membranes" Nature Structure Biol 3:842 (1996).

Suitably, each hydrophobic domain comprises at least 3, at least 4 hydrophobic amino acid residues.

Suitably, each hydrophobic domain comprises phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline and glutamine residues. Suitably, each hydrophobic domain consists of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline and/or glutamine residues.

In one embodiment, each hydrophobic domain consists of phenylalanine, leucine, isoleucine, tyrosine and/or glutamine residues.

In one embodiment, each hydrophobic domain consists of tryptophan and/or proline residues.

Suitably, the peptide comprises a hydrophobic domain. Suitably, the or each hydrophobic domain is located at the centre of the peptide. Suitably, the hydrophobic domain may therefore be referred to as a core hydrophobic domain. Suitably, the or each hydrophobic core domain is flanked on either side by arm domains. Suitably, the arm domain may comprise one or more cationic domains and one or more further hydrophobic domains. Suitably, each arm domain comprises a cationic domain.

In one embodiment, the peptide comprises two arm domains flanking a hydrophobic core domain, wherein each arm domain comprises a cationic domain.

In one embodiment, the peptide consists of two cationic arm domains flanking a hydrophobic core domain.

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

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

Suitably, the or each hydrophobic domain consists of one of the following sequences: FQILY (SEQ ID NO:21), YQFLI (SEQ ID NO:20), ILFQY (SEQ ID NO: 22).

Suitably, the or each hydrophobic domain consists of FQILY (SEQ ID NO: 21).

Suitably, each hydrophobic domain in the peptide may have the same sequence or a different sequence.

Peptides

The present invention relates to short cell penetrating peptides for the transport of therapeutic cargo molecules in the treatment of medical conditions.

The sequence of the peptide is a contiguous single molecule, and thus the domains of the peptide are contiguous. Suitably, the peptide comprises several domains in a linear arrangement between the N-terminus and the C-terminus. Suitably, the domain is selected from the cationic domains and hydrophobic domains described above. Suitably, the peptide consists of a cationic domain and a hydrophobic domain, wherein the domains are as defined above.

Each domain has the consensus sequence characteristics described in the relevant portions above, but the exact sequence of each domain can be varied and modified. Thus, each domain may have a series of sequences. The combination of each possible domain sequence results in a series of peptide structures, each of which forms part of the present invention. The characteristics of the peptide structure are described below.

Suitably, the hydrophobic domain separates any two cationic domains. Suitably, each hydrophobic domain is flanked on either side thereof by a cationic domain.

Suitably, no cationic domain is adjacent to another cationic domain.

In one embodiment, the peptide comprises one hydrophobic domain flanked by two cationic domains, in the following arrangement:

[ cationic Domain ] - [ hydrophobic Domain ] - [ cationic Domain ]

Thus, suitably, the hydrophobic domain may be referred to as a core domain and each cationic domain may be referred to as an arm domain. Suitably, the hydrophobic arm domain flanks the cationic core domain, flanking it.

In one embodiment, the peptide consists of two cationic domains and one hydrophobic domain.

In one embodiment, the peptide consists of one hydrophobic core domain flanked by two cationic arm domains.

In one embodiment, the peptide consists of a hydrophobic core domain comprising a sequence selected from the group consisting of: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25) and WWPW (SEQ ID NO:26), the hydrophobic core domain is flanked by two cationic arm domains, each of which comprises a sequence selected from the group consisting of: RBRRBRR (SEQ ID NO:1), RBRBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBHBH (SEQ ID NO:14), BH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB [ Hyp ] B (SEQ ID NO:17), R [ Hyp ] H [ Hyp ] HB (SEQ ID NO:18), and R [ HyRR ] Hyp ] R (SEQ ID NO: 19).

In one embodiment, the peptide consists of a hydrophobic core domain comprising a sequence selected from the group consisting of: FQILY (SEQ ID NO:21), YQFLI (SEQ ID NO:20) and ILFQY (SEQ ID NO:22), the hydrophobic core domain is flanked by two cationic arm domains comprising a sequence selected from: RBRRBRR (SEQ ID NO:1), RBRBRBR (SEQ ID NO:2), RBRRBR (SEQ ID NO:4), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO: 9).

In one embodiment, the peptide consists of a hydrophobic core domain comprising the sequence: FQILY (SEQ ID NO:21), flanked by two cationic arm domains comprising a sequence selected from the group consisting of: RBRRBRR (SEQ ID NO:1), RBRBRBR (SEQ ID NO:2), RBRRBR (SEQ ID NO:4), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO: 8).

In any such embodiment, other groups may be present, such as linkers, terminal modifications, and/or therapeutic molecules.

Suitably, the peptide is N-terminally modified.

Suitably, the peptide is N-acetylated, N-methylated, N-trifluoroacetylated, N-trifluoromethylsulfonylated or N-methylsulfonylated. Suitably, the peptide is N-acetylated.

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

In one embodiment, the peptide is N-acetylated.

Suitably, the peptide is C-terminally modified.

Suitably, the peptide comprises a C-terminal modification selected from: carboxy-, thioacid-, aminooxy-, hydrazino-, thioester-, azide, strained alkyne, strained alkene, aldehyde-, thiol, or haloacetyl.

Advantageously, the C-terminal modification provides a means for attaching the peptide to a therapeutic molecule.

Thus, the C-terminal modification may comprise a linker and vice versa. Suitably, the C-terminal modification may consist of a linker, or vice versa. Suitable linkers are described elsewhere herein.

Suitably, the peptide comprises a C-terminal carboxyl group.

Suitably, the C-terminal carboxyl group is provided by a glycine or β -alanine residue.

In one embodiment, the C-terminal carboxyl group is provided by a beta-alanine residue.

Suitably, the C-terminal β -alanine residue is a linker.

Suitably, therefore, each cationic domain may further comprise an N-or C-terminal modification. Suitably, the cationic domain comprises a C-terminal modification at the C-terminus. Suitably, the cationic domain comprises an N-terminal modification at the N-terminus. Suitably, the cationic domain comprises a linker group at the C-terminus, suitably the cationic domain comprises a C-terminal β -alanine at the C-terminus. Suitably, the cationic domain is N-acetylated at the N-terminus.

The peptide of the present invention is defined as having an overall length of 40 amino acid residues or less. Thus, the peptide may be considered an oligopeptide.

Suitably, the total length of the peptide is from 3 to 30 amino acid residues, suitably from 5 to 25 amino acid residues, from 10 to 25 amino acid residues, from 13 to 23 amino acid residues, from 15 to 20 amino acid residues.

Suitably, the total length of the peptide is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 amino acid residues.

Suitably, the peptide is capable of penetrating a cell. Thus, the peptide may be considered a cell penetrating peptide.

Suitably, the peptide is for attachment to a therapeutic molecule. Suitably, the peptide is for use in the transport of a therapeutic molecule into a target cell. Suitably, the peptide is for delivery of a therapeutic molecule into a target cell. Thus, the peptide may be considered a carrier peptide.

Suitably, the peptide is capable of penetrating into cells and tissues, suitably into the nucleus of a cell. Suitably into muscle tissue.

Suitably, the peptide may be selected from any of the following sequences:

RBRRBRRFQILYRBRBR (SEQ ID NO:27)

RBRRBRRFQILYRBRR (SEQ ID NO:28)

RBRRBRFQILYRRBRBR (SEQ ID NO:29)

RBRBRFQILYRBRRBRR (SEQ ID NO:30)

RBRRBRRYQFLIRBRBR (SEQ ID NO:31)

RBRRBRRILFQYRBRBR (SEQ ID NO:32)

RBRRBRFQILYRBRBR (SEQ ID NO:33)

RBRRBFQILYRBRRBR (SEQ ID NO:34)

RBRRBRFQILYBRBR (SEQ ID NO:35)

RBRRBFQILYRBRBR (SEQ ID NO:36)

RBRRBRRFQILYRBHBH (SEQ ID NO:37)

RBRRBRRFQILYHBHBR (SEQ ID NO:38)

RBRRBRRFQILYHBRBH (SEQ ID NO:39)

RBRRBRRYQFLIRBHBH (SEQ ID NO:40)

RBRRBRRILFQYRBHBH (SEQ ID NO:41)

RBRHBHRFQILYRBRBR (SEQ ID NO:42)

RBRBBHRFQILYRBHBH (SEQ ID NO:43)

RBRRBRFQILYRBHBH (SEQ ID NO:44)

RBRRBRFQILYHBHBH (SEQ ID NO:45)

RBRRBHFQILYRBHBH (SEQ ID NO:46)

HBRRBRFQILYRBHBH (SEQ ID NO:47)

RBRRBFQILYRBHBH (SEQ ID NO:48)

RBRRBRFQILYBHBH (SEQ ID NO:49)

RBRRBRYQFLIHBHBH (SEQ ID NO:50)

RBRRBRILFQYHBHBH (SEQ ID NO:51)

RBRRBRRFQILYHBHBH (SEQ ID NO:52)。

suitably, the peptide may be selected from any of the following additional sequences:

RBRRBRFQILYBRBS (SEQ ID NO:53)

RBRRBRFQILYBRB[Hyp] (SEQ ID NO:54)

RBRRBRFQILYBR[Hyp]R (SEQ ID NO:55)

RRBRRBRFQILYBRBR (SEQ ID NO:56)

BRRBRRFQILYBRBR (SEQ ID NO:57)

RBRRBRWWWBRBR (SEQ ID NO:58)

RBRRBRWWPWWBRBR (SEQ ID NO:59)

RBRRBRWPWWBRBR (SEQ ID NO:60)

RBRRBRWWPWBRBR (SEQ ID NO:61)

RBRRBRRWWWRBRBR (SEQ ID NO:62)

RBRRBRRWWPWWRBRBR (SEQ ID NO:63)

RBRRBRRWPWWRBRBR (SEQ ID NO:64)

RBRRBRRWWPWRBRBR (SEQ ID NO:65)

RBRRBRRFQILYBRBR (SEQ ID NO:66)

RBRRBRRFQILYRBR (SEQ ID NO:67)

BRBRBWWPWWRBRRBR (SEQ ID NO:68)

RBRRBRRFQILYBHBH (SEQ ID NO:69)

RBRRBRRFQIYRBHBH (SEQ ID NO:70)

RBRRBRFQILYBRBH (SEQ ID NO:71)

RBRRBRFQILYR[Hyp]H[Hyp]H (SEQ ID NO:72)

R[Hyp]RR[Hyp]RFQILYRBHBH (SEQ ID NO:73)

R[Hyp]RR[Hyp]RFQILYR[Hyp]H[Hyp]H (SEQ ID NO:74)

RBRRBRWWWRBHBH (SEQ ID NO:75)

RBRRBRWWPRBHBH (SEQ ID NO:76)

RBRRBRPWWRBHBH (SEQ ID NO:77)

RBRRBRWWPWWRBHBH (SEQ ID NO:78)

RBRRBRWWPWRBHBH (SEQ ID NO:79)

RBRRBRWPWWRBHBH (SEQ ID NO:80)

RBRRBRRWWWRBHBH (SEQ ID NO:81)

RBRRBRRWWPWWRBHBH (SEQ ID NO:82)

RBRRBRRWPWWRBHBH (SEQ ID NO:83)

RBRRBRRWWPWRBHBH (SEQ ID NO:84)

RRBRRBRFQILYRBHBH (SEQ ID NO:85)

BRRBRRFQILYRBHBH (SEQ ID NO:86)

RRBRRBRFQILYBHBH (SEQ ID NO:87)

BRRBRRFQILYBHBH (SEQ ID NO:88)

RBRRBHRFQILYRBHBH (SEQ ID NO:89)

RBRRBRFQILY[Hyp]R[Hyp]R (SEQ ID NO:101)

R[Hyp]RR[Hyp]RFQILYBRBR (SEQ ID NO:102)

R[Hyp]RR[Hyp]RFQILY[Hyp]R[Hyp]R (SEQ ID NO:103)

RBRRBRWWWBRBR (SEQ ID NO:104)

RBRRBRWWPWWBRBR (SEQ ID NO:105)。

suitably, the peptide consists of one of the following sequences:

RBRRBRRFQILYRBRBR (SEQ ID NO:27)

RBRRBRRYQFLIRBRBR (SEQ ID NO:31)

RBRRBRRILFQYRBRBR (SEQ ID NO:32)

RBRRBRFQILYBRBR (SEQ ID NO:35)

RBRRBRRFQILYRBHBH (SEQ ID NO:37)

RBRRBRRFQILYHBHBR (SEQ ID NO:38)

RBRRBRFQILYRBHBH (SEQ ID NO:44)。

in one embodiment, the peptide consists of the sequence: RBRRBRFQILYBRBR (SEQ ID NO: 35).

In one embodiment, the peptide consists of the sequence: RBRRBRRFQILYRBHBH (SEQ ID NO: 37).

In one embodiment, the peptide consists of the sequence: RBRRBRFQILYRBHBH (SEQ ID NO: 44).

Conjugates

The peptides of the invention may be covalently linked to a therapeutic molecule to provide a conjugate.

The therapeutic molecule may be any molecule useful for treating a disease. The therapeutic molecule may be selected from: nucleic acids, peptide nucleic acids, antisense oligonucleotides (e.g., PNA, PMO), mRNA, grnas (e.g., when using CRISPR/Cas9 technology), short interfering RNAs, micrornas, antagomirnas, peptides, cyclic peptides, proteins, pharmaceuticals, drugs, or nanoparticles.

In one embodiment, the therapeutic molecule is an antisense oligonucleotide.

Suitably, the antisense oligonucleotide consists of Phosphorodiamidate Morpholino Oligonucleotides (PMO).

Alternatively, the oligonucleotide may be a modified PMO or any other charge neutral oligonucleotide, for example a Peptide Nucleic Acid (PNA), a chemically modified PNA such as γ -PNA (Bahal, nat. comm.2016), an oligonucleotide phosphoramidate (in which the non-bridging oxygen of the phosphate is replaced by an amine or alkylamine) such as those described in WO2016028187a1, or any other partially or fully charge neutralized oligonucleotide.

The therapeutic antisense oligonucleotide sequence may be selected from any available sequence, for example, antisense oligonucleotides for exon skipping IN DMD, described IN https:// research-reproduction.uwa. edu.au/en/publication/antisense-oligonucleotide-induced-exon-skiping-across-the-human, or therapeutic antisense oligonucleotides complementary to ISSN1 or IN7 sequences for the treatment of SMA, described IN Zhou, HGT, 2013; and Hammond et al, 2016; and Osman et al, HMG, 2014.

Suitably, the antisense oligonucleotide sequence is for use in inducing exon skipping for the treatment of DMD.

Suitably, the antisense oligonucleotide sequence is for use in inducing exon skipping in the dystrophin gene for the treatment of DMD. Suitably, the antisense oligonucleotide sequence may induce exon skipping of one or more exons.

In one embodiment, the antisense oligonucleotide sequence is for inducing exon skipping of a single exon of a dystrophin gene for the treatment of DMD. Suitably, the single exon is selected from any exon associated with DMD, which may be any exon in the dystrophin gene, such as exons 45, 51 or 53. PMO oligonucleotides of any sequence can be purchased (e.g., from Gene Tools Inc. U.S.A.).

In one embodiment, the therapeutic molecule of the conjugate is an oligonucleotide complementary to a pre-mRNA of a gene target.

Suitably, the oligonucleotide complementary to the pre-mRNA of the gene target causes a steric blocking event which alters the pre-mRNA, resulting in an altered mRNA and hence an altered sequence protein. Suitably, the gene target is a dystrophin gene. Suitably, the spatial occlusion event may be exon retention (exon inclusion) or exon skipping. In one embodiment, the spatial blocking event is exon skipping, suitably exon skipping of a single exon of the dystrophin gene.

Optionally, lysine residues may be added to one or both ends of the therapeutic molecule (e.g., PMO or PNA) prior to attachment to the peptide to improve water solubility.

Suitably, the therapeutic molecule has a molecular weight of less than 5000Da, suitably less than 3000Da or suitably less than 1000 Da.

Suitably, the peptide is covalently linked at the C-terminus to the therapeutic molecule.

Suitably, the peptide is covalently linked to the therapeutic molecule via a linker, if desired. The linker may act as a spacer to separate the peptide sequence from the therapeutic molecule.

The linker may be selected from any suitable sequence.

Suitably, the linker is present between the peptide and the therapeutic molecule. Suitably, the linker is a spacer group for peptides and therapeutic molecules. Thus, the linker may comprise an artificial amino acid.

In one embodiment, the conjugate comprises a peptide covalently linked to a therapeutic molecule through a linker. In one embodiment, the conjugate comprises the following structure:

[ peptide ] - [ linker ] - [ therapeutic molecule ].

In one embodiment, the conjugate consists of the following structure:

[ peptide ] - [ linker ] - [ therapeutic molecule ].

Suitably, any of the peptides listed herein may be used in the conjugates according to the invention. In one embodiment, the conjugate comprises a peptide selected from one of the following sequences: RBRRBRFQILYBRBR (SEQ ID NO:35), RBRRBRRFQILYRBHBH (SEQ ID NO:37) and RBRRBRFQILYRBHBH (SEQ ID NO: 44).

Suitably, in any case, the peptide may further comprise an N-terminal modification as described above.

Suitable linkers include, for example, the C-terminal cysteine residue, which can form a disulfide, thioether, or thiol-maleimide linkage; including a C-terminal aldehyde to form an oxime, a click reaction with a basic amino acid on a peptide or a carboxylic acid moiety on a peptide covalently conjugated to an amino group to form a carboxamide linkage, or to form a morpholino linkage.

Suitably, the linker is 1-5 amino acids in length. Suitably, the linker may comprise any linker known in the art.

Suitably, the linker is selected from any one of the following sequences: G. BC, XC, C, GGC, BBC, BXC, XBC, X, XX, B, BB, BX, and XB. Suitably, wherein X is 6-aminocaproic acid.

Suitably, the linker may be a polymer, for example PEG.

In one embodiment, the linker is β -alanine.

In one embodiment, the peptide is conjugated to the therapeutic molecule through a carboxamide linkage.

The linker of the conjugate may form part of the therapeutic molecule to which the peptide is attached. Alternatively, the therapeutic molecule attachment may be directly linked to the C-terminus of the peptide. Suitably, in such embodiments, no linker is required.

Alternatively, the peptide may be chemically conjugated to the therapeutic molecule. Chemical linkages may be attached, for example, by disulfide, alkenyl, alkynyl, aryl, ether, thioether, triazole, amide, carboxamide, urea, thiourea, semicarbazide, carbazide, hydrazine, oxime, phosphate, phosphoramidate, phosphorothioate, boranophosphate, iminophosphate, or thiol-maleimide.

Optionally, a cysteine may be added at the N-terminus of the therapeutic molecule to allow for disulfide bond formation with the peptide, or the N-terminus may be bromoacetylated to conjugate the thioether to the peptide.

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

Suitably, the imaging molecule may be any molecule capable of visualizing the conjugate. Suitably, the imaging molecule may be indicative of the location of the conjugate. Suitably, the conjugate is in vitro or in vivo. Suitably, there is provided a method of monitoring the position of a conjugate comprising an imaging molecule, the method comprising: administering the conjugate to a subject and imaging the subject to locate the conjugate.

Examples of such imaging molecules include detection molecules, contrast molecules (contrast molecules) or enhancement molecules. Suitable imaging molecules may be selected from radionuclides; a fluorophore; nanoparticles (e.g., nanoshells); a nanocage; chromogenic agents (e.g., enzymes), radioisotopes, dyes, radiopaque materials, fluorescent compounds, and combinations thereof.

Suitably, 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, imaging cytometry, fluorescence microscopy, phase contrast microscopy, SEM, TEM. Suitable medical imaging techniques include, for example, X-ray, fluoroscopy, MRI, scintigraphy, SPECT, PET, CT, CAT, FNRI.

In certain instances, the imaging molecule can be considered a diagnostic molecule. Suitably, the diagnostic molecule is capable of diagnosing a disease using the conjugate. Suitably, diagnosis of a disease may be achieved by determining the location of the conjugate using an imaging molecule. Suitably, there is provided a method of diagnosing a disease, the method comprising administering to a subject an effective amount of a conjugate comprising an imaging molecule and monitoring the location of the conjugate.

Suitably, further details such as the attachment of the conjugate comprising the imaging molecule are the same as those described above in relation to the conjugate comprising the therapeutic molecule.

Suitably, the peptides of the invention may be covalently linked to a therapeutic molecule and an imaging molecule to provide a conjugate.

Suitably, the conjugate is capable of penetrating into cells and tissues, suitably into the nucleus of a cell. Suitably into muscle tissue.

Pharmaceutical composition

The conjugates of the invention may be formulated as pharmaceutical compositions.

Suitably, the pharmaceutical composition comprises a conjugate of the invention.

Suitably, the pharmaceutical composition may further comprise a pharmaceutically acceptable diluent, adjuvant or carrier.

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

As used herein, the phrase "pharmaceutically acceptable" refers to those ligands, materials, formulations, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, formulation or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the conjugate from one organ or site of the body to another organ or site of the body. Each cell penetrating peptide must be "acceptable" in the sense of being compatible with the other components of the composition (e.g., peptides and therapeutic molecules) and not harmful to the individual. Lyophilized compositions that can be reconstituted and administered are also within the scope of the compositions of the invention.

The pharmaceutically acceptable carrier may be, for example, an excipient, vehicle, diluent, and combinations thereof. For example, when the compositions are administered orally, they may be formulated into tablets, capsules, granules, powders or syrups; or for parenteral administration, they may be formulated as injections, instillation preparations or suppositories. These compositions can be prepared by conventional methods, and if desired, the active compound (i.e., conjugate) can be mixed with any conventional additives, such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspending agents, emulsifiers, coating agents, or combinations thereof.

It is to be understood that the pharmaceutical compositions of the present disclosure may further include other known therapeutic agents, drugs, modifications of compounds into prodrugs, and the like, for use in the alleviation, mediation, prevention, and treatment of the diseases, disorders, and conditions described herein in medical applications.

Suitably, the pharmaceutical composition is for use as a medicament. Suitably, in the same manner as described herein for the conjugate, for use as a medicament. All features described herein in relation to medical treatment using the conjugates apply to the pharmaceutical composition.

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

Medical use

The conjugates comprising the peptides of the invention can be used as medicaments for the treatment of diseases.

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

Also provided is a method of treating a patient or subject in need of treatment for a disease condition, the method comprising the step of administering to the patient or subject a therapeutically effective amount of the conjugate.

Suitably, the medical treatment entails delivery of the therapeutic molecule into the cell, suitably into the nucleus of the cell.

The disease to be treated may include any disease in which improved penetration of the cells and/or nuclear membranes by the therapeutic molecule may result in improved therapeutic efficacy.

Suitably, the conjugate is for use in the treatment of a disease of the neuromuscular system.

Conjugates comprising the peptides of the invention are useful for treating genetic diseases of the neuromuscular system. Conjugates comprising the peptides of the invention are useful for treating neuromuscular genetic diseases. In a suitable embodiment, there is provided a conjugate according to the second aspect for use in the treatment of a genetic disease of the neuromuscular system. Suitably, the conjugate is for use in the treatment of a genetic disease. Suitably, the conjugate is for use in the treatment of a genetic disease of the neuromuscular system. Suitably, the conjugate is for use in the treatment of a genetic neuromuscular genetic disease. Suitably, the conjugate is for use in the treatment of a genetic X-linked genetic disease of the neuromuscular system. Suitably, the conjugate is for use in the treatment of a genetic X-linked neuromuscular disease.

Suitably, the conjugate is for use in the treatment of a disease caused by a splicing defect. In such embodiments, the therapeutic molecule may comprise an oligonucleotide capable of preventing or correcting splicing defects and/or increasing the production of correctly spliced mRNA molecules.

Suitably, the conjugate is for use in the treatment of any one of the following diseases: duchenne Muscular Dystrophy (DMD), Bucher Muscular Dystrophy (BMD), Menkes ' disease, beta thalassemia, dementia, Parkinson's disease, Spinal Muscular Atrophy (SMA), myotonic Dystrophy (DM), Huntington's chorea, Hutchinson-Gilford progeria, ataxia telangiectasia or cancer.

In one embodiment, the conjugate is used to treat DMD.

In one embodiment, there is provided a conjugate according to the second aspect for use in the treatment of DMD. Suitably, in such embodiments, the therapeutic molecule of the conjugate can be used to increase expression of a dystrophin protein. Suitably, in such embodiments, the therapeutic molecule of the conjugate can be used to increase the expression of a functional dystrophin protein.

Suitably, the conjugate increases dystrophin expression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. Suitably, the conjugate increases dystrophin expression by up to 50%.

Suitably, the conjugate restores dystrophin expression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. Suitably, the conjugate restores dystrophin expression by up to 50%.

Suitably, the conjugate restores dystrophin function by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. Suitably, the conjugate restores dystrophin function by up to 50%.

Suitably, the therapeutic molecule of the conjugate may achieve this by causing skipping of one or more exons during dystrophin transcription.

Suitably, the therapeutic molecule of the conjugate causes skipping of one or more exons of the dystrophin gene by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%. Suitably, the therapeutic molecule of the conjugate causes skipping of one or more exons of up to 50% of the dystrophin gene.

Suitably, the patient or subject to be treated may be any animal or human. Suitably, the patient or subject may be a non-human mammal. Suitably, the patient or subject may be male or female. In one embodiment, the subject is male.

Suitably, the patient or subject to be treated may be of any age. Suitably, the patient or subject to be treated is 0-40 years of age, suitably 0-30 years of age, suitably 0-25 years of age, suitably 0-20 years of age.

Suitably, the conjugate is for systemic administration to a subject, for example, by the intramedullary, intrathecal, intraventricular, intravitreal, enteral, parenteral, intravenous, intraarterial, intramuscular, intratumoral, subcutaneous, oral or nasal route.

In one embodiment, the conjugate is for intravenous administration to a subject.

In one embodiment, the conjugate is for intravenous administration to a subject by injection.

Suitably, the conjugate is administered to a subject in a "therapeutically effective amount", by which is meant an amount sufficient to show benefit to the individual. The amount actually administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease being treated. The decision on dosage is within the responsibility of general practitioners and other physicians. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences,20th Edition,2000, pub.

Exemplary doses may be 0.01mg/kg-50mg/kg, 0.05mg/kg-40mg/kg, 0.1mg/kg-30mg/kg, 0.5mg/kg-18mg/kg, 1mg/kg-16mg/kg, 2mg/kg-15mg/kg, 5mg/kg-10mg/kg, 10mg/kg-20mg/kg, 12mg/kg-18mg/kg, 13mg/kg-17 mg/kg.

Advantageously, the dosage of the conjugates of the invention is one order or magnitude lower than the dosage required to be effective by administration of the therapeutic molecule alone.

Suitably, after administration of the conjugate of the invention, one or more toxicity markers are significantly reduced compared to existing conjugates using currently available peptide carriers.

Suitable toxicity markers may be renal toxicity markers.

Suitable toxicity markers include KIM-1, NGAL, BUN, creatinine, alkaline phosphatase, alanine transferase and aspartate transaminase.

Suitably, the level of at least one of KIM-1, NGAL and BUN is reduced after administration of the conjugate of the invention when compared to existing conjugates using currently available peptide carriers.

Suitably, the level of each of KIM-1, NGAL and BUN is reduced after administration of the conjugate of the invention when compared to existing conjugates using currently available peptide carriers.

Suitably, the level of the or each marker is significantly reduced when compared to existing conjugates using currently available peptide carriers.

Suitably, the level of the or each marker is reduced by up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% after administration of the conjugate of the invention when compared to existing conjugates using currently available peptide carriers.

Advantageously, the toxicity of the peptide, and thus of the resulting conjugate, is significantly reduced compared to existing cell penetrating peptides and conjugates. In particular, KIM-1 and NGAL-1 are markers of toxicity, which are significantly reduced by up to 120-fold compared to existing conjugates using currently available peptide carriers.

Nucleic acids and hosts

The peptides of the invention may be prepared by any standard protein synthesis method (e.g., chemical synthesis, semi-chemical synthesis) or by using an expression system.

The invention therefore also relates to a nucleotide sequence comprising or consisting of a DNA encoding said peptide, an expression system (e.g.a vector comprising said sequence and sequences required for expression and control) and host cells and host organisms transformed by said expression system.

Accordingly, also provided are nucleic acids encoding the peptides according to the invention.

Suitably, the nucleic acid may be provided in isolated or purified form.

Also provided are expression vectors comprising nucleic acids encoding the peptides according to the invention.

Suitably, the vector is a plasmid.

Suitably, the vector comprises a regulatory sequence (e.g. a promoter) operably linked to the nucleic acid encoding the peptide according to the invention. Suitably, the expression vector is capable of expressing the peptide when transfected into a suitable cell (e.g. a mammalian, bacterial or fungal cell).

Host cells comprising the expression vectors of the invention are also provided.

The expression vector may be selected according to the host cell into which the nucleic acid of the present invention can be inserted. Such transformation of host cells involves conventional techniques, such as those taught in Sambrook et al [ Sambrook, J., Russell, D. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, USA ]. The selection of suitable vectors is within the ability of the person skilled in the art. Suitable vectors include plasmids, phages, cosmids and viruses.

The produced peptide may be isolated and purified from the host cell by any suitable method, such as precipitation or chromatographic separation, e.g. affinity chromatography.

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

In the present specification, the term "operably linked" may include situations where a selected nucleotide sequence and a regulatory nucleotide sequence are covalently linked in such a way that expression of the nucleotide coding sequence is under the control of the regulatory sequence, such that the regulatory sequence is capable of affecting transcription of the nucleotide coding sequence forming part or all of the selected nucleotide sequence. The resulting transcript may then be translated into the desired peptide, where appropriate.

Drawings

Certain embodiments of the invention will now be described with reference to the following figures and tables, in which:

FIG. 1 shows the in vitro exon 23 skipping efficacy of certain peptides of the DPEP1 series conjugated with antisense therapeutic PMOs at 0.25. mu.M, 0.5. mu.M and 1. mu.M in H2K-mdx cells (error bars: standard deviation, n.gtoreq.3), as measured by densitometric analysis of nested RT-PCR.

FIG. 2 shows the in vitro exon 23 skipping efficacy of certain peptides of the DPEP3 series conjugated with antisense therapeutic PMOs at 0.25. mu.M, 0.5. mu.M and 1. mu.M in H2K-mdx cells (error bars: standard deviation, n.gtoreq.3), as measured by densitometric analysis of nested RT-PCR.

Figure 3 shows the in vivo efficacy of certain peptides of the DPEP1 series conjugated with antisense therapeutic PMO in (a) tibialis anterior, (B) diaphragm muscle and (C) heart muscle after a single intravenous dose of 10mg/kg as measured by western blot and qRT-PCR in mdx mice (error bars: standard deviation, n ═ 3).

Figure 4 shows the in vivo efficacy of certain peptides of the DPEP3 series conjugated with antisense therapeutic PMO in (a) tibialis anterior, (B) diaphragm and (C) heart muscle after a single intravenous dose of 10mg/kg as measured by western blot and qRT-PCR in mdx mice (error bars: standard deviation, n-3).

Figure 5 shows the relative KIM-1 levels measured in the urine of C57BL/6 mice 2 and 7 days after administration of a single dose of 30mg/kg of each DPEP peptide conjugated to an antisense therapeutic PMO compared to administration of currently available peptide vectors conjugated to the same antisense therapeutic PMO and saline (error bars: standard deviation, n ═ 6).

Figure 6 shows the relative NGAL levels measured in the urine of C57BL/6 mice 2 and 7 days after administration of a single dose of 30mg/kg of each DPEP peptide conjugated to an antisense therapeutic PMO compared to those currently available with the same antisense therapeutic PMO conjugated peptide carrier and with saline (error bars: standard deviation, n-6).

Figure 7 shows BUN serum levels measured in C57BL/6 mice 7 days after administration of a single dose of 30mg/kg of each DPEP peptide conjugated to an antisense therapeutic PMO, compared to administration of currently available peptide vectors conjugated to the same antisense therapeutic PMO and saline (error bars: standard deviation, n ═ 6).

Figure 8 shows creatinine serum levels measured 7 days after administration of a single dose of 30mg/kg of each DPEP peptide conjugated to an antisense therapeutic PMO in C57BL/6 mice compared to those currently available with the same antisense therapeutic PMO conjugated peptide vehicle and saline (error bar: standard deviation, n-6).

Figure 9 shows the (a) alanine transferase, (B) alkaline phosphatase and (C) aspartate transaminase serum levels measured in C57BL/6 mice 7 days after administration of a single dose of 30mg/kg of each DPEP peptide conjugated to an antisense therapeutic PMO compared to those administered with the currently available peptide vector conjugated to the same antisense therapeutic PMO and with saline (error bars: standard deviation, n ═ 6).

Figure 10 shows the in vivo efficacy of exon 23 skipping assessed by qRT-PCR in (a) tibialis anterior, (B) diaphragm and (C) heart of C57BL/6 mice following a single intravenous administration of 30mg/kg of various DPEP peptides conjugated to an antisense therapeutic PMO, compared to administration of currently available peptide vectors conjugated to the same antisense therapeutic PMO and administration of saline.

Fig. 11A and B: urinary KIM-1 levels were assessed at day 2 or day 7 after single dose administration of between 2.5-50mg/kg of peptide-PMO at different doses to 8-10 week old C57BL6 mice (n-4-6) compared to administration of currently available peptide vectors conjugated to the same antisense therapeutic PMO. KIM-1 levels were determined by ELISA and normalized to urinary creatinine levels. Data are expressed as fold-changes relative to saline-injected mouse control KIM-1 levels (n-10).

Figure 12 dose-response comparison studies of in vivo exon skipping efficacy of peptide-PMO were performed after single dose administration of increasing doses of peptide-PMO between 2.5-50mg/kg to 8-10 week old C57BL6 mice (n-3-6) compared to administration of currently available peptide vectors conjugated to the same antisense therapeutic PMO. qPCR analysis to assess exon 23 exclusion in (a) tibialis anterior, (B) diaphragm muscle, and (C) heart 7 days after administration.

FIG. 13: shows that different DPEP1/3- [ CAG]₇PMO conjugates corrected in vitro the splicing defect of the Mbnl1 transcript at different concentrations in myoblasts from DM1 patients with DM1, with 2600 repeats in the DMPK gene (n-1-3);

FIG. 14: shows that different DPEP1/3- [ CAG]₇PMO conjugates at different concentrations in myoblasts from DM1 patients from DM1 patientsSplicing defects of DMD transcripts were corrected in vitro for 2600 repeats in the DMPK gene (error bars: mean ± SEM, n ═ 1-3);

FIG. 15: it is shown that the C57BL6 female mice were injected with different DPEP1/3- [ CAG-]₇Relative KIM-1 levels assessed in urine at day 2 and day 7 post PMO conjugate, as measured by ELISA with samples diluted to fit a standard curve. Values were normalized to urine creatinine levels to calculate urine protein concentrations. KIM-1 levels were similar to saline control injections compared to the fold increase induced by the current Pip series peptide vectors (error bars: mean. + -. SEM, n-4-10).

FIG. 16: it is shown that the C57BL6 female mice were injected with different DPEP1/3- [ CAG-]₇Relative NGAL levels measured in urine at day 2 and day 7 after PMO conjugate, as measured by ELISA with samples diluted to fit a standard curve. Values were normalized to urine creatinine levels to calculate urine protein concentrations. NGAL levels were similar to those of saline control injections compared to the fold increase induced by the current Pip series peptide vectors (error bars: mean ± SEM, n-4-10).

FIG. 17: it was shown that the C57BL6 female mice were injected with different DPEP1/3- [ CAG]₇BUN levels assessed in serum 7 days after PMO conjugate. BUN levels were similar to saline control injections compared to the fold increase induced by the current Pip series peptide vector (error bars: mean ± SEM, n-4-10).

FIG. 18: it was shown that the C57BL6 female mice were injected with different DPEP1/3- [ CAG]₇Creatinine levels assessed in serum 7 days after PMO conjugate. Creatinine levels were similar to saline control injections (error bars: mean ± SEM, n-4-10) compared to the fold increase induced by the current Pip series peptide carrier.

FIG. 19: shows that different DPEP1/3- [ CAG ] were administered by bolus IV (tail vein) injection]₇Serum of C57BL6 female mice of PMO conjugate assessed levels of (A) alanine transferase (ALT), (B) alkaline phosphatase (ALP) and (C) aspartate transaminase (AST), serum collected 7 days after injection,and compared to saline injections. ALP, ALT, AST levels were similar to those of saline control injections, compared to the fold increase induced by the current Pip series peptide vectors.

Throughout the description and claims of this specification, the words "comprise" and "comprise", and variations of the words "comprise" and "comprising", mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular forms "a", "an", and "the" include plural referents unless the context requires otherwise. In particular, where the indefinite article is used, the invention is to be understood as embracing both the plural and the singular, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any 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 where at least some of such features and/or steps are mutually exclusive.

The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

Examples

1. Materials and methods

1.1 Synthesis and preparation of P-PMO

9-fluorenylmethoxycarbonyl (Fmoc) -protected L-aminoAcid, benzotriazol-1-yl-oxy-tripyrrolidinophosphonium (PyBOP), 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium Hexafluorophosphate (HBTU), and Fmoc-beta-Ala-OH Pre-assembled Wang's resin (0.19 or 0.46mmol g)^-1) Obtained from Merck (Hohenbrunn, Germany). HPLC grade acetonitrile, methanol and synthetic grade N-methyl-2-pyrrolidone (NMP) were purchased from Fisher Scientific (Loughborough, UK). Peptide synthesis grade N, N-Dimethylformamide (DMF) and diethyl ether were obtained from VWR (Leicestershire, uk). Piperidine and trifluoroacetic acid (TFA) were obtained from Alfa Aesar (Heysham, uk). PMO was purchased from Gene Tools Inc. (Philograph, USA). Chick embryo extracts and horse serum were obtained from Sera Laboratories International Ltd (West Sussex, UK). Interferons were purchased from Roche Applied Science (Penzberg, Germany). All other reagents were obtained from Sigma-Aldrich (st. louis, MO, usa) unless otherwise stated. MALDI-TOF mass spectrometry was performed using a Voyager DE Pro BioSpectrometry workstation. 10mg mL in 50% aqueous acetonitrile^-1Is used as a substrate, or a stock solution of sinapic acid or alpha-cyano-4-hydroxycinnamic acid. Error bars are ± 0.1%.

1.2 Synthesis of P-PMO peptides for screening in H2k mdx cells

a) Preparation of peptide variant libraries

Fmoc-beta-Ala-OH preloaded Wang resin (0.19 or 0.46mmol g) was used by applying standard Fmoc chemistry and following the manufacturer's recommendations^-1Merck Millipore), using an Intavis parallel peptide synthesizer to prepare peptides on a 10. mu. mol scale, or using CEM Liberty Blue^TMPeptides were prepared on a 100 μmol scale using a peptide synthesizer (Buckingham, UK). In the case of synthesis using the Intavis parallel peptide synthesizer, the double coupling step was used with a PyBOP/NMM coupling mix, followed by acetic anhydride capping after each step. For the synthesis using the CEM Liberty Blue peptide synthesizer, a single standard coupling was used for all amino acids (except arginine, which was performed by double coupling). The coupling was performed once, at 60 watts microwave power at 75 ℃ for 5 minutes; except for arginine residues, each of which was coupled twice. Each deprotection reaction was carried out twice at 75 c, once for 30 seconds, then for 3 minutes,at 35 watts microwave power. Once the synthesis was complete, the resin was washed with DMF (3x50mL) and the N-terminus of the solid phase bound peptide was acetylated with acetic anhydride in the presence of DIPEA at room temperature. After N-terminal acetylation, the peptide resin was washed with DMF (3x20mL) and DCM (3x20 mL). The peptides were separated from the solid support by treatment with a separation mixture (a clean cocktail) consisting of: trifluoroacetic acid (TFA): h₂O: triisopropylsilane (TIPS) (95%: 2.5%: 2.5%: 3-10 mL). After peptide release, excess TFA was removed by purging with nitrogen. The crude peptide was precipitated by addition of cold ether (15-40mL, depending on the scale of synthesis) and centrifuged at 3200rpm for 5 minutes. The crude peptide precipitate was washed three times with cold diethyl ether (3X 15mL) and purified by RP-HPLC using a Varian 940-LC HPLC system equipped with a 445-LC amplification module and a 440-LC fraction collector. Peptides were purified by semi-preparative HPLC on RP-C18 column (10X250mm, Phenomenex Jupiter) using a linear gradient at 0.1% TFA/H₂CH in O₃CN flow rate of 15mL min^-1. Detection was performed at 220nm and 260 nm. Fractions containing the desired peptide were combined and lyophilized to give the peptide as a white solid.

Table 1: peptides synthesized for the tests in the examples, with N-terminal acetylation and C-terminal β -alanine linkers. Pip9b2 and R6Gly are comparative peptides. R6Gly uses C-terminal glycine as linker.

b) Synthesis of PMO-peptide conjugate libraries

A25-mer PMO antisense sequence against mouse dystrophin exon 23 was used (GGCCAAACCTCGGCTTACCTGAAAT (SEQ ID NO: 90)). The peptide is conjugated to the 3' end of the PMO via its C-terminal carboxyl group. This was achieved by using 2.3 and 2 equivalents of PyBOP and HOAt, respectively, in NMP, where 2.3 equivalents of DIPEA relative to the peptide and 2.5 fold excess of peptide relative to PMO dissolved in DMSO were present. In some embodiments, 2.3 equivalent HBTU generation is usedThe PyBOP was substituted for activating the C-terminal carboxyl group of the peptide. Typically, to a solution of the peptide (2500nmol) in N-methylpyrrolidinone (NMP, 80 μ L) was added PyBOP (19.2 μ L of a 0.3M solution of NMP), HOAt (16.7 μ L of a 0.3M solution of NMP), DIPEA (1.0 μ L) and PMO (100 μ L of a 10mM solution in DMSO). The mixture was left at 40 ℃ for 2.5 hours and washed with H by addition of 0.1% TFA₂The reaction was quenched with O solution (300. mu.L). The solution was purified by ion exchange chromatography using a modified Gilson HPLC system. The PMO-peptide conjugate was purified on an ion exchange column (Resource S4 mL, GE Healthcare) using a linear gradient (0 to 1M) of sodium chloride in sodium phosphate buffer (25mM, pH 7.0) containing 20% CH3CN at a flow rate of 4mL min-1. Fractions containing the desired compound were combined and lyophilized to give the peptide-PMO derivative as a white solid. By usingExcess salts may be removed from the peptide-PMO conjugate by filtration of the fractions collected after ion exchange using an ultra-153K centrifugal filtration device. The conjugates were lyophilized and analyzed by MALDI-TOF. Before use, the conjugate was dissolved in sterile water and filtered through a 0.22 μm cellulose acetate membrane. The concentration of peptide-PMO was determined by molar absorption of the conjugate at 265nm in 0.1N HCl solution.

(see Table 2 for yield)

TABLE 2 yield of P-PMO conjugates for cell culture assays. (the yield is based on dry weight of lyophilized purified ppmo. purity of P-PMO is greater than 95% as determined by normal phase HPLC at 220nm and 260 nm. (a) P-PMO was synthesized using HBTU activation instead of PyBOP).

1.3 cell culture

Mouse H2k mdx myoblasts were plated in gelatin (0.01%) coated flasks at 33 ℃ with 10% CO₂The cells were then cultured in Duchen modified Eagle Medium (DMEM PAA laboratories) supplemented with 20% heat-inactivated fetal bovine serum (FBS Gold, PAA laboratories), 2% chick embryo extract (Seralab), 1% penicillin-streptomycin-neomycin antibiotic cocktail (PSN, Gibco) and 3 pg/. mu.L of interferon-gamma (Roche). Cells were plated at 2X10⁵Individual cells/mL were seeded in gelatin (0.01%) coated 24-well plates at 33 ℃ with 10% CO₂The mixture is placed for 2 days. For differentiation into myotubes, cells were maintained in DMEM supplemented with 5% horse serum (Sigma) and 1% PSN at 37 ℃ with 5% CO₂Further growth was performed for 2 days.

1.4 transfection of cells

Cells were incubated with peptide-PMO conjugates prepared as described above (which were made in Opti-MEM without serum) and 350 μ L was added to each well in duplicate and incubated for 4 hours at 37 ℃. The transfection medium was then replaced with DMEM supplemented with 5% horse serum and 1% PSN and the cells were incubated for an additional 20 hours at 37 ℃. Cells were washed with PBS and 0.5mL of TRI RNA (Sigma) isolation reagent was added to each well. Cells were frozen at-80 ℃ for 1 hour.

