WO2009101399A1 - Treatment of muscular dystrophy using peptide nucleic acid ( pna) - Google Patents

Treatment of muscular dystrophy using peptide nucleic acid ( pna) Download PDF

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WO2009101399A1
WO2009101399A1 PCT/GB2009/000379 GB2009000379W WO2009101399A1 WO 2009101399 A1 WO2009101399 A1 WO 2009101399A1 GB 2009000379 W GB2009000379 W GB 2009000379W WO 2009101399 A1 WO2009101399 A1 WO 2009101399A1
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pna
exon
method according
sequence
gene
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PCT/GB2009/000379
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Matthew Wood
Haifang Yin
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Isis Innovation Limited
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/11Antisense
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/31Chemical structure of the backbone
    • C12N2310/318Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181Peptide nucleic acid, PNA
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    • C12N2320/00Applications; Uses
    • C12N2320/30Special therapeutic applications
    • C12N2320/33Alteration of splicing

Abstract

A method of correcting expression of a gene in a human cell having a muscle disease or muscular dystrophy phenotype, wherein without correction the gene fails to express functional protein due to one or more mutations, said method comprising delivering to the cell a peptide nucleic acid (PNA) comprising a sequence capable of targeting a sequence responsible for exon skipping in the mutated pre-mRNA at an exon to be skipped or included, wherein expression is corrected by the PNA inducing exon skipping or inclusion and thereby correcting the expression of said mutated pre-mRNA.

Description

TREATMENT

Field of the Invention

The present invention relates to methods of correcting expression of a gene in a human cell.

Background of the Invention

Muscular dystrophy (MD) refers to a group of more than 30 inherited diseases that cause muscle weakness and muscle loss. Some forms of MD appear in infancy or childhood, while others may not appear until middle age or later. The different muscular dystrophies vary in who they affect and the symptoms. All forms of MD grow worse as the person's muscles get weaker. Some types of MD affect heart muscles, other types affect involuntary muscles and other organs. Most people with MD eventually lose the ability to walk. Muscles, primarily voluntary muscles, become progressively weaker. The most common types of MD are due to a genetic deficiency of the muscle protein dystrophin.

Duchenne muscular dystrophy (DMD) is an X-Hnