HK40030260A - Antisense oligonucleotides that bind to exon 51 of human dystrophin pre-mrna - Google Patents

Description

Antisense oligonucleotides that bind to exon 51 of human dystrophin precursor mRNA

Cross Reference to Related Applications

The present application claims priority from GB 1711809.2 filed on 21/7/2017, the content of GB 1711809.2 being incorporated herein by reference.

Technical Field

The present invention relates to therapeutic antisense oligonucleotides that bind to exon 51 of human dystrophin precursor mRNA to induce exon skipping (exon skipping), and conjugates and compositions thereof. The invention also relates to methods and uses of said antisense oligonucleotides for treating muscle disorders, in particular Duchenne Muscular Dystrophy.

Background

Disruption of alternative splicing (alternative splicing) is the basis of many diseases, and the use of antisense oligonucleotides to regulate splicing can be of therapeutic interest. Splice-switching antisense oligonucleotides (SSOs) are emerging treatments for neuromuscular diseases, and several SSOs are currently undergoing clinical trials for conditions such as Spinal Muscular Atrophy (SMA) and Duchenne Muscular Dystrophy (DMD), where antisense-mediated exon skipping can restore the open reading frame and allow synthesis of proteins with partial or full function rather than non-functional proteins.

Duchenne Muscular Dystrophy (DMD) is one of the most common lethal genetic disorders in men worldwide, with an incidence of about 3,600-9,337 live-born men. DMD is caused by a dystrophin deficiency resulting from a mutation in the dystrophin (DMD) gene. The gene encoding the protein contains 79 exons, distributed over more than 200 ten thousand nucleotides of DNA. Any exon mutation that alters the reading frame of an exon or introduces a stop codon, or is characterized by the complete removal of one or more exons from the reading frame or the repetition of one or more exons, may disrupt the production of functional dystrophin, resulting in DMD. One less severe form of muscular dystrophy, Becker Muscular Dystrophy (BMD), has been found to occur in: the mutation (usually the deletion of one or more exons) results in the correct reading frame along the entire dystrophin transcript so that mRNA to protein translation is not prematurely terminated. If the joining of upstream and downstream exons maintains the correct reading frame of the gene in the processing of the mutated dystrophin precursor mRNA, the result is that the mRNA encodes a protein with short internal deletions that retains some activity, resulting in a beck phenotype. Deletion of one or more exons that do not alter the reading frame of dystrophin results in the BMD phenotype, while deletion of exons that cause a shift in the reading frame (frame-shift) will result in DMD (Monaco, Bertelson et al, 1988). Generally, dystrophin mutations include point mutations and exon deletions that alter the reading frame and thus interrupt proper protein translation, resulting in DMD.

One of the most promising therapeutic approaches at present is exon skipping using Antisense Oligonucleotides (AO). Exon skipping can restore the reading frame by removing the mutated exon and/or its flanking exons from the DMD pre-mRNA, enabling the production of truncated but partially functional dystrophin. Most DMD patients have deletion mutations and 20% of them belong to exon 51 skipping.

The first DMD antisense drug eteplirsen (exonds 51) was conditionally approved by the united states Food and Drug Administration (FDA) in 2016, developed to exclude exon 51 from mutant DMD. eteplirsen is an AO modified with phosphorodiamidate morpholino oligomer (morpholino or PMO), which is an antisense chemical substance that has been well established in terms of its safety and efficacy. However, eteplirsen is still controversial, as only weak evidence supports the effectiveness of this drug, both in restoring dystrophin to therapeutically beneficial levels and improving clinical outcomes. The FDA has previously rejected another drug candidate for DMD exon 51 skipping: 2' -O-methyl-phosphorothioate-based AO "drisapersen". Although the therapeutic agent must ensure the highest possible benefit at the lowest risk level, no significant improvement in muscle function is shown after treatment with drisapersen, and its use leads to concerns over safety.

Thus, exon skipping therapies currently face significant challenges because, despite the fact that they have shown significant therapeutic effects in many animal studies, their observed efficacy in patients is very low.

Exon skipping efficiency is mainly dependent on the AO target sequence; however, there is little debate or discussion that the sequences targeted by eteplirsen and drisapersen may not be the optimal choice for exon skipping therapy. Several groups have performed extensive AO screening work to computationally and empirically determine effective AO sequences. However, the exon skipping effectiveness of designed AOs has not been quantitatively and statistically evaluated. Although restoration of dystrophin expression is essential for improving dystrophic (dystrophic) muscle function, the ability of AO to rescue dystrophin expression has not been reported in a sufficient quantitative way in previous AO screening studies. Other studies were highly dependent on RT-PCR from primary DMD myocytes. Notably, the AO sequences of eteplirsen and drisapersen were determined only in this case.

Thus, the effectiveness of exon 51 skipping therapy can be improved by selecting better AO sequences, and by performing more stringent AO screens using more reliable and direct biological measurements, such as dystrophin rescued in DMD, to validate the best antisense oligonucleotides to be employed in clinical trials.

It is an object of one or more aspects of the present invention to address one or more of such problems in the art.

Summary of The Invention

According to a first aspect of the invention there is provided an antisense oligonucleotide capable of binding to exon 51 of a human dystrophin precursor mRNA, wherein the binding of the antisense oligonucleotide occurs entirely within the region between 0 and +89 of the precursor mRNA sequence and wherein the antisense oligonucleotide comprises at least 27 bases.

According to a second aspect of the invention there is provided a conjugate comprising an antisense oligonucleotide according to the first aspect and a carrier, wherein the carrier is conjugated to the antisense oligonucleotide.

According to a third aspect of the invention there is provided a cell loaded with the conjugate of the second aspect.

According to a fourth aspect of the invention, there is provided a pharmaceutical composition comprising an antisense oligonucleotide according to the first aspect and/or a conjugate according to the second aspect and a pharmaceutically acceptable excipient.

According to a fifth aspect of the invention, there is provided a method of treating a muscle disorder in a subject, the method comprising administering to the subject an effective amount of an antisense oligonucleotide capable of binding to exon 51 of a human dystrophin precursor mRNA, wherein the binding of the antisense oligonucleotide occurs entirely within the region between 0 and +89 of the precursor mRNA sequence, and wherein the antisense oligonucleotide comprises at least 27 bases.

According to a sixth aspect of the invention there is provided an antisense oligonucleotide capable of binding to exon 51 of a human dystrophin precursor mRNA for use in the treatment of a muscle disorder in a subject, wherein the binding of the antisense oligonucleotide occurs entirely within the region between 0 and +89 of the precursor mRNA sequence and wherein the antisense oligonucleotide comprises at least 27 bases.

According to a seventh aspect of the invention there is provided a method of increasing expression of human dystrophin in a cell, said method comprising contacting the cell with an effective amount of an antisense oligonucleotide capable of binding to exon 51 of a human dystrophin precursor mRNA, wherein binding of the antisense oligonucleotide occurs entirely within the region between 0 and +89 of the precursor mRNA sequence, and wherein the antisense oligonucleotide comprises at least 27 bases.

Detailed Description

The present inventors have developed a series of antisense oligonucleotides that bind in the early region 0 to +89 of exon 51 of the dystrophin pre-mRNA sequence and that have at least 27 bases longer than usual, each with significant efficiency and effectiveness.

To generate the antisense oligonucleotides, the inventors conducted a study that quantitatively evaluated the effectiveness of morpholino based antisense oligonucleotides for exon 51 skipping using a systematic screening method, including in silico, in vitro and in vivo testing.

The present inventors performed a combinatorial screen using computational analysis to predict exon skipping efficiency of designed antisense oligonucleotide sequences, followed by in vitro testing of morpholino antisense oligonucleotides in immortalized DMD patient-derived muscle cell lines. This study revealed that the initiator region of the human dystrophin exon 51 sequence is a very promising target region for inducing exon skipping, in particular the region 0 to +89 of this sequence. This is in marked contrast to the internal regions targeted by known eteplirsen and drisapersen antisense therapies.

The antisense oligonucleotides identified from this region were then optimized to most efficiently restore dystrophin production in muscle cells. Various factors were studied, including the length of the antisense oligonucleotide. Surprisingly, the inventors found that antisense oligonucleotides bound in this early region were more effective when they were longer than many known antisense oligonucleotide sequences directed against exon 51. In particular, the inventors identified an increasing trend that correlated effectiveness with antisense oligonucleotide lengths from 27 bases and longer. The inventors have demonstrated that only a few base differences means that antisense oligonucleotides have significantly different efficiencies. As demonstrated herein, the effect of a 30-mer antisense oligonucleotide is 1.5 times that of the same sequence of a 25-mer (jump efficiency 42% vs 65%). Without wishing to be bound by theory, this may be because longer sequences may be more specific for the target sequence and less likely to cause off-target effects.

It is demonstrated herein that optimization of these identified antisense oligonucleotide sequences by the present inventors increases the efficiency of exon 51 skipping and the efficiency of dystrophin rescue by up to more than 12-fold and 7-fold, respectively, compared to the industry standard "eteplirsen" sequence. Furthermore, using transgenic mice with the human DMD gene, it was demonstrated that the most potent antisense oligonucleotides identified by these in vitro screens induced statistically significant exon 51 skipping in vivo, which has never been shown for eteplirsen or drisapersen antisense oligonucleotides. Accordingly, the antisense oligonucleotides described herein have been demonstrated to provide effective therapies and treatments for muscle disorders, particularly for the treatment of DMD. These antisense oligonucleotides not only provide an alternative therapy for the medical field, but only one such drug is approved for the market in the medical field. They also provide improved treatment options that are several-fold effective in increasing dystrophin expression. This is expected to provide a viable treatment option for patients suffering from DMD and other muscle disorders, and there is strong evidence to support the effectiveness of this therapy.

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 unless such combination is explicitly excluded or explicitly avoided.

It is to be noted that where an aspect of the invention may relate to a method or use comprising an antisense oligonucleotide, it may also comprise a conjugate or pharmaceutical composition comprising an antisense oligonucleotide as defined herein.

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

Antisense oligonucleotides

The present invention relates to antisense oligonucleotides having a length of at least 27 bases that bind to the region from 0 to +89 of exon 51 of the human dystrophin precursor mRNA, which may be used for the treatment of muscle disorders.

Suitably, an "antisense oligonucleotide" may be referred to herein as an "AO" or an "oligonucleotide (oligo)" or an "oligomer (oligomer)".

Suitably, the antisense oligonucleotide induces skipping of exon 51 of the human dystrophin gene.

Suitably, the antisense oligonucleotide enhances skipping of exon 51 of the human dystrophin gene.

Suitably, the antisense oligonucleotide allows for the expression of functional human dystrophin.

Suitably, the antisense oligonucleotide enhances the expression of functional human dystrophin.

Suitably, the antisense oligonucleotide comprises at least 28 bases, suitably at least 29 bases, suitably at least 30 bases.

Suitably, the antisense oligonucleotide comprises between 27 and 30 bases.

In one embodiment, the antisense oligonucleotide comprises 30 bases.

In one embodiment, the antisense oligonucleotide consists of 30 bases.

Suitably, binding of the antisense oligonucleotide occurs entirely within the region between 0 and +88, between 0 and +87, between 0 and +86, between 0 and +85, between 0 and +84, between 0 and +83, between 0 and +82, between 0 and +81, between 0 and +80, between 0 and +79, or between 0 and +78 of the precursor mRNA sequence.

In one embodiment, the binding of the antisense oligonucleotide occurs entirely within the region between 0 and +78 of the precursor mRNA sequence.

Suitably, the antisense oligonucleotide comprises at least 27 bases of one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide comprises at least 28 bases of one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide comprises at least 29 bases of one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide comprises at least 27 consecutive bases of one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide comprises at least 28 consecutive bases of one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide comprises at least 29 consecutive bases of one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide shares at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide shares between 90% and 100% identity with one of the following sequences: SEQ ID No.1(Ac0), SEQ ID No.2(Ac5), SEQ ID No.3(Ac26), SEQ ID No.4(Ac30) or SEQ ID No.5(Ac 48).

Suitably, the antisense oligonucleotide shares at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide shares between 90% and 100% homology with one of the following sequences: SEQ ID No.1(Ac0), SEQ ID No.2(Ac5), SEQ ID No.3(Ac26), SEQ ID No.4(Ac30) or SEQ ID No.5(Ac 48).

Suitably, the antisense oligonucleotide may comprise a variant antisense oligonucleotide differing by 1, 2, 3,4, 5,6, 7,8, 9 or 10 bases from one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide may comprise a variant antisense oligonucleotide differing by up to 3 bases from one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide may comprise a variant antisense oligonucleotide differing by up to 2 bases from one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide may comprise a variant antisense oligonucleotide differing by a single base from one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide comprises one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide consists of one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

Suitably, the antisense oligonucleotide comprises SEQ ID No.1(Ac0) or SEQ ID No.5(Ac 48).

In one embodiment, the antisense oligonucleotide comprises SEQ ID No.1(Ac 0).

Suitably, the antisense oligonucleotide consists of SEQ ID No.1(Ac0) or SEQ ID No.5(Ac 48).

In one embodiment, the antisense oligonucleotide consists of SEQ ID NO.1(Ac 0).

It is to be understood that the invention may also include aspects directed to each individual antisense oligonucleotide sequence listed in table 3, i.e., antisense oligonucleotides comprising or consisting of any of the sequences listed in table 3. Furthermore, according to the second aspect of the invention, conjugates comprising antisense oligonucleotides as listed in table 3 are envisaged. Furthermore, according to a fourth aspect of the present invention, pharmaceutical compositions are envisaged comprising antisense oligonucleotides as listed in table 3 or conjugates thereof. Furthermore, according to a fifth aspect of the invention, a medical use is envisaged comprising antisense oligonucleotides as listed in table 3 for treating a muscle disorder. Furthermore, according to a sixth aspect, a method of treatment is envisaged comprising antisense oligonucleotides as listed in table 3. Furthermore, according to a seventh aspect, a method of increasing expression of human dystrophin in a cell is contemplated, the method comprising antisense oligonucleotides as listed in table 3.

Suitably, the antisense oligonucleotide is synthetic, rather than natural.

Suitably, antisense oligonucleotides may be prepared conventionally by well-known solid phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Pat. No.4,458,066. Any other means for such synthesis known in the art may additionally or alternatively be employed.

Suitably, the antisense oligonucleotide is an antisense oligonucleotide analogue.

Suitably, the terms "oligonucleotide analogue" and "nucleotide analogue" refer to any modified synthetic analogue of an oligonucleotide or nucleotide, respectively, as is known in the art.

Suitable examples of oligonucleotide analogs include Peptide Nucleic Acids (PNAs), morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, acylphosphonate oligonucleotides, and phosphoramidite oligonucleotides.

Suitably, the antisense oligonucleotide comprises morpholino subunits. Thus, suitably, the antisense oligonucleotide is a morpholino antisense oligonucleotide.

Suitably, the antisense oligonucleotide comprises morpholino subunits linked together by phosphorus-containing linkages (links). Thus, suitably, the antisense oligonucleotide is a phosphoramidite or phosphorodiamidate morpholino antisense oligonucleotide.

The term "morpholino antisense oligonucleotide" or "PMO" (phosphoramidite or phosphorodiamidate morpholino oligonucleotides) refers to an antisense oligonucleotide analog comprising morpholino subunit structures wherein (i) these structures are linked together by a phosphorus-containing bond, suitably 1 to 3 atoms in length, suitably 2 atoms in length, and suitably uncharged or cationic, linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring has a purine or pyrimidine base pairing moiety which is operative to bind to a base in a polynucleotide by base-specific hydrogen bonding.

Suitably, the antisense oligonucleotide comprises a phosphorus-containing intersubunit linkage linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit.

Suitably, the antisense oligonucleotide comprises a phosphorus-containing intersubunit linkage according to the following structure (I):

wherein:

y1 is-O-, -S-, -NH-or-CH 2-;

z is O or S;

pj is a purine or pyrimidine base-pairing moiety that binds effectively to a base in a polynucleotide via base-specific hydrogen bonding; and

x is fluorine, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, amino, optionally substituted alkylamino, or optionally substituted heterocyclyl.

Optionally, alterations can be made to the intersubunit linkages, provided that the alterations do not interfere with binding or activity. For example, the oxygen attached to the phosphorus may be replaced by sulfur (thiophosphordiamide). The 5' oxygen may be substituted with amino or amino substituted with lower alkyl. The pendant nitrogen attached to the phosphorus may be unsubstituted, mono-substituted or di-substituted with an (optionally substituted) lower alkyl group.

Suitably, the synthesis, structure and binding characteristics of morpholino oligonucleotides are described in detail in the following: U.S. patent nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063 and 5,506,337, and PCT application No. PCT/US 07/11435.

Binding of antisense oligonucleotides

The present invention relates to antisense oligonucleotides capable of binding to the 0 to +89 region of exon 51 of human dystrophin precursor mRNA.

By "capable of binding" is meant that the antisense oligonucleotide comprises a sequence capable of binding to the region of human dystrophin precursor mRNA.

Suitably, the antisense oligonucleotide is complementary to a sequence in said region of human dystrophin precursor mRNA.

Suitably, the antisense oligonucleotide comprises a sequence complementary to a sequence in said region of human dystrophin precursor mRNA.

When a sufficient number of corresponding positions in each molecule are occupied by nucleotides, the antisense oligonucleotide and the sequence within the region 0 to +89 of exon 51 of the human dystrophin pre-mRNA are complementary to each other, and both can hydrogen bond to each other and thereby cause exon skipping, suitably of exon 51. Thus, "hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or pairing such that stable and specific binding occurs between the antisense oligonucleotide and a sequence within the 0 to +89 region of exon 51 of the human dystrophin pre-mRNA. Suitably, the antisense oligonucleotide is sufficiently hybridizable and/or complementary to a sequence within the region 0 to +89 of exon 51 of the human dystrophin pre-mRNA to induce exon skipping, suitably exon 51 skipping.

Suitably, the antisense oligonucleotide may not be 100% complementary to a sequence within the region 0 to +89 of exon 51 of human dystrophin precursor mRNA. However, suitably, the antisense oligonucleotides are sufficiently complementary to avoid non-specific binding.

Suitably, the antisense oligonucleotide is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% complementary to a sequence within the 0 to +89 region of exon 51 of human dystrophin pre-mRNA.

It will be appreciated that in order for the antisense oligonucleotide to be able to bind, it is not required that the entire length of the antisense oligonucleotide binds to human dystrophin precursor mRNA. It is understood that a portion of the antisense oligonucleotide may not bind to human dystrophin precursor mRNA, e.g., the 5 'or 3' end of the antisense oligonucleotide. However, according to the first aspect, the portion of the antisense oligonucleotide that binds to human dystrophin pre-mRNA must fall within the region 0 to +89 of exon 51.

Thus, suitably, the antisense oligonucleotide may hybridise to a sequence within the region 0 to +89 of exon 51 of the human dystrophin precursor mRNA. Suitably, the antisense oligonucleotide can hybridize sufficiently to a sequence within the region 0 to +89 of exon 51 of human dystrophin pre-mRNA to cause exon skipping of exon 51.

Human dystrophin

The present invention relates to therapeutic antisense oligonucleotides for treating muscle disorders, in particular dystrophin disorders such as DMD.

Dystrophin is a rod-like cytoplasmic protein and is an important part of the protein complex that connects the cytoskeleton of muscle fibers to the surrounding extracellular matrix through the cell membrane.

A DMD gene encoding dystrophin is one of the longest known human genes, covering 2.3 megabases at the Xp21 locus (0.08% of the human genome.) the primary transcript in muscle is about 2,100 kilobases long and takes 16 hours to transcribe, the mature mRNA is 14.0 kilobases long.79 exon muscle transcripts encode 3685 amino acid residue protein dystrophin comprises an actin binding domain and a central rod domain (rod domain). this large central domain consists of 24 spectrin-like triple helix elements of about 109 amino acids which have homology to α -actin and spectrin.the repeat sequence is usually interrupted by four proline rich non-repeat segments also called hinge regions. each repeat sequence is encoded by two exons, typically by one intron between amino acids 47 and 48 of the first part of α -helix 2. the interrupt is seen at a different position in the dystrophin, i.e. the cysteine rich repeat region is also found in cysteine rich regions (cysteine rich region 15).

Under normal conditions, the amino terminus of dystrophin binds to F-actin, while the carboxy terminus binds to the dystrophin-associated protein complex (DAPC) at the sarcolemma. DAPC contains dystrophins, sarcoglycan, integrins and lacuna, and mutations in any of these components cause autosomal inherited muscular dystrophy. Normal skeletal muscle tissue contains only a small amount of dystrophin (about 0.002% of total muscle protein), but its deficiency (or abnormal expression) results in the development of severe and currently incurable symptoms that are most easily characterized by several abnormal intracellular signaling pathways, ultimately leading to significant myofiber necrosis and progressive muscle weakness and fatigability. In the absence of dystrophin, DAPC becomes unstable, which results in decreased levels of member proteins and, in turn, progressive fiber damage and membrane leakage. In various forms of muscular dystrophy, such as Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD), myocytes produce altered and functionally deficient forms of dystrophin, or no dystrophin at all, mainly due to mutations in the gene sequences that result in incorrect splicing. The predominant expression of defective dystrophins, or the complete absence of dystrophin or dystrophin-like protein, results in a rapid progression of muscle degeneration.

Dystrophin patients typically contain an out-of-frame mutation (e.g., a deletion, insertion, or splice site mutation) in the mRNA encoding the dystrophin protein, resulting in a shift in the reading frame or premature termination of the translation process, and thus no functional dystrophin is produced in most muscle fibers.

Suitably, the antisense oligonucleotide triggers exon skipping to restore the reading frame of the dystrophin mRNA. Suitably, the antisense oligonucleotide triggers exon skipping of exon 51 to restore the reading frame of the dystrophin mRNA. Suitably, restoration of the reading frame restores the production of partially functional dystrophin.

Suitably, the partially functional dystrophin is a truncated dystrophin.

Suitably, the truncated dystrophin is the same dystrophin produced in a patient suffering from a less severe muscle disorder BMD.

Muscular disorders

The present invention relates to the use of therapeutic antisense oligonucleotides in the treatment of muscle disorders.

Suitably, the muscle disorder is selected from any muscle disorder resulting from a gene mutation.

Suitably, the muscle disorder is selected from any muscle disorder resulting from a genetic mutation in a gene associated with muscle function.

Suitably, the muscle disorder is selected from any muscle disorder resulting from a genetic mutation in a human dystrophin gene.

Suitably, the muscle disorder is selected from any muscular dystrophy disorder.

Suitably, the muscle disorder is selected from duchenne muscular dystrophy, becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy.

Suitably, the muscle disorder is Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD).

In one embodiment, the muscle disorder is DMD.

Carrier and conjugate

The invention also relates to conjugates of antisense oligonucleotides with carriers.

Suitably, the vector may comprise any molecule operable to transport the antisense oligonucleotide into a target cell, suitably a muscle cell.

Suitable carriers may include: peptides, small molecule chemicals, polymers, nanoparticles, lipids, liposomes, exosomes, etc.

Suitably, the carrier is a peptide. Peptides may be selected, for example, from viral proteins such as VP22 (derived from herpes virus envelope proteins), snake venom proteins such as CyLOP-1 (derived from crotamin), cell adhesion glycoproteins such as pVEC (derived from murine vascular endothelial cadherin), cell-penetrating peptides (pennetratin, antennapedia homeodomain), Tat (human immunodeficiency virus transactivation regulatory protein), or reverse Tat.

Suitably, the peptide is a cell penetrating peptide.

Suitably, the peptide is an arginine-rich cell penetrating peptide.

The use of arginine-rich peptide vectors is particularly useful. Certain arginine-based peptide vectors have been demonstrated to be highly effective in delivering antisense compounds to primary cells, including muscle cells (Marshall, Oda et al, 2007; Jearawiriyapa isarn, Moulton et al, 2008; Wu, Moulton et al, 2008). Furthermore, arginine peptide vectors exhibit enhanced ability to alter splicing of several gene transcripts when conjugated to antisense oligonucleotides compared to other peptides (Marshall, Oda et al, 2007).

Suitably, the arginine-rich cell penetrating peptide may be selected from, for example, those described in WO2015075747, WO2013030569, WO2009147368, US20120289457 or US 20160237426.

In one embodiment, the arginine-rich cell penetrating peptide is selected from those described in WO2013030569 or WO 2009147368.

Suitably, the vector has the ability to induce cellular penetration of the antisense oligonucleotide in at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the cells of a particular cell culture population. Suitably, the vector has the ability to induce cellular penetration of the antisense oligonucleotide in at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the myocytes of a myocyte culture.

Suitably, the conjugation of the vector to the antisense oligonucleotide may be at any position suitable for forming a covalent bond between the vector and the antisense oligonucleotide or between the linker moiety and the antisense oligonucleotide. For example, conjugation of the carrier may be at the 3' end of the antisense oligonucleotide. Alternatively, conjugation of the vector to the antisense oligonucleotide may be located at the 5' end of the oligonucleotide. Alternatively, the carrier may be conjugated to the antisense oligonucleotide through any intersubunit linkage.

Suitably, the carrier is covalently coupled at its N-terminal or C-terminal residue to the 3 'or 5' end of the antisense oligonucleotide.

Suitably, the carrier is coupled at its C-terminal residue to the 5' end of the antisense oligonucleotide.

Optionally, where the antisense oligonucleotide comprises phosphorus-containing intersubunit linkages and the carrier is a peptide, the peptide may be conjugated to the antisense oligonucleotide by covalent bonding to the phosphorus of a terminal linker.

Alternatively, when the carrier is a peptide and the antisense oligonucleotide is morpholino, the peptide can be conjugated to the nitrogen atom of the morpholino group at the 3' end of the oligomer.

Optionally, the linker moiety may comprise one or more of an optionally substituted piperazinyl moiety, β alanine, glycine, proline, and/or a 6-aminocaproic acid residue, in any combination.

Alternatively, the carrier may be conjugated directly to the antisense oligonucleotide without a linker moiety.

Suitably, the conjugate may also comprise a homing moiety.

Suitably, the homing moiety is selective for a selected mammalian tissue, i.e. the same tissue targeted by the antisense oligonucleotide. Suitably, the homing portion is selective for muscle tissue.

Suitably, the homing moiety is a homing peptide.

In, for example, "Effective dystrin Restoration by a Novel Muscle-localization peptide-Morpholino Conjugate in dystrin-discovery mdx Mice" Gao et al, MolTher.2014 Jul; suitable homing peptides are disclosed in 22(7) 1333-1341.

Suitably, the carrier peptide and homing peptide may be formed as a chimeric fusion protein.

Suitably, the conjugate may comprise a chimeric peptide formed from a cell penetrating peptide and a muscle specific homing peptide.

Optionally, the conjugate may be in the form of: a carrier peptide-homing peptide-antisense oligonucleotide, or the following forms: homing peptide-carrier peptide-antisense oligonucleotide.

Suitably, the antisense oligonucleotide may be conjugated to a carrier that enhances the solubility of the antisense oligonucleotide, suitably solubility in aqueous media. Suitably, in addition to a vector operable to transport the antisense oligonucleotide, a solubility-enhancing vector may be conjugated to the antisense oligonucleotide. Suitably, the solubility enhancing vector and the antisense oligonucleotide transporting vector may be formed as a chimeric fusion protein.

Suitable carriers for enhancing the solubility of antisense oligonucleotides are polymers such as polyethylene glycol or triethylene glycol.

Pharmaceutically acceptable excipients

The invention also relates to pharmaceutical compositions comprising the antisense oligonucleotides or conjugates thereof of the invention, and further comprising one or more pharmaceutically acceptable excipients.

Suitably, the Pharmaceutical compositions are prepared in a manner known in the art using pharmaceutically inert inorganic and/or organic excipients (as described in Remingtons Pharmaceutical Sciences, Mack pub. co., Easton, PA (1985)). The term "pharmaceutically acceptable" refers to molecules and compositions that are physiologically tolerable and do not typically produce allergic or similar untoward reactions when administered to a patient.

Suitably, the pharmaceutical composition may be formulated as a pill, tablet, coated tablet, hard gelatin capsule, soft gelatin capsule and/or suppository, solution and/or syrup, injectable solution, microcapsule, implant and/or rod (rod), and the like.

In one embodiment, the pharmaceutical composition may be formulated as an injection solution.

Suitably, the pharmaceutically acceptable excipients used in the preparation of pills, tablets, coated tablets and hard gelatine capsules may be selected from any of the following: lactose, corn starch and/or derivatives thereof, talc, stearic acid and/or its salts, and the like.

Suitably, the pharmaceutically acceptable excipients for the preparation of soft gelatin capsules and/or suppositories may be selected from fats, waxes, semi-solid and liquid polyols, natural and/or hardened oils and the like.

Suitably, the pharmaceutically acceptable excipients for the preparation of solutions and/or syrups may be selected from water, sucrose, invert sugar, glucose, polyols and the like.

Suitably, the pharmaceutically acceptable excipients used for the preparation of injectable solutions may be selected from water, saline, alcohols, glycerol, polyols, vegetable oils and the like.

Suitably, the pharmaceutically acceptable excipients used for the preparation of microcapsules, implants and/or rods may be selected from mixed polymers such as glycolic acid and lactic acid and the like.

In addition, pharmaceutical compositions may comprise liposomal formulations, which are described in N.Weiner (Drug Deverop Inpharm 15(1989)1523), "Liposome Dermatics" (Springer Verlag 1992) and Hayashi (Gene therapy 3(1996) 878).

Optionally, the pharmaceutical composition may comprise two or more different antisense oligonucleotides or conjugates thereof. Optionally, the pharmaceutical composition may further comprise one or more antisense oligonucleotides or conjugates thereof targeting different exons, suitably different exons of human dystrophin precursor mRNA. Optionally, one or more additional antisense oligonucleotides or conjugates thereof may target an exon adjacent to exon 51 of the human dystrophin precursor mRNA, e.g., exon 50 or exon 52. Suitably, one or more antisense oligonucleotides or conjugates thereof targeting different exons of human dystrophin precursor mRNA may be manipulated with the antisense oligonucleotides of the invention to restore the reading frame of the dystrophin mRNA.

Optionally, the pharmaceutical composition may further comprise one or more antisense oligonucleotides or conjugates thereof targeting different genes. For example, one or more additional antisense oligonucleotides or conjugates thereof can be targeted to a myostatin. Such Dual targeting is described in "Dual exon scraping in myostatin and dysphin for Duchenne molecular dynamics" Kemaladewi et al, BMC Med genomics.2011 Apr 20; 4:36.

Optionally, the one or more additional antisense oligonucleotides may be linked together and/or to the antisense oligonucleotide of the first aspect.

Optionally, the antisense oligonucleotide and/or conjugate may be present in the pharmaceutical composition as a physiologically tolerable salt. Suitably, the physiologically tolerable salt retains the desired biological activity of the antisense oligonucleotide and/or conjugate thereof and does not impart undesired toxic effects. For antisense oligonucleotides, suitable examples of pharmaceutically acceptable salts include (a) salts formed with the following cations: such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, and the like; (b) acid addition salts formed with the following inorganic acids: such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; (c) salts with organic acids having: such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

The one or more therapeutically active ingredients may be selected from, for example, corticosteroids, upregulators of dystrophin (utrophin), TGF- β inhibitors, and myostatin inhibitors.

Suitably, the pharmaceutical composition may comprise, in addition to the active ingredient and the excipients, additives such as fillers, extenders, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, dyes, flavourings or fragrances, thickeners, diluents or buffer substances, and additionally solvents and/or solubilizers and/or agents for achieving a slow-release effect, and also salts for varying the osmotic pressure, coating agents and/or antioxidants. Suitable additives may include Tris-HCl, acetate, phosphate, Tween 80, polysorbate 80, ascorbic acid, sodium metabisulfite, thimerosal, benzyl alcohol, lactose, mannitol, and the like.

Administration of

The present invention relates to therapeutic antisense oligonucleotides and pharmaceutical compositions comprising the same for administration to a subject.

Suitably, the antisense oligonucleotide and/or the pharmaceutical composition may be for topical, enteral or parenteral administration.

Suitably, the antisense oligonucleotides and/or pharmaceutical compositions can be used for oral, transdermal, intravenous, intrathecal, intramuscular, subcutaneous, intranasal, transmucosal, and the like administration.

In one embodiment, the antisense oligonucleotide and/or the pharmaceutical composition is for intramuscular administration.

In one embodiment, the antisense oligonucleotide and/or the pharmaceutical composition is for intramuscular administration by injection.

An "effective amount" or "therapeutically effective amount" refers to an amount of antisense oligonucleotide administered to a subject as a single dose or as part of a series of doses effective to produce a desired physiological response or therapeutic effect in the subject.

Suitably, the desired physiological response comprises an increased expression of a relatively functional or biologically active form of dystrophin, suitably in a muscle tissue or cell containing a defective dystrophin or not containing a dystrophin.

Suitably, the desired therapeutic effect includes ameliorating a symptom or pathology of the muscle disorder, reducing the progression of a symptom or pathology of the muscle disorder, and slowing the onset of a symptom or pathology of the muscle disorder. Examples of such symptoms include fatigue, mental retardation, muscle weakness, difficulty in motor skills (e.g., running, one-foot jumping, jumping), frequent falls, and difficulty walking.

Suitably, the antisense oligonucleotide or conjugate thereof is administered at a dose ranging from about 0.0001mg to about 100mg per kilogram body weight per day.