1.5 RNA extraction and nested RT-PCR analysis

Total cellular RNA was extracted with TRI reagent and further precipitated with ethanol. Use ofThe purified RNA was quantified using ND-1000(Thermo Scientific). RT-PCR was performed using the OneStep RT-PCR kit (Roche, Indianapolis, USA) using the RNA (400ng) as a template. For primer sequences, see table 4. The cycling conditions for the initial reverse transcription were 50 ℃ for 30 minutes and 94 ℃ for 7 minutes for 1 cycle, followed by 30 cycles of 94 ℃ for 20 seconds, 55 ℃ for 40 seconds, and 68 ℃ for 80 seconds. One microliter of RT-PCR product was used as template for the second PCR. Amplification was performed in 25 cycles using 0.5U of SuperTAQ, each cycle being 94 ℃ for 30 seconds, 55 ℃ for 1 minute and 72 ℃ for 1 minute. 1.5% agarose gel was usedThe gel separated the products by electrophoresis. Images of the agarose gels were visualized on Molecular Imager ChemiDoc^TM XRS⁺Images were taken on an imaging system (BioRad, uk) and analyzed using Image Lab (V4.1). Exon skipping experimental data, expressed as a percentage of exon 23 skipping from at least three independent experiments, were analyzed and plotted using Microsoft Excel.

1.6 Synthesis of PMO-peptide conjugates for testing in H2k mdx mice

a) Synthesis of peptide variants

Using CEM Liberty Blue^TMMicrowave peptide synthesizer (platinum han, uk) and Fmoc chemistry, following the manufacturer's recommendations, peptides were synthesized on a 100 μmol scale. The side chain protection groups used were unstable to trifluoroacetic acid treatment and the peptides were synthesized using a 5-fold excess of Fmoc-protected amino acid (0.25mmol) activated with PyBOP (5-fold excess) in the presence of DIPEA. Piperidine (20% v/v in DMF) was used to remove the N-Fmoc protecting group. The coupling was performed once, at 60 watts microwave power at 75 ℃ for 5 minutes; except for arginine residues, each of which was coupled twice. Each deprotection reaction was carried out twice at 75 ℃ for 30 seconds and then 3 minutes at 35W microwave power. Once the synthesis was complete, the resin was washed with DMF (3x50mL) and the N-terminus of the solid phase bound peptide was acetylated with acetic anhydride in the presence of DIPEA at room temperature. After N-terminal acetylation, the peptide resin was washed with DMF (3x20mL) and DCM (3x20 mL). Separating the peptides from the solid support by treatment with a separation mixture at room temperature for 3 hours, the separation mixture consisting of: trifluoroacetic acid (TFA): water: triisopropylsilane (TIPS) (95%: 2.5%: 2.5%: 10 mL). Excess TFA was removed by purging with nitrogen. The isolated peptide was precipitated by addition of ice cold diethyl ether and centrifuged at 3000rpm for 5 minutes. The crude peptide precipitate was washed three times with cold diethyl ether (3X 40mL) and purified by RP-HPLC using a Varian 940-LC HPLC system equipped with a 445-LC amplification module and a 440-LC fraction collector. Purification by semi-preparative HPLC on RP-C18 column (10X250mm, Phenomenex Jupiter)Peptide conversion using CH₃CN in 0.1% TFA/H₂Linear gradient in O, flow rate 15mL min^-1. Detection was performed at 220nm and 260 nm.

b) Synthesis of PMO-peptide conjugates

A25-mer PMO antisense sequence against mouse dystrophin exon 23 was used (GGCCAAACCTCGGCTTACCTGAAAT (SEQ ID NO: 90)). The peptide is conjugated to the 3' end of the PMO via its C-terminal carboxyl group. This was achieved by using 2.3 and 2 equivalents of PyBOP and HOAt, respectively, in NMP, where 2.3 equivalents of DIPEA relative to the peptide and 2.5 fold excess of peptide relative to PMO dissolved in DMSO were present. In some embodiments, HBTU (2.3 eq) was used instead of PyBOP for activating the C-terminal carboxyl group of the peptide. Typically, to a solution of the peptide (10. mu. mol) in N-methylpyrrolidinone (NMP, 100. mu.L) was added PyBOP (76.6. mu.L of a 0.3M solution of NMP), HOAt (66.7. mu.L of a 0.3M solution of NMP), DIPEA (4.0. mu.L) and PMO (400. mu.L of a 10mM solution in DMSO). The mixture was left at 40 ℃ for 2 hours and the reaction was quenched by the addition of 0.1% TFA (1 mL). The reaction was purified on a cation exchange chromatography column (Resource S6 mL, GE Healthcare) using a linear gradient (0 to 1M) of sodium chloride in sodium phosphate buffer (25mM, pH 7.0) containing 20% CH3CN at a flow rate of 6mL min-1. By usingExcess salts may be removed from the peptide-PMO conjugate by filtration of the fractions collected after ion exchange using an ultra-153K centrifugal filtration device. The conjugates were lyophilized and analyzed by MALDI-TOF. Before use, the conjugate was dissolved in sterile water and filtered through a 0.22 μm cellulose acetate membrane. The concentration of peptide-PMO was determined by molar absorption of the conjugate at 265nm in 0.1N HCl solution. The overall yield (Table 3) was 25-36% based on PMO.

Peptide-PMO	Yield of the product
		D-Pep 1.1-PMO	36％
D-Pep 1.3-PMO	25％a
		D-Pep 1.4-PMO	24％a
D-Pep 1.5-PMO	25％a
		D-Pep 1.6-PMO	25％a
D-Pep 3.1-PMO	28％
		D-Pep 3.2-PMO	33％
D-Pep 3.7-PMO	26％
		D-pep 3.8-PMO	34％
D-Pep 3.9-PMO	28％
		D-Pep 3.10-PMO	28％

Table 3. yield of large scale synthesized P-PMO conjugate for in vivo analysis (based on dry weight of lyophilized purified PPMO. purity of PPMO as determined by normal phase HPLC at 220nm and 260nm > 95% > (a) PPMO was synthesized using HBTU activation instead of PyBOP).

1.7 in vivo assessment of dystrophin recovery by P-PMO

After the ethical examination of the institution, the experiment was performed in department of biomedical sciences of oxford university according to the project authorization of the department of interior administration. Housing the mice in minimal disease facilities; the environment was temperature controlled with a 12 hour bright and dark cycle. All animals were freely available rodent chow and water.

The experiments were performed in female mdx mice 10-12 weeks old. Mdx mice were restrained prior to a single tail vein injection of 10mg/kg P-PMO. 1 week after injection, mice were sacrificed, TA, heart and diaphragm were removed and snap frozen in dry ice cooled isopentane and stored at-80 ℃.

1.8 Western blot analysis

To assess the duration of dystrophin recovery after a single administration, one third of the muscle (TA and diaphragm) or ninety transverse frozen sections (heart) 7 μm thick were dissolved in 300 μ l buffer (50mM Tris pH 8, 150mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 10% SDS and protease/phosphatase inhibitor) and then centrifuged at 13000rpm (Heraeus, #3325B) for 10 minutes. The supernatant was collected and heated at 100 ℃ for 3 minutes. The protein was quantified by the BCA method as described previously (19), and 40. mu.g of protein per sample was dissolved in NuPage 3-8% Tris-acetate gel. Proteins were transferred to 0.45 μm pore size PVDF membranes for 1h at 30V and then 1h at 100V as described previously (37), and then probed with monoclonal anti-dystrophin (1:200, NCL-DYS1, Novocastra) and anti-Vinculin (Vincultin) (loading control, 1: 100000, hVIN-1, Sigma) antibodies. Secondary antibody IRDye 800CW goat anti-mouse was used at 1: 20000 (LiCOR).

The dystrophin recovery levels in P-PMO treated mdx mice are expressed relative to the levels of C57BL/10 wild-type control mice, which are considered to be 100%. To this end, a standard curve was generated by including 5 serial dilutions of C57BL/10 protein parallel to the P-PMO treated mdx sample. The dilution series were as follows: of the 40 μ g total protein loaded in each lane, 75%, 40%, 15%, 5% or 0% was from C57BL/10 protein lysate, respectively, and the remainder was from untreated mdx protein lysate. These standards were aliquoted and used in parallel with the treated mdx samples in each Western blot. For all standard and treated samples, dystrophin intensity quantification was performed by the fluorescence Odyssey imaging system and normalized by calculating the ratio to the Fluorescent intensity of vinculin in all samples. The standard normalized values are plotted against their known dystrophin concentrations to obtain the best-fit mathematical expression used to interpolate the normalized values for each sample of P-PMO treated mdx mice.

1.9 in vivo RT-qPCR analysis of Dmd exon 23 skipping

Quantitative analysis of mouse Dmd transcript exon 23 exclusion was performed on skeletal muscle and heart tissues treated with peptide-PMO. Briefly, RNA was extracted from homogenized tissue using a Trizol-based extraction method, and cDNA was synthesized using random primers. Primers/probes were synthesized by Integrated DNA Technologies and were intended to amplify regions spanning exons 23-24, representing non-skipped products (mDMDM 23-24, see Table 4), or to specifically amplify transcripts lacking exon 23 using probes spanning the boundaries of exons 22 and 24 (mDMDM 22-24). The level of each transcript was determined by calibration with a standard curve made using known transcript quantities and skip percentages derived from skip/skip + unskip.

Table 4: primer and probe sequences for exon 23 skipping were quantified by either nested RT-PCR or quantitative RT-PCR methods.

1.10 toxicological assessment of peptide-PMO

8-10 week old female C57BL/6 mice were administered a single 30mg/kg dose of peptide-PMO in 0.9% saline by intravenous bolus tail vein injection. After 20 hours of placement in metabolic cages (Tecniplast, uk), urine was collected non-invasively under refrigerated conditions on days 2 and 7 after administration. On day 7, serum was collected from the jugular vein at necropsy, as were tibialis anterior, diaphragm and heart tissue.

The same procedure was performed with different single doses of peptide-PMO ranging from 2.5mg/kg to 50mg/kg in 0.9% saline by intravenous tail vein injection.

After appropriate dilution of the urine to fit the standard curve, the urine levels of KIM-1 (renal injury molecule-1) and NGAL (neutrophil gelatinase-associated lipocalin) were quantified by ELISA (KIM-1R & D cat # MKM100, NGAL R & D cat # MLCN 20). Values were normalized to urine creatinine levels quantified at the MRC Harwell institute, Mary Lyon Centre, Oxfordshire, uk. Serum blood urea nitrogen levels were quantified at the MRC Harwell institute, Mary Lyon Centre, Oxfordshire, uk.

All water levels were quantified on the AU680 clinical chemistry analyzer Beckman Coulter.

Quantification of exon skipping efficacy was determined by quantitative RT-PCR of exon 23 skipped and non-skipped transcripts and expressed as the percentage of skipped transcripts relative to the total (skipped and non-skipped) transcripts (see table 4 for sequence).

2. Results

The results provided herein demonstrate that the peptide-PMO conjugates produced herein have a significant dose-responsive effect in intracellular exon skipping activity (fig. 1, 2 and 12). These figures also highlight that all DPEP1 and DPEP3 series (i.e. the peptides of the invention) have sufficient cell penetration efficacy in cells considered for therapeutic use.

The results provided herein further highlight the in vivo activity of the peptide-PMO conjugates in a mouse model of the associated disease (fig. 3-4). Overall, the results indicate that the activity of this conjugate is greatest in the tibialis anterior, with the order of magnitude of the activity: tibialis anterior > diaphragm > heart. These figures demonstrate that DPEP peptide conjugates of the invention have good exon skipping activity in vivo and provide an increase in dystrophin expression in vivo. Furthermore, DPEP conjugates of the invention have advantages over previous cell penetrating peptides (such as 'PIP' peptide and R6Gly, when used in the same conjugate) in both aspects.