Suitably, the antisense oligonucleotide or conjugate thereof is administered daily, once every 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or once every 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months.

Suitably, the dose and frequency of administration may be determined by a physician as needed to maintain the desired expression of functional dystrophin.

Suitably, the antisense oligonucleotides or conjugates thereof may be administered separately as 2, 3,4, 5,6 or more sub-doses, optionally in unit dosage form, at appropriate intervals per day.

Test subject

The invention also relates to the treatment of muscle disorders by administering to a subject in need thereof a therapeutically effective amount of an antisense oligonucleotide or a conjugate thereof.

Suitably, the subject suffers from a muscle disorder, as defined above.

Suitably, the subject is a mammal. Suitably, the subject is a human.

Suitably, the subject may be male or female. Suitably, however, the subject is male.

Suitably, the subject is of any age. Suitably, however, the subject is between 1 month and 50 years of age, suitably between 1 and 30 years of age, suitably between 2 and 27 years of age, suitably between 4 and 25 years of age.

Increased exon skipping and dystrophin expression

The present invention relates to therapeutic antisense oligonucleotides that restore functional dystrophin expression by inducing exon skipping in human dystrophin precursor mRNA for the treatment of muscle disorders.

Suitably, a "functional" dystrophin protein refers to a dystrophin protein having sufficient biological activity to reduce the progressive degradation of muscle tissue that would otherwise be characteristic of a muscular dystrophy when compared to a defective dystrophin protein present in a subject having a muscle disorder such as DMD.

Suitably, the functional dystrophin may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the biological activity of a wild-type dystrophin in vitro or in vivo.

Suitably, the functional dystrophin has at least 10% to 20% of the biological activity of a wild-type dystrophin in vitro or in vivo.

Suitably, the activity of dystrophin in muscle culture in vitro can be measured in terms of myotube size, myofibrillar tissue, contractile activity and spontaneous aggregation of acetylcholine receptors (see, e.g., Brown et al, Journal of cell science.112:209-216, 1999).

Animal models are also a valuable resource for studying disease pathogenesis and provide a means to test dystrophin-related activity. The two most widely used animal models for the DMD study are mdx mice and Golden Retriever Muscular Dystrophy (GRMD) dogs, both of which are dystrophin negative (see, e.g., Collins & Morgan, Int J Exp Pathol84: 165-. These and other animal models can be used to measure the functional activity of various dystrophins.

Suitably, "exon skipping" refers to the process of removing the entire exon or a portion thereof from a particular pre-processed RNA (precursor RNA) and thereby excluding its presence in mature RNA translated into protein.

Suitably, the portion of the protein originally encoded by the skipped exon is not present in the expressed form of the protein.

Thus, suitably, exon skipping results in a truncated, but still functional, form of the protein as defined above.

Suitably, the skipped exon is an exon from the human dystrophin gene, which sequence may contain mutations or other alterations that would otherwise cause aberrant splicing.

Suitably, the exon that is skipped is exon 51 of the dystrophin gene.

Suitably, the antisense oligonucleotide is operable to induce exon skipping in the dystrophin precursor mRNA.

Suitably, the antisense oligonucleotide is operable to induce exon skipping of exon 51 in the dystrophin precursor mRNA.

Suitably, the antisense oligonucleotide is operable to increase expression of a functional form of dystrophin in muscle tissue, and is operable to increase muscle function in muscle tissue.

Suitably, the antisense oligonucleotide is operable to increase muscle function by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to the muscle function of a subject having a muscle disorder, such as DMD, who does not receive the antisense oligonucleotide.

Suitably, the antisense oligonucleotide is operable to increase the percentage of muscle fibers expressing functional dystrophin in about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the muscle fibers as compared to a subject having a muscle disorder, such as DMD, who does not receive the antisense oligonucleotide.

Suitably, the antisense oligonucleotide is operable to induce expression of a functional form of dystrophin to a level of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the expression of dystrophin in wild type cells and/or in a subject.

Suitably, the antisense oligonucleotide is operable to induce expression of a functional form of dystrophin to a level of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20% of the expression of dystrophin in the wild type cell and/or the subject.

Suitably, the antisense oligonucleotide is operable to induce expression of a functional form of dystrophin to a level of at least 10%, 15% or 20% of the expression of dystrophin in the wild type cell and/or the subject.

Suitably, the antisense oligonucleotide is operable to induce exon 51 skipping in dystrophin precursor mRNA to a level of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

Suitably, the antisense oligonucleotide is operable to induce exon 51 skipping in dystrophin precursor mRNA to a level of at least 60%, 70%, 80%, 90% or 100%.

Suitably, the antisense oligonucleotide is operable to induce exon 51 skipping in dystrophin pre-mRNA to a level of between 60% and 80%.

An "increased" or "enhanced" amount can include an increase of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more, of the amount that would result if the antisense oligonucleotide compound (absent the agent) or a control compound were administered under the same conditions.

Suitably, an "increased" or "enhanced" amount is a statistically significant amount.

Brief Description of Drawings

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

FIG. 1: in vitro screening of Antisense Oligonucleotides (AO) and AO analogues of eteplirsen (aete) and drisapersen (ardri) at 10 μ M in DMD skeletal muscle cells with exon 52 deletion of immortalized clones (KM571) is shown. Differentiated myotubes were harvested on day 5 post-transfection, (a) efficiency of exon 51 skipping as measured by one-step RT-PCR. A representative image is shown. M, 100bp marker; blank, no RNA template. (B) Efficiency of induction of truncated dystrophin as measured by quantitative western blot with anti-dystrophin C-terminal antibody. Use healthCalibration curves for kang 8220 cells calculate salvaged dystrophin levels. Data represent mean ± SD from 3-4 independent experiments. P<0.01, compared to aEte;andcompared to aDri; p § p<0.01, compared to all AOs in (a) and Ac0 in (B).

FIG. 2: shown is the time course analysis of dystrophin exon 51 skipping and protein in DMD-KM571 cell line transfected with 5 μ M Ac0, Ac48 and the AO analogue of eteplirsen and the AO analogue of drisapersen with exon 52 deletion. Samples were collected at 2 and 11 days post-transfection, (a) RT-PCR analysis of exon 51 skipping. M, 100bp marker; r, repeating sample numbering; blank, no RNA template. (B) Induced dystrophin was quantified by western blotting with an anti-dystrophin C-terminal antibody. Representative replicate samples from 3 independent experiments are shown.

FIG. 3: dose-dependent effects of Ac0, Ac48, as well as AO analogs of eteplirsen and AO analogs of drisapren in immortalized DMD skeletal muscle cells as measured by one-step RT-PCR and quantitative western blot are shown. DMD skeletal muscle cells were transfected with 1 μ M, 3 μ M, and 10 μ M AO and harvested on day 5 post-transfection. (A) And (B) show the efficiency of exon 51 skipping and the expression level of rescued dystrophin, respectively, in DMD myocytes with exon 52 deletion mutation (ID KM 571). The efficacy of skipping exon 51 and rescuing dystrophin expression in DMD myocytes with exon 48-50 deletion mutations (ID6594) is shown in (C) and (D), respectively. Data represent mean ± SD from 3-7 independent experiments performed in the KM571 cell line and from 3-4 independent experiments performed in the 6594 cell line. P<0.05，**p<0.01, compared to aEte;andcompared to Ac 48; p § p<0.05，§§p<0.01, NS, compared to aDri (at the same concentration), was not significant compared to Ac0 at the next dose; ns, no significance compared to Ac0 at 10 μ M. (E) Dose responsiveness to AO was analyzed by regression models. The statistical significance of the regression equations for jumping and generating dystrophin, respectively, is p<0.008 and p<0.014. The graph represents values for exon skipping or dystrophin levels predicted in a regression analysis. Regression slopes and 95% Confidence Intervals (CI) for individual AOs are shown.

FIG. 4: immunocytochemistry in immortalized DMD patient-derived skeletal muscle cells with exon 52 deletion mutation (ID KM571) and exon 48-50 deletion mutations (ID6594) is shown. Cells at day 5 after transfection with 10 μ M Ac0, Ac48, and eteplirsen analog (aEte) were stained with anti-dystrophin C-terminal antibody. Grey lines indicate dystrophin positive myotubes. White points indicate nuclei counterstained with DAPI. Representative false positive myotubes due to their contraction or detachment from the culture plate. Representative images from 3 independent experiments are shown. Scale bar: 100 μm.

FIG. 5: length optimization for Ac0 morpholino antisense oligonucleotides is shown. Immortalized DMD myocytes were transfected with Ac0 morpholinos comprising lengths of 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, and 30-mer. Representative images and quantification of exon 51 skipping induced by Ac0 morpholinos at 1 μ M (a and B) and 3 μ M (C and D) in DMD myocytes with exon 52 deletion (KM571) are shown, as presented by RT-PCR. (E-H) shows the results for immortalized DMD cells with exon 48-50 deletions. Data from 3 independent experiments are shown.

FIG. 6: exon 51 skipping efficiencies induced by Ac0, Ac48, the AO analog of eteplirsen (aEte) and the AO analog of drisapersen (aDri) in primary DMD and healthy skeletal muscle cells are shown. Differentiated myotubes were transfected with 10 μ M of Ac0, Ac48, and eteplirsen and drisapersen analogs and then harvested after 3 days. The prepro-mutations in the exon 45-50 deletion mutations (ID4546) (A and B) or exon 49-50 deletion mutations (ID4555) (C and D) are shownExon 51 skipping efficiency in primary DMD cells as well as primary healthy muscle cells (E and F), as demonstrated by one-step RT-PCR. Data represent mean ± SD from at least triplicate wells under each condition. M, 100bp marker. P<0.05 and x p<0.01, compared to Ac 48;compared to aEte; p § p<0.01, compared to aDri.

FIG. 7: the in vivo efficacy of 30-mer Ac0 antisense morpholino oligonucleotides in the hDMD/no Dmd mouse model (hDMD/Dmd-null mouse model) is shown. Exon skipping efficacy was analyzed by RT-PCR with tibialis anterior muscle 2 weeks after intramuscular injection of Ac0 morpholino or eteplirsen analog aEte (50 μ g in 30 μ L saline). (A) Densitometric analysis of exon 51 skipping as presented by a microchip-based capillary electrophoresis system. (B) Mean percentage of exon 51 skipping efficiency (mean ± SE). N in each group is 7. M, a marker; NT, untreated muscle; UM, upper marker dye; LM, bottom marker dye.

FIG. 8: the in vivo efficacy of 30-mer Ac48 antisense morpholino oligonucleotides in the hDMD/no Dmd mouse model is shown. Exon skipping efficacy was analyzed by RT-PCR with tibialis anterior, 2 weeks after intramuscular injection of Ac48 morpholino or drisapersen analog aldri (50 μ g in 30 μ L saline). Densitometric analysis of exon 51 skipping as presented by a microchip-based capillary electrophoresis system. M, a marker; NT, untreated muscle; UM, upper marker dye; LM, bottom marker dye.

Throughout the description and claims of this specification, the words "comprise/comprises/containing" and "comprise/including/containing" and variations thereof 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 encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, 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 design and computer simulation screening of AO.

The AO of the 30-mer and 25-mer of 413 targeted exons 51 were designed and analyzed using our recently developed AO prediction algorithm (see Table 3). Table 3 shows in the column from left to right: exon numbering, distance from acceptor splice sites, AO sequences (5 'to 3'), predicted skipping%, and ranking in the screen. The left AO is a 30-mer, and the right AO is a 25-mer. Based on the predicted exon skipping efficiencies, 8 AOs spaced at least 4 bases apart were selected for in vitro screening (table 2). The specificity of The target sequences of The selected AO, eteplirsen and drisapersen was analyzed using The University of California, Santa Cruz Genome Browser (http:// Genome. ucsc. edu/index. html) and confirmed that these AO sequences did not theoretically bind to any non-target RNA sequences with 100% identity.

1.2 antisense morpholinos.

All antisense sequences, including the AO analog of eteplirsen and the AO analog of drisapren, were synthesized by Gene Tools (Philomanth, OR) via morpholino chemistry.

1.3 cells.

As previously described, immortalized human skeletal muscle cells derived from three healthy subjects (ID 8220, CHQ and KM155) and two DMD patients having exon 52 deletion mutation (ID KM571) and exon 48-50 deletion mutation (ID6594), respectively, in the DMD gene were generated by transduction with a human telomerase expression vector and a cyclin-dependent kinase 4 expression vector in the Institute of pathology human cell immortalization platform. Three immortalized healthy muscle cell lines were characterized 33 and the clone line 8220 showing the highest expression of dystrophin was selected as a positive control to prevent overestimation of the expression of dystrophin salvaged in immortalized DMD cells. Primary Skeletal Muscle cells derived from DMD patients and healthy subjects with exon 45-50(ID4546) and exon 49-50(ID 4555) deletion mutations were prepared from BioBank of Skelet Muscle, Nerve Tissue, DNA and Cell Lines.

1.4 AO transfection.

To mimic as closely as possible the in vivo effects of AO-mediated exon skipping therapy, in vitro screening was performed using mature, differentiated myotubes expressing sufficient levels of DMD mRNA. Growth Medium (GM): cells were cultured in proliferation conditions in DMEM/F12 with skeletal muscle supplement mixture (Promocell, Heidelberg, Germany), 20% fetal bovine serum (Life Technologies, Waltham, Mass.), and antibiotics (50 units penicillin and 50. mu.g/ml streptomycin, Life Technologies, Waltham, Mass.). Immortalized and primary DMD skeletal muscle cells at 1.7x10, respectively⁴/cm²And 2.2x10⁴/cm²Seeded in type I collagen coated 12-well or 24-well culture plates. Two days after inoculation, at about 80% -90% confluence, the cells were treated with Differentiation Medium (DM): DMEM/F12 with 2% horse serum (GE Healthcare, Chicago, IL), 1x ITS solution (Sigma, st. After 3 days in DM, the cells were incubated with a medium containing 6. mu.M Endo-porter transfection reagent (GeneTools, Philomath, OR) 1. mu.M, 3. mu.M, 5. mu.M OR 10. mu.M AO transfected cells (1 mM concentrated AO was incubated at 65 ℃ for 10min just before dilution with DM). Two days after AO transfection, the DM containing AO was replaced with regular DM. Cells were harvested at day 2, day 5, or day 11 (day 5, day 8, or day 14 post-differentiation) after AO transfection.

1.5 mice.

Animal studies were approved by the University of Alberta (University of Alberta) Committee for Animal Care and Use Committee (Animal Care and Use Committee), the National Center for Children's medicine (Children's National medical Center), and the National Center for Neurology and psychiatry (National Center of Neurology and Psychiatry; NCNP). Male and female Dmd exon 52-deficient mdx 5242 and wild type mice (Jackson Laboratory, Bar Harbor, ME) with a C57BL/6J background were prepared at 4-8 weeks of age. The Dmd mutation was confirmed in the affected mice by genotyping with PCR. A transgenic mouse model with a human DMD gene and lacking the mouse Dmd gene (hDMD/null Dmd mice) was generated by crossing male hDMD mice (Jackson Laboratory, BarHarbor, ME) with female Dmd-free mice (Dmd-null mouse).

1.6 intramuscular injection.

As previously described, 5 μ g or 20 μ g of morpholino Ac0, Ac48, eteplirsen or drisapersen in mouse form in 40 μ L saline was injected intramuscularly into the Tibialis Anterior (TA) muscle under inhalation anesthesia with isoflurane. 43 mu.g of Ac0 morpholino and eteplirsen analogs in 30. mu.L saline were injected into TA muscle of hDMD/Dmd-free mice. All muscle samples were harvested 2 weeks after intramuscular injection.

1.7 exon skipping analysis by RT-PCR.

Total RNA was extracted with Trizol (Invitrogen, Waltham, MA) as previously described. RT-PCR was performed on 200ng and 320ng total RNA in immortalized and primary skeletal muscle cells using the SuperScriptIII One-Step RT-PCR system (Invitrogen, Waltham, Mass.) and 0.2. mu.M forward and reverse primers (see Table 1), respectively, to detect dystrophin mRNA. Primers were designed using Primer3Plus software and their specificity was confirmed in healthy human skeletal muscle cells (line 8220). RT-PCR conditions were as follows: 50 ℃ for 5 minutes; at 94 ℃ for 2 minutes; 35 cycles of 94 ℃ for 15 seconds, 60 ℃ for 30 seconds and 68 ℃ for 35 seconds; and 68 ℃ for 5 minutes. The PCR products were separated on a 1.5% agarose Gel and visualized by SYBR Safe DNA Gel Stain (Invitrogen, Waltham, MA). The efficiency of exon 51 skipping was calculated using ImageJ software (NIH) or MCE-202 MultiNA System (Shimadzu, Kyoto, Japan) using the following formulaThe unknown top band above the native band (possibly from an accidental splicing event) was excluded from quantification of skipping efficiency. The sequence of the PCR product was confirmed using Big Dye terminator v3.1(Applied Biosystems, Waltham, Mass.). GAPDH or 18S ribosomal RNA was used as internal control.