It is also demonstrated herein that both the levels of KIM-1 and NGAL, which are indicators of renal toxicity, are significantly lower after administration of DPEP peptide conjugate compounds than conjugates with the previous cell penetrating peptides. DPEP 1.9 and 3.8 conjugates showed the lowest levels of such markers (fig. 5, 6 and 11). Serum blood urea nitrogen levels (another marker of renal insufficiency) were also only elevated for the conjugate with Pip9b2, but not for the conjugate with the DPEP peptide of the invention (fig. 7). The second major finding was that, 7 days after administration, for all DPEP peptide conjugates, KIM-1 and NGAL levels were reduced to near normal saline levels, indicating that there was also some reversal and improvement in kidney-related toxicity. This effect was not observed with the previous conjugates of cell penetrating peptides. The effect of toxicity reversal was still seen with the DPEP peptides of the present invention when administered at high doses of 50mg/kg (fig. 11). The previous cell penetrating peptides showed no reduction in toxicity after 7 days and the toxic markers remained at much higher levels throughout the process.

It was further demonstrated that at higher doses of 30 and 50mg/kg, exon skipping activity of all DPEP peptide conjugates in TA and diaphragm was still high (fig. 10 and 12), which indicates a broader therapeutic index for these compounds as evidenced by a reduced level of kidney injury markers, since toxicity markers were reduced many-fold. It is also noteworthy that all DPEP peptide conjugates have higher activity than the known R6Gly comparator in the conjugate while keeping at least similar levels of toxicity markers and similar activity as the known PIP peptide comparator in the conjugate while having much lower levels of toxicity markers. In certain instances, DPEP peptide conjugates of the invention not only exhibit increased activity, but also exhibit reduced toxicity markers as compared to known R6Gly conjugates.

Thus, the DPEP1 and 3 peptides of the present invention provide promising cell penetrating peptides for improving the efficacy and reducing the toxicity of therapeutic conjugates for the treatment of human neuromuscular diseases.

3. Further embodiments

Synthesis and preparation of P-PMO

9-fluorenylmethoxycarbonyl (Fmoc) -protected L-amino acids, benzotriazol-1-yl-oxy-trispyrrolidinophosphonium (PyBOP), 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium Hexafluorophosphate (HBTU) and Fmoc-. beta. -Ala-OH Realgar resin (0.19 or 0.46mmol g-1) were obtained from Merck (Hohenbrun, Germany). 1-hydroxy-7-azabenzotriazole (HOAt) was obtained from Sigma-Aldrich. HPLC grade acetonitrile, methanol and synthetic grade N-methyl-2-pyrrolidone (NMP) were purchased from Fisher Scientific (Loughborough, UK). Peptide synthesis grade N, N-Dimethylformamide (DMF) and diethyl ether were obtained from VWR (Leicestershire, uk). Piperidine and trifluoroacetic acid (TFA) were obtained from Alfa Aesar (Heysham, uk). PMO was purchased from Gene Tools Inc. (Philograph, USA). All other reagents were obtained from Sigma-Aldrich (st. louis, MO, usa) unless otherwise stated. MALDI-TOF mass spectrometry was performed using a Voyager DE Pro BioSpectrometry workstation. 10mg mL in 50% aqueous acetonitrile^-1Is used as a substrate, or a stock solution of sinapic acid or alpha-cyano-4-hydroxycinnamic acid. Error bars are ± 0.1%.

Synthesis of P-PMO peptides for screening in cells

a) Preparation of peptide variant libraries

Peptides were prepared on the 10. mu. mol scale using the Fmoc-. beta. -Ala-OH preloaded Wang resin (0.19 or 0.46mmol g-1, Merck Millipore), using the Intavis parallel peptide synthesizer, or on the 100. mu. mol scale using the CEM Liberty blue (TM) peptide synthesizer (Buckingham, UK) by applying standard Fmoc chemistry and following the manufacturer's recommendations. In the case of synthesis using the Intavis parallel peptide synthesizer, the double coupling step was used with a PyBOP/NMM coupling mix, followed by acetic anhydride capping after each step. For the synthesis using the CEM Liberty Blue peptide synthesizer, a single standard coupling was used for all amino acids (except arginine, which was performed by double coupling). The coupling was performed once, at 60 watts microwave power at 75 ℃ for 5 minutes; except for arginine residues, each of which was coupled twice. Each deprotection reaction was carried out twice at 75 ℃ for 30 seconds and then 3 minutes at 35W microwave power. Once the synthesis was complete, the resin was washed with DMF (3x50mL) and the N-terminus of the solid phase bound peptide was acetylated with acetic anhydride in the presence of DIPEA at room temperature. After N-terminal acetylation, the peptide resin was washed with DMF (3x20mL) and DCM (3x20 mL). Separating the peptides from the solid support by treatment with a separation mixture at room temperature for 3 hours, the separation mixture consisting of: trifluoroacetic acid (TFA): H2O: triisopropylsilane (TIPS) (95%: 2.5%: 2.5%: 3-10 mL). After peptide release, excess TFA was removed by purging with nitrogen. The crude peptide was precipitated by addition of cold ether (15-40mL, depending on the scale of synthesis) and centrifuged at 3200rpm for 5 minutes. The crude peptide precipitate was washed three times with cold diethyl ether (3X 15mL) and purified by RP-HPLC using a Varian 940-LC HPLC system equipped with a 445-LC amplification module and a 440-LC fraction collector. Peptides were purified by semi-preparative HPLC on RP-C18 chromatography column (10X250mm, Phenomenex Jupiter) using a linear gradient of CH3CN in 0.1% TFA/H2O at a flow rate of 15mL min-1. Detection was performed at 220nm and 260 nm. Fractions containing the desired peptide were combined and lyophilized to give the peptide as a white solid (see table 5 for yield).

Table 5: the peptides synthesized for the test in the examples have an N-terminal acetylated (Ac) and a C-terminal β -alanine linker (B), S being a glycosylated serine residue. DPEP5.7, Pip9b2 and Pip6a are comparative peptides.

b) Synthetic peptide-PMO conjugate libraries

A21-mer PMO antisense sequence CAGCAGCAGCAGCAGCAGCAG (SEQ ID NO.107), also known as [ CAG ], for the triplet repeat sequence was used]7. The peptide is conjugated to the 3' end of the PMO via its C-terminal carboxyl group. This was achieved by using 2.5 and 2 equivalents of PyBOP and HOAt, respectively, in NMP, with 2.5 equivalents of DIPEA and a 2.5-fold excess of peptide over PMO dissolved in DMSO. Typically, to a solution of the peptide (2500nmol) in N-methylpyrrolidinone (NMP, 80. mu.L) was added PyBOP (19.2. mu.L of a 0.3M solution of NMP), HOAt (16.7. mu.L of a 0.3M solution of NMP), DIPEA (1.0mL) and PMO (180. mu.L of a 10mM solution in DMSO). The mixture was left at 40 ℃ for 2.5H and the reaction was quenched by addition of 0.1% TFA in H2O (300. mu.L). The solution was purified by ion exchange chromatography using a modified Gilson HPLC system. The PMO-peptide conjugate was purified on an ion exchange column (Resource S4 mL, GE Healthcare) using a linear gradient of sodium phosphate buffer (25mM, pH 7.0) containing 20% CH3 CN. The conjugate was eluted from the column using sodium chloride solution (1M) at a flow rate of 4mL min-1 or 6mL min-1. Fractions containing the desired compound were immediately combined and desalted. By usingExcess salts may be removed from the peptide-PMO conjugate by filtration of the fractions collected after ion exchange using an ultra-153K centrifugal filtration device. The conjugates were lyophilized and analyzed by MALDI-TOF. Before use, the conjugate was dissolved in sterile water and filtered through a 0.22 μm cellulose acetate membrane. The concentration of peptide-PMO was determined by molar absorption of the conjugate at 265nm in 0.1N HCl solution. (see Table 6 for yields)

Peptides	Yield of
		D-Pep 1.1	36％
D-Pep 1.7	41％
		D-pep 1.8	38％
D-Pep 1.9	40％
		D-Pep 1.9W3	43％
D-Pep 1.9W4P	23％
		D-Pep 3.1	31％
D-Pep 3.8	36％
		D-Pep 5.70	31％

Table 6. yield of P-PMO conjugate for cell culture analysis and in vivo experiments (based on dry weight of lyophilized purified P-PMO. purity of P-PMO was greater than 95% as determined by normal phase HPLC at 220nm and 260 nm.

Synthesis of peptide-PMO conjugates

Peptides were synthesized and conjugated to PMO as described previously. PMO sequences targeting the CUG/CTG extension repeat (5'-CAGCAGCAGCAGCAGCAGCAG-3' (SEQ ID NO: 107)) were purchased from Gene Tools LLC. This is the [ CAG ]7PMO mentioned elsewhere herein.

Cell culture and peptide-PMO treatment.

Immortalized myoblasts from healthy individuals or DM1 patients with 2600 CTG repeats were cultured in growth medium consisting of M199: DMEM mix (ratio of 1:4, Life Technologies) supplemented with 20% fbs (Life Technologies), 50 μ g/ml gentamicin (Life Technologies), 25 μ g/ml fetuin, 0.5ng/ml bFGF, 5ng/ml EGF and 0.2 μ g/ml dexamethasone (Sigma-Aldrich). For myoblasts, myoblast differentiation was induced by switching confluent cell cultures to DMEM medium supplemented with 5 μ g/ml insulin (Sigma-Aldrich). For treatment, WT or DM1 cells were differentiated for 4 days. The medium was then replaced with fresh differentiation medium containing peptide-PMO conjugate at a concentration of 1, 2, 5, 10, 20 or 40 μ M. Cells were harvested 48 hours after treatment for analysis. Cell viability was quantified using a fluorescence-based assay 2 days after transfection of peptide-PMO at a concentration of 40uM in human hepatocytes or 1, 2, 5, 10, 20, or 40 μ M in human myoblasts.

RNA isolation, RT-PCR and qPCR analysis.

For mouse tissues: prior to RNA extraction, muscles were destroyed in TriReagent (Sigma-Aldrich) using the Fastprep system and lysine Matrix D tubes (MP biomedicals). For human cells: prior to RNA extraction, cells were lysed in proteinase K buffer (500mM NaCl, 10mM Tris-HCl, pH 7.2, 1.5mM MgCl2, 10mM EDTA, 2% SDS, and 0.5mg/ml proteinase K) for 45 min at 55 ℃. Total RNA was isolated using TriReagent according to the manufacturer's protocol. One microgram of RNA was reverse transcribed using the M-MLV first strand synthesis system (Life Technologies) in a total volume of 20. mu.L according to the manufacturer's instructions. One microliter of the cDNA preparation was then used in a semi-quantitative PCR assay according to standard protocols (ReddyMix, Thermo Scientific). Within the range of linear amplification of each gene, PCR amplification was performed for 25-35 cycles. The PCR products were separated on a 1.5-2% agarose gel, stained with ethidium bromide, and quantified using ImageJ software. The ratio of exon retention was quantified as the percentage of retention relative to the total isoform signal intensity. The primers are shown in table 7 below:

table 7: primers for PCR

Toxicology

Toxicology assessments were performed as described above in section 1.10.