Table 1:

1.8 Western blot

Cells were harvested with RIPA buffer (Thermo Scientific, Waltham, MA) containing clomplete, Mini, EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland) and then homogenized 10 times through a 21-gauge needle, supernatant was prepared as a sample by centrifugation at 14,000g for 15min at 4 ℃ as previously described to prepare protein from muscle tissue using Bradford assay, supernatant was diluted 100-fold with distilled water protein in sample buffer was heated at 70 ℃ for 10min, the sample buffer contained 10% SDS, 70mM Tris-HCl, pH 6.8, 5mM EDTA, 20% glycerol, 0.004% bromophenol blue and 5% 2-mercaptoethanol, protein blots were then run as previously described using 12 μ g and 30 μ g of cell and tissue for sodium dodecyl sulfate-amide gel electrophoresis, western blot, 32,43,44 were incubated with 12 μ g and 30 μ g of anti-myoglobin protein from cells and tissues in a blocking solution as well as anti-myoglobin-protein-staining with anti-rat-protein-staining antibody (rabbit-rat-protein-expressing protein) as described by staining with anti-rat-protein-rabbit protein-expressing protein-staining (rabbit-rat-protein-expressing protein, rat-antibody, rabbit protein, rat-expressing protein, rat-expressing protein, rat-expressing protein, rat-expressing, protein, rat-expressing, protein, rat-expressing, protein, rat-expressing, protein, rat-expressing, protein, and cell, rat-expressing, and cell-expressing, rat.

1.9 immunocytochemistry.

Cells were fixed with 4% paraformaldehyde for 5min at room temperature. After washing with PBS containing 0.01% Triton X-100, cells were blocked with 10% goat serum (Life Technologies, Waltham, MA) in PBS with 0.05% Triton X-100 for 20min and then incubated with anti-dystrophin C-terminal antibody (ab15278) or rod domain antibody (DYS1) diluted 1:50 in blocking solution overnight at 4 ℃. Dystrophin signals were detected with either Alexa 488-conjugated or Alexa 594-conjugated secondary antibody (1: 500). Desmin (desmin) (1:80, Abcam, Cambridge, United Kingdom) and Rapid MyHC (1:30, Leica Biosystems, Buffalo Grove, IL) were tested to confirm myogenic differentiation of cells. Cells were stored in a SlowFade Gold antibody mountain with DAPI at 4 ℃ until analysis (Invitrogen, Waltham, Mass.).

1.10 immunohistochemistry.

Dystrophin-positive muscle fibers on frozen sections of TA muscle from untreated and treated mdx52 mice were detected with ab15278 antibody as previously described. The signal intensity of dystrophin in treated mice was compared to that of wild type mice using a neutral density filter (Eclipse TE 2000-U, Nikon, Tokyo, Japan).

1.11 statistical analysis

To determine the significance of the efficiency of exon skipping and dystrophin rescue, data sets were prepared from at least three independent experiments performed in immortalized cells, triplicate wells of primary cells, and 3-7 mice. Statistical analysis between the AO treatment groups was performed by one-way ANOVA followed by post hoc Tukey-Kramer multiple comparison test. A simple linear regression analysis was performed to determine dose responsiveness to AO. Statistical significance for all analyses was set at p < 0.05.

2. Results

2.1 in silico screening of AO for exon 51 skipping.

We designed a total of 413 AOs: 204 and 209 AOs with 30-mer and 25-mer lengths, respectively, covering all possible target sites in DMD exon 51 (see table 3). Our exon skipping efficiency algorithm ("InSilico Screening Based on Predictive exons as a Design Tool for Exon skiping Oligonucleotides in Duchenne Muscumular dynamics" Echigoya et al, PLOSONE March 2015) predicts that the highest efficiency of exon 51 skipping for 30-mer AO is 80.5% and 41.2% for 25-mer AO in the initial 5' site of exon 51. In silico screening showed that the exon skipping efficiency of the region of 30 bases targeted by eteplirsen was very low (23.7%), which ranked 92 th among all 413 candidate AOs tested. Note that the target site of the 30-mer eteplirsen fully encompasses the drisapersen target site.

2.2 characterization of immortalized cloned healthy skeletal muscle cell line and DMD skeletal muscle cell line

Important issues for preclinical testing with primary DMD myocytes include low myocyte purity and insufficient amounts of mutant dystrophin mRNA, which pose a problem when attempting to test AO potency. To overcome these obstacles, we generated immortalized cloned skeletal muscle cells from 3 healthy subjects and 2 DMD patients (ID KM571 and 6594, respectively) with exon 52(ex52) and ex48-50 deletion (del.) mutations. All immortalized skeletal muscle cell lines tested expressed readily detectable dystrophin mRNA from day 3 after induced differentiation. To avoid overestimating the dystrophin levels induced by AO in DMD cells, we selected the cell line with the highest level of dystrophin (as determined by western blot) among the three immortalized healthy skeletal muscle cell lines (ID 8220) as positive control. Dystrophin expression in 8220 cell lines was also confirmed by immunocytochemistry.

2.3 in vitro screening of exon 51 skipping AO

Based on in silico screening results, we selected 8 30-mer AO sequences, including high-ranked and low-ranked sequences, spaced at least 4 bases apart from each other for in vitro screening (table 2). In this study, all tested AOs, including eteplirsen and drisapersen sequences, were synthesized using morpholino chemistry, which has been demonstrated to be well tolerated in patients participating in clinical trials. Here, we refer to control morpholino oligonucleotides (produced by Gene Tools) having the same sequence as eteplirsen and drisapersen as "eteplirsen analogs" and "drisapersen analogs". In RT-PCR, 10 μ M of 5 our morpholinoaos (Ac0, Ac5, Ac26, Ac30, and Ac48) showed significantly higher skipping efficiencies in immortalized DMD skeletal muscle cells with exon 52 deletion compared to eteplirsen analogs and drisapersen analogs (fig. 1A). Of the tested AOs, Ac0 in particular had the highest jump efficiency, up to 72%, which was 4-fold and 25-fold higher than the eteplirsen and drisapersen analogues, respectively. In western blot, Ac0 also induced the highest level of dystrophin, up to 16% of the level of healthy control cell line, followed by Ac48, which was 13% (fig. 1B). Interestingly, the two AOs with the highest jump efficiencies under test, Ac0 and Ac48, were not the best AOs predicted by the algorithm.

Table 2:

ac, acceptor splice site (acceptor splice site). Non-upper case nucleotides represent intron sequences.^aRanking in 25-mer AO.

2.4 time course analysis of Ac0, Ac48 and AO analogs of eteplirsen and of drisapersen

The sustained effect of 5 μ M of Ac0, Ac48 and eteplirsen and drisapersen analogs was examined in KM571 cells with exon 52 deletion. The advantages of the oligonucleotides Ac0 and Ac48 of the invention compared to the AO analogue of eteplirsen and the AO analogue of drisapersen in terms of exon skipping efficiency and dystrophin rescue were observed at day 2 and day 11 post-transfection (figure 2).

Dose-dependent effects of 5 Ac0, Ac48 and eteplirsen and drisapersen analogs

RT-PCR showed that the highest concentration of Ac0 of 10 μ M induced up to 74% and 64% skipping of exon 51 in DMD KM571 cells (exon 52 deletion) and 6594 cells (exon 48-50 deletion), respectively, which was significantly higher than eteplirsen and drisapersen analogs (fig. 3). At the lowest concentration (1 μ M), Ac0 showed 12-fold and 10-fold higher exon skipping efficiencies in KM571 and 6594 cells, respectively, compared to eteplirsen analogues. Interestingly, even a 1 μ M concentration of Ac0 induced a higher level of exon 51 skipping than the 10 μ M eteplirsen analog (24% efficiency vs 15% in KM571 and 24% efficiency vs 21% in 6594, respectively). Quantitative western blot revealed that 10 μ M Ac0 rescued dystrophin expression in DMD cell lines up to 21% of the level of healthy cell lines (fig. 3A to D). Even at 1 μ M, the relative ratios of Ac0 to eteplirsen analogues exhibited 7.1-fold and 3.3-fold higher efficiencies in the production of dystrophin in KM571 and 6594 cell lines, respectively. Ac0 at 1 μ M was able to produce salvaged dystrophin at levels above or comparable to the eteplirsen analogue at 10 μ M (10% vs 6% in KM571 and 11% vs 10% in 6594, respectively), confirming that Ac0 was more than 10-fold more effective in producing dystrophin than eteplirsen analogue in terms of concentration (concentration-wise). In DMD muscle cell lines, the drisapersen analog did not work efficiently for both exon skipping and dystrophin production. Exon skipping responses to Ac0 and Ac48 occurred in a dose-dependent manner to a greater extent than both the eteplirsen and drisapersen analogs (fig. 3A and C). In both DMD cell lines, the dose responsiveness of Ac0 was also higher for dystrophin production than the control analogs (fig. 3E).