Results

Treated muscle cells (myoblasts) derived from DM1 patients showed DPEP1 or 3 peptide- [ CAG]₇The PMO conjugates specifically target the mutated CUGexp-DMPK transcript, thereby eliminating the deleterious sequestration (sequestration) of the nuclear RNA cluster (foci) to the MBNL1 splicing factor and the resulting loss of function of MBNL1, which caused the splicing defect and muscle dysfunction by MBNL1. DPEP1/3 peptide- [ CAG]₇The PMO conjugate penetrated the cells and induced normalization of splicing with high efficiency (fig. 13). These new generations of so-called "DPEP 1 and DPEP 3" peptides, when conjugated with the CAG7 repeat antisense oligonucleotide PMO, showed a high efficacy in correcting splicing defects in vitro, indicating a potential therapeutic use for the treatment of DM 1.

Furthermore, preliminary toxicological assessments of the conjugates formed with DPEP1/3 indicate that ALP, ALT, AST, KIM-1, BUN, NGAL and creatinine levels are similar to those of saline control injections, in contrast to the fold increase typically caused by currently available peptide vectors from the Pip series. With these preliminary data we demonstrate that the peptide DPEP is conjugated to [ CAG ]]₇The conjugates formed with PMO were as effective as those formed with the existing peptides (e.g., Pip6a), but had a broader therapeutic window due to their lower toxicity (fig. 15-19).

Sequence listing

<110> Oxford university science and technology Innovation Co., Ltd

British research and innovation organization

<120> cell-penetrating peptide

<130> P236820WO

<150> GB 1812972.6

<151> 2018-08-09

<160> 114

<170> PatentIn version 3.5

<210> 1

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<400> 1

Arg Xaa Arg Arg Xaa Arg Arg

1 5

<210> 2

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is bAla

<400> 2

Arg Xaa Arg Xaa Arg

1 5

<210> 3

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<400> 3

Arg Xaa Arg Arg

1

<210> 4

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<400> 4

Arg Xaa Arg Arg Xaa Arg

1 5

<210> 5

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (3)..(3)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<400> 5

Arg Arg Xaa Arg Xaa Arg

1 5

<210> 6

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<400> 6

Arg Xaa Arg Arg Xaa

1 5

<210> 7

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> X is bAla

<220>

<221> MOD_RES

<222> (3)..(3)

<223> X is bAla

<400> 7

Xaa Arg Xaa Arg

1

<210> 8

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is bAla

<400> 8

Arg Xaa His Xaa His

1 5

<210> 9

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is bAla

<400> 9

His Xaa His Xaa Arg

1 5

<210> 10

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<400> 10

Arg Xaa Arg His Xaa His Arg

1 5

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(5)

<223> X is bAla

<400> 11

Arg Xaa Arg Xaa Xaa His Arg

1 5

<210> 12

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<400> 12

Arg Xaa Arg Arg Xaa His

1 5

<210> 13

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<400> 13

His Xaa Arg Arg Xaa Arg

1 5

<210> 14

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is bAla

<400> 14

His Xaa His Xaa His

1 5

<210> 15

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> X is bAla

<220>

<221> MOD_RES

<222> (3)..(3)

<223> X is bAla

<400> 15

Xaa His Xaa His

1

<210> 16

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> X is bAla

<220>

<221> MOD_RES

<222> (3)..(3)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<400> 16

Xaa Arg Xaa Ser Xaa

1 5

<210> 17

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> X is bAla

<220>

<221> MOD_RES

<222> (3)..(3)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is hydroxyproline

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<400> 17

Xaa Arg Xaa Xaa Xaa

1 5

<210> 18

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is hydroxyproline

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is hydroxyproline

<220>

<221> MOD_RES

<222> (6)..(6)

<223> X is bAla

<400> 18

Arg Xaa His Xaa His Xaa

1 5

<210> 19

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is hydroxyproline

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is hydroxyproline

<400> 19

Arg Xaa Arg Arg Xaa Arg

1 5

<210> 20

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 20

Tyr Gln Phe Leu Ile

1 5

<210> 21

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 21

Phe Gln Ile Leu Tyr

1 5

<210> 22

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 22

Ile Leu Phe Gln Tyr

1 5

<210> 23

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 23

Phe Gln Ile Tyr

1

<210> 24

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 24

Trp Trp Pro Trp Trp

1 5

<210> 25

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 25

Trp Pro Trp Trp

1

<210> 26

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 26

Trp Trp Pro Trp

1

<210> 27

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 27

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr Arg Xaa Arg Xaa

1 5 10 15

Arg

<210> 28

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 28

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr Arg Xaa Arg Arg

1 5 10 15

<210> 29

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 29

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Arg Arg Xaa Arg Xaa

1 5 10 15

Arg

<210> 30

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 30

Arg Xaa Arg Xaa Arg Phe Gln Ile Leu Tyr Arg Xaa Arg Arg Xaa Arg

1 5 10 15

Arg

<210> 31

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 31

Arg Xaa Arg Arg Xaa Arg Arg Tyr Gln Phe Leu Ile Arg Xaa Arg Xaa

1 5 10 15

Arg

<210> 32

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 32

Arg Xaa Arg Arg Xaa Arg Arg Ile Leu Phe Gln Tyr Arg Xaa Arg Xaa

1 5 10 15

Arg

<210> 33

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 33

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Arg Xaa Arg Xaa Arg

1 5 10 15

<210> 34

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 34

Arg Xaa Arg Arg Xaa Phe Gln Ile Leu Tyr Arg Xaa Arg Arg Xaa Arg

1 5 10 15

<210> 35

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 35

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa Arg

1 5 10 15

<210> 36

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 36

Arg Xaa Arg Arg Xaa Phe Gln Ile Leu Tyr Arg Xaa Arg Xaa Arg

1 5 10 15

<210> 37

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 37

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr Arg Xaa His Xaa

1 5 10 15

His

<210> 38

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 38

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr His Xaa His Xaa

1 5 10 15

Arg

<210> 39

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 39

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr His Xaa Arg Xaa

1 5 10 15

His

<210> 40

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 40

Arg Xaa Arg Arg Xaa Arg Arg Tyr Gln Phe Leu Ile Arg Xaa His Xaa

1 5 10 15

His

<210> 41

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 41

Arg Xaa Arg Arg Xaa Arg Arg Ile Leu Phe Gln Tyr Arg Xaa His Xaa

1 5 10 15

His

<210> 42

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 42

Arg Xaa Arg His Xaa His Arg Phe Gln Ile Leu Tyr Arg Xaa Arg Xaa

1 5 10 15

Arg

<210> 43

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 43

Arg Xaa Arg Xaa Xaa His Arg Phe Gln Ile Leu Tyr Arg Xaa His Xaa

1 5 10 15

His

<210> 44

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 44

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Arg Xaa His Xaa His

1 5 10 15

<210> 45

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 45

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr His Xaa His Xaa His

1 5 10 15

<210> 46

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 46

Arg Xaa Arg Arg Xaa His Phe Gln Ile Leu Tyr Arg Xaa His Xaa His

1 5 10 15

<210> 47

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 47

His Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Arg Xaa His Xaa His

1 5 10 15

<210> 48

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 48

Arg Xaa Arg Arg Xaa Phe Gln Ile Leu Tyr Arg Xaa His Xaa His

1 5 10 15

<210> 49

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 49

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa His Xaa His

1 5 10 15

<210> 50

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 50

Arg Xaa Arg Arg Xaa Arg Tyr Gln Phe Leu Ile His Xaa His Xaa His

1 5 10 15

<210> 51

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 51

Arg Xaa Arg Arg Xaa Arg Ile Leu Phe Gln Tyr His Xaa His Xaa His

1 5 10 15

<210> 52

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 52

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr His Xaa His Xaa

1 5 10 15

His

<210> 53

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 53

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa Ser

1 5 10 15

<210> 54

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> x hydroxyproline

<400> 54

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa Xaa

1 5 10 15

<210> 55

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is hydroxyproline

<400> 55

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa Arg

1 5 10 15

<210> 56

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (3)..(3)

<223> X is bAla

<220>

<221> MOD_RES

<222> (6)..(6)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 56

Arg Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa Arg

1 5 10 15

<210> 57

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 57

Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa Arg

1 5 10 15

<210> 58

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (10)..(10)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<400> 58

Arg Xaa Arg Arg Xaa Arg Trp Trp Trp Xaa Arg Xaa Arg

1 5 10

<210> 59

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 59

Arg Xaa Arg Arg Xaa Arg Trp Trp Pro Trp Trp Xaa Arg Xaa Arg

1 5 10 15

<210> 60

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (11)..(11)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<400> 60

Arg Xaa Arg Arg Xaa Arg Trp Pro Trp Trp Xaa Arg Xaa Arg

1 5 10

<210> 61

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (11)..(11)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<400> 61

Arg Xaa Arg Arg Xaa Arg Trp Trp Pro Trp Xaa Arg Xaa Arg

1 5 10

<210> 62

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 62

Arg Xaa Arg Arg Xaa Arg Arg Trp Trp Trp Arg Xaa Arg Xaa Arg

1 5 10 15

<210> 63

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 63

Arg Xaa Arg Arg Xaa Arg Arg Trp Trp Pro Trp Trp Arg Xaa Arg Xaa

1 5 10 15

Arg

<210> 64

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 64

Arg Xaa Arg Arg Xaa Arg Arg Trp Pro Trp Trp Arg Xaa Arg Xaa Arg

1 5 10 15

<210> 65

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 65

Arg Xaa Arg Arg Xaa Arg Arg Trp Trp Pro Trp Arg Xaa Arg Xaa Arg

1 5 10 15

<210> 66

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 66

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa Arg

1 5 10 15

<210> 67

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 67

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr Arg Xaa Arg

1 5 10 15

<210> 68

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> X is bAla

<220>

<221> MOD_RES

<222> (3)..(3)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 68

Xaa Arg Xaa Arg Xaa Trp Trp Pro Trp Trp Arg Xaa Arg Arg Xaa Arg

1 5 10 15

<210> 69

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 69

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr Xaa His Xaa His

1 5 10 15

<210> 70

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 70

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Tyr Arg Xaa His Xaa His

1 5 10 15

<210> 71

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 71

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa His

1 5 10 15

<210> 72

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is hydroxyproline

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is hydroxyproline

<400> 72

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Arg Xaa His Xaa His

1 5 10 15

<210> 73

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is hydroxyproline

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is hydroxyproline

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 73

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Arg Xaa His Xaa His

1 5 10 15

<210> 74

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is hydroxyproline

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is hydroxyproline

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is hydroxyproline

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is hydroxyproline

<400> 74

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Arg Xaa His Xaa His

1 5 10 15

<210> 75

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (11)..(11)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<400> 75

Arg Xaa Arg Arg Xaa Arg Trp Trp Trp Arg Xaa His Xaa His

1 5 10

<210> 76

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (11)..(11)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<400> 76

Arg Xaa Arg Arg Xaa Arg Trp Trp Pro Arg Xaa His Xaa His

1 5 10

<210> 77

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (11)..(11)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<400> 77