2.6 immunocytochemical evaluation of dystrophin rescue

Immunocytochemistry revealed that in DMD skeletal muscle cell line with exon 52 and exon 48-50 deletion mutations, 10 μ M of Ac0 and Ac48 produced more dystrophin-positive myotubes and showed stronger signal intensity compared to eteplirsen analogues (figure 4).

2.7 optimization of Ac0 morpholino length

In silico simulations and in vitro screens revealed that the initial 5' region of exon 51 between 0 and +89 is an important region affecting exon 51 skipping. To optimize the sequence length of Ac0 targeting this region, we compared the hopping efficiencies of Ac0 morpholinos of different lengths (25-mer to 30-mer), where the nucleotides at the 5' site were systematically removed one at a time (see table 2). In vitro testing of immortalized DMD myocytes treated with 1 μ M of these AOs showed that 25-mer to 30-mer Ac0 morpholino produced efficient exon skipping (> 20%) (fig. 5), an effect not observed for the same dose of eteplirsen and drisapersen analogs (fig. 3). However, the efficiency of exon skipping increases with increasing AO length. Statistically significant efficacy of 30-mer Ac0 compared to shorter Ac0 morpholinos, even those with AOs only 1 or 2 bases shorter, was demonstrated at doses of 1 μ M and 3 μ M in both cell lines.

2.8 Effect of Ac0, Ac48 and eteplirsen and drisapersen analogs on Primary DMD patient-derived skeletal muscle cells

We also tested AO in primary DMD skeletal muscle cells with either exon 45-50 deletion mutation (ID4546) or exon 49-50 deletion mutation (ID4555) to verify whether the superior potency of 30-mer Ac0 is consistent for other muscle cell types and deletion mutation patterns. RT-PCR showed that Ac0 achieved significantly higher exon skipping efficiencies compared to eteplirsen or drisapersen analogs in two primary DMD myocytes (fig. 6A to D): efficiencies as high as 5-fold and 7-fold, respectively, were observed compared to eteplirsen and drisapersen analogs. Significant efficiency of Ac 0-mediated exon 51 skipping was also demonstrated in primary healthy skeletal muscle cells (fig. 6E and F). Interestingly, with increasing efficiency of exon 51 skipping, spontaneous exon 52 skipping without disruption of the reading frame was observed in primary healthy muscle cells and DMD muscle cells as well as in immortalized DMD muscle cell lines with exon 48-50 deletions (6594).

2.10 in vivo efficacy of Ac0 morpholino and eteplirsen analogs in hDMD/Dmd-free mice

One major obstacle in the development of exon skipping therapies is the inability to always test human-specific AO in an appropriate animal model. This limits the assessment of the in vivo efficacy of an AO designed for a patient. Here, we developed a new mouse model with the full-length human DMD gene but lacking the entire mouse Dmd gene (hDMD/none Dmd) to test the in vivo efficacy of human AO. This mouse model was used to avoid cross-reactivity between human and mouse sequences (note that conventional mdx mice still have mouse dystrophin mRNA, which can cross-react with human targeted AO) and was obtained by cross breeding between hDMD mice 34 and Dmd-free mice 35. Ac0, Ac48, eteplirsen analog, or drisapersen analog were injected into the TA muscle of these mice, and the effectiveness of exon 51 skipping in vivo was analyzed 2 weeks after injection. The results show that the exon skipping efficiency of mice treated with Ac0 was significantly higher compared to eteplirsen analogues (fig. 7). A visible band of exon 51 skipping was found in Ac 48-treated mice with a mean exon skipping efficiency of 1.11% (± 0.46%, SE). On the other hand, no band that could quantify exon 51 skipping was observed in mice treated with drisapersen analogs (fig. 8).

Sequence of

Table 3:

Claims (24)

1. an antisense oligonucleotide capable of binding to exon 51 of a human dystrophin pre-mRNA, wherein binding of said antisense oligonucleotide occurs entirely within the region between 0 and +89 of said pre-mRNA sequence, and wherein said antisense oligonucleotide comprises at least 27 bases.

2. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises at least 28 bases, at least 29 bases, or at least 30 bases.

3. The antisense oligonucleotide of claim 1 or 2, wherein the antisense oligonucleotide consists of 30 bases.

4. The antisense oligonucleotide according to any preceding claim, wherein binding of the antisense oligonucleotide occurs entirely within the region between 0 and +88, between 0 and +87, between 0 and +86, between 0 and +85, between 0 and +84, between 0 and +83, between 0 and +82, between 0 and +81, between 0 and +80, between 0 and +79, or between 0 and +78 of exon 51 of the human dystrophin pre-mRNA.

5. The antisense oligonucleotide according to any preceding claim, wherein the binding of the antisense oligonucleotide occurs entirely within the region between 0 and +78 of the pre-mRNA sequence.

6. The antisense oligonucleotide according to any preceding claim, wherein the antisense oligonucleotide is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% complementary to a sequence of exon 51 of human dystrophin pre-mRNA that falls within said region.

7. An antisense oligonucleotide according to any preceding claim, wherein the antisense oligonucleotide is capable of hybridising to a sequence of exon 51 of human dystrophin pre-mRNA that falls within said region.

8. The antisense oligonucleotide according to any preceding claim, wherein the antisense oligonucleotide comprises at least 27 bases of one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

9. The antisense oligonucleotide according to any preceding claim, wherein the antisense oligonucleotide shares at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity and/or homology with one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

10. The antisense oligonucleotide according to any preceding claim, wherein the antisense oligonucleotide comprises one of the following sequences: SEQ ID NO.1(Ac0), SEQ ID NO.2(Ac5), SEQ ID NO.3(Ac26), SEQ ID NO.4(Ac30) or SEQ ID NO.5(Ac 48).

11. The antisense oligonucleotide of any preceding claim, wherein the antisense oligonucleotide comprises SEQ ID No.1(Ac0) or SEQ ID No.5(Ac 48).

12. A conjugate comprising the antisense oligonucleotide of any one of claims 1-11 and a carrier, wherein the carrier is conjugated to the antisense oligonucleotide.

13. The conjugate of claim 12, wherein the carrier is operable to transport the antisense oligonucleotide into a target cell.

14. The conjugate of claim 12 or 13, wherein the carrier is selected from a peptide, a small molecule chemical, a polymer, a nanoparticle, a lipid, a liposome, or an exosome.

15. The conjugate of any one of claims 12-14, wherein the carrier is a cell penetrating peptide.

16. The conjugate of any one of claims 12-15, wherein the carrier is an arginine-rich cell penetrating peptide.

17. A cell loaded with the conjugate of any one of claims 13-16.

18. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1-11 and/or a conjugate according to any one of claims 12-16, and a pharmaceutically acceptable excipient.

19. An antisense oligonucleotide capable of binding to exon 51 of a human dystrophin pre-mRNA for use in the treatment of a muscle disorder in a subject, wherein binding of said antisense oligonucleotide occurs entirely within a region between 0 and +89 of said pre-mRNA sequence, and wherein said antisense oligonucleotide comprises at least 27 bases.

20. The antisense oligonucleotide for use according to claim 19, wherein said antisense oligonucleotide comprises the features defined in any one of claims 1 to 11.

21. The antisense oligonucleotide for use according to claim 19 or 20, wherein the muscle disorder is a disorder caused by a genetic mutation in a gene associated with muscle function.

22. The antisense oligonucleotide for use according to any one of claims 19-21, wherein the muscle disorder is duchenne muscular dystrophy or becker muscular dystrophy.

23. A method of increasing expression of human dystrophin in a cell, said method comprising contacting said cell with an effective amount of an antisense oligonucleotide capable of binding to exon 51 of a human dystrophin precursor mRNA, wherein binding of said antisense oligonucleotide occurs entirely within the region between 0 and +89 of said precursor mRNA sequence, and wherein said antisense oligonucleotide comprises at least 27 bases.

24. The method of claim 23, wherein the antisense oligonucleotide comprises the features defined in any one of claims 1-11.