Arg Xaa Arg Arg Xaa Arg Pro Trp Trp Arg Xaa His Xaa His

1 5 10

<210> 78

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 78

Arg Xaa Arg Arg Xaa Arg Trp Trp Pro Trp Trp Arg Xaa His Xaa His

1 5 10 15

<210> 79

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 79

Arg Xaa Arg Arg Xaa Arg Trp Trp Pro Trp Arg Xaa His Xaa His

1 5 10 15

<210> 80

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 80

Arg Xaa Arg Arg Xaa Arg Trp Pro Trp Trp Arg Xaa His Xaa His

1 5 10 15

<210> 81

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 81

Arg Xaa Arg Arg Xaa Arg Arg Trp Trp Trp Arg Xaa His Xaa His

1 5 10 15

<210> 82

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 82

Arg Xaa Arg Arg Xaa Arg Arg Trp Trp Pro Trp Trp Arg Xaa His Xaa

1 5 10 15

His

<210> 83

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 83

Arg Xaa Arg Arg Xaa Arg Arg Trp Pro Trp Trp Arg Xaa His Xaa His

1 5 10 15

<210> 84

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 84

Arg Xaa Arg Arg Xaa Arg Arg Trp Trp Pro Trp Arg Xaa His Xaa His

1 5 10 15

<210> 85

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (3)..(3)

<223> X is bAla

<220>

<221> MOD_RES

<222> (6)..(6)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 85

Arg Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Arg Xaa His Xaa

1 5 10 15

His

<210> 86

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 86

Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr Arg Xaa His Xaa His

1 5 10 15

<210> 87

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (3)..(3)

<223> X is bAla

<220>

<221> MOD_RES

<222> (6)..(6)

<223> X is bAla

<220>

<221> MOD_RES

<222> (13)..(13)

<223> X is bAla

<220>

<221> MOD_RES

<222> (15)..(15)

<223> X is bAla

<400> 87

Arg Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa His Xaa His

1 5 10 15

<210> 88

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> X is bAla

<220>

<221> MOD_RES

<222> (4)..(4)

<223> X is bAla

<220>

<221> MOD_RES

<222> (12)..(12)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<400> 88

Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr Xaa His Xaa His

1 5 10 15

<210> 89

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MOD_RES

<222> (2)..(2)

<223> X is bAla

<220>

<221> MOD_RES

<222> (5)..(5)

<223> X is bAla

<220>

<221> MOD_RES

<222> (14)..(14)

<223> X is bAla

<220>

<221> MOD_RES

<222> (16)..(16)

<223> X is bAla

<400> 89

Arg Xaa Arg Arg Xaa His Arg Phe Gln Ile Leu Tyr Arg Xaa His Xaa

1 5 10 15

His

<210> 90

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> 25-mer PMO antisense sequence against mouse dystrophin exon 23

<400> 90

ggccaaacct cggcttacct gaaat 25

<210> 91

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> exon 20Fo primer

<400> 91

cagaattctg ccaattgctg ag 22

<210> 92

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> exon 26Ro primer

<400> 92

ttcttcagct tgtgtcatcc 20

<210> 93

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> exon 20Fi primers

<400> 93

cccagtctac caccctatca gagc 24

<210> 94

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> exon 26Ri primer

<400> 94

cctgccttta aggcttcctt 20

<210> 95

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> mDMD23-24 primer 1

<400> 95

caggccattc ctctttcagg 20

<210> 96

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> mDMD23-24 primer 2

<400> 96

gaaactttcc tcccagttgg t 21

<210> 97

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> mDMD23-24 Probe

<220>

<221> misc_feature

<222> (1)..(1)

<223> labeling with FAM

<220>

<221> misc_feature

<222> (9)..(10)

<223> residues 9 and 10 are linked by an internal quencher ZEN

<220>

<221> misc_feature

<222> (29)..(29)

<223> labeling with IABkFQ

<400> 97

tcaacttcag ccatccattt ctgtaaggt 29

<210> 98

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> mDMD22-24 primer 1

<400> 98

ctgaatatga aataatggag gagagactcg 30

<210> 99

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> mDMD22-24 primer 2

<400> 99

cttcagccat ccatttctgt aaggt 25

<210> 100

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> mDMD22-24 Probe

<220>

<221> misc_feature

<222> (1)..(1)

<223> labeling with FAM

<220>

<221> misc_feature

<222> (9)..(10)

<223> residues 9 and 10 are linked by an internal quencher ZEN

<220>

<221> misc_feature

<222> (19)..(19)

<223> labeling with IABkFQ

<400> 100

atgtgattct gtaatttcc 19

<210> 101

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X is bAa

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is bAa

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X is hydroxyproline

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> X is hydroxyproline

<400> 101

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa Arg

1 5 10 15

<210> 102

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X is hydroxyproline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is hydroxyproline

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> X is bAla

<400> 102

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa Arg

1 5 10 15

<210> 103

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X is hydroxyproline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is hydroxyproline

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X is hydroxyproline

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> X is hydroxyproline

<400> 103

Arg Xaa Arg Arg Xaa Arg Phe Gln Ile Leu Tyr Xaa Arg Xaa Arg

1 5 10 15

<210> 104

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X is bAla

<400> 104

Arg Xaa Arg Arg Xaa Arg Trp Trp Trp Xaa Arg Xaa Arg

1 5 10

<210> 105

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> X is bAla

<400> 105

Arg Xaa Arg Arg Xaa Arg Trp Trp Pro Trp Trp Xaa Arg Xaa Arg

1 5 10 15

<210> 106

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Glucoylated serine residue

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X is bAla

<400> 106

Arg Xaa Arg Xaa Arg Ser Arg Xaa Arg Xaa Arg

1 5 10

<210> 107

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> 21 mer PMO antisense sequences

<400> 107

cagcagcagc agcagcagca g 21

<210> 108

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer Mbnl1.F

<400> 108

gctgcccaat accaggtcaa c 21

<210> 109

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer Mbnl1.R

<400> 109

tggtgggaga aatgctgtat gc 22

<210> 110

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer DMD.F

<400> 110

ttagaggagg tgatggagca 20

<210> 111

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer DMD.R

<400> 111

gatactaagg actccatcgc 20

<210> 112

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X is aminocaproic acid

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X is aminocaproic acid

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> X is aminocaproic acid

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> X is aminocaproic acid

<400> 112

Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Tyr Gln Phe Leu Ile Arg Xaa

1 5 10 15

Arg Xaa Arg Xaa Arg

20

<210> 113

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X is aminocaproic acid

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> X is bAla

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> X is aminocaproic acid

<400> 113

Arg Xaa Arg Arg Xaa Arg Arg Phe Gln Ile Leu Tyr Arg Xaa Arg Xaa

1 5 10 15

Arg

<210> 114

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 114

Arg Arg Arg Arg Arg Arg

1 5

Claims (25)

1. A peptide having a total length of 40 amino acid residues or less, the peptide comprising:

two or more cationic domains, each cationic domain comprising at least 4 amino acid residues; and

one or more hydrophobic domains, each hydrophobic domain comprising at least 3 amino acid residues;

wherein the peptide does not comprise artificial amino acid residues.

2. The peptide of claim 1, wherein the peptide does not comprise an amino caproic acid (X) residue, preferably wherein the peptide does not comprise a 6-amino caproic acid residue.

3. The peptide of claim 1 or 2, wherein the peptide consists of natural amino acid residues.

4. The peptide according to any preceding claim, wherein each cationic domain is 4 to 12 amino acid residues, preferably 4 to 7 amino acid residues in length.

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

6. The peptide according to any of claims 1 to 4, 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. The peptide according to any preceding claim, wherein each cationic domain comprises an arginine, histidine, β -alanine, hydroxyproline and/or serine residue, preferably wherein each cationic domain consists of an arginine, histidine, β -alanine, hydroxyproline and/or serine residue.

8. The peptide according to 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%, at least 70% arginine and/or histidine residues.

9. The peptide of any preceding claim, wherein the peptide comprises two cationic domains.

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

11. The peptide of any preceding claim, wherein each hydrophobic domain is 3-6 amino acids in length, preferably each hydrophobic domain is 5 amino acids in length.

12. The peptide of any preceding claim, 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%, 100% hydrophobic amino acids.

13. The peptide of any preceding claim, wherein each hydrophobic domain comprises phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline and glutamine residues; preferably wherein each hydrophobic domain consists of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline and/or glutamine residues.

14. The peptide of any preceding claim, wherein the peptide comprises one hydrophobic domain.

15. The peptide of any preceding claim, wherein the or each hydrophobic domain comprises one of the following sequences: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26), or any combination thereof; preferably, the or each hydrophobic domain consists of one of the following sequences: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26), or any combination thereof.

16. The peptide of any preceding claim, wherein the peptide consists of two cationic domains and one hydrophobic domain, preferably wherein the peptide consists of one hydrophobic core domain and two cationic arm domains flanking it.

17. The peptide of any preceding claim, wherein the peptide consists of one hydrophobic core domain comprising a sequence selected from: YQFLI (SEQ ID NO:20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25) and WWPW (SEQ ID NO:26), each cationic arm domain comprising a sequence selected from: RBRRBRR (SEQ ID NO:1), RBRBRBR (SEQ ID NO:2), RBRR (SEQ ID NO:3), RBRRBR (SEQ ID NO:4), RRBRBR (SEQ ID NO:5), RBRRB (SEQ ID NO:6), BRBR (SEQ ID NO:7), RBHBH (SEQ ID NO:8), HBHBR (SEQ ID NO:9), RBRHBR (SEQ ID NO:10), RBRBBHR (SEQ ID NO:11), RBRRBH (SEQ ID NO:12), HBRRBR (SEQ ID NO:13), HBHBHBH (SEQ ID NO:14), BH (SEQ ID NO:15), BRBSB (SEQ ID NO:16), BRB [ Hyp ] B (SEQ ID NO:17), R [ Hyp ] H [ Hyp ] HB (SEQ ID NO:18), and R [ HyRR ] Hyp ] R (SEQ ID NO: 19).

18. The peptide of any preceding claim, wherein the peptide consists of one of the following sequences: RBRRBRRFQILYRBR (SEQ ID NO:27), RBRRBRRYQFLERRBR (SEQ ID NO:31), RBRRBRRILFQYRRBR (SEQ ID NO:32), RBRRBRFQILYBRBR (SEQ ID NO:35), RBRRBRRFQILYRBHBH (SEQ ID NO:37), RBRRBRRFQILYHBR (SEQ ID NO:38), RBRRBRFQILYRBHBH (SEQ ID NO: 44).

19. A conjugate comprising the peptide of any one of claims 1-18 covalently linked to a therapeutic molecule.

20. The conjugate of claim 19, further comprising a linker, preferably wherein the linker connects the conjugate to the therapeutic molecule.

21. The conjugate of claim 19 or 20, wherein the linker is selected from G, BC, XC, C, GGC, BBC, BXC, XBC, X, XX, B, BB, BX, and XB.

22. The conjugate of any one of claims 19-21, wherein the therapeutic molecule is selected from the group consisting of: a nucleic acid, a peptide nucleic acid, an antisense oligonucleotide (e.g. PNA, PMO), a short interfering RNA, a microrna, a peptide, a cyclic peptide, a protein, a drug or a pharmaceutical product, preferably wherein the therapeutic molecule is an antisense oligonucleotide.

23. Use of a conjugate according to any of claims 19-22 in the manufacture of a medicament.

24. Use according to claim 23, wherein the medicament is for the treatment of a disease of the neuromuscular or musculoskeletal system, preferably a genetic disease of the neuromuscular or musculoskeletal system, preferably a genetic X-linked genetic disease of the neuromuscular or musculoskeletal system.

25. The use of claim 23 or 24, wherein the medicament is for the treatment of DMD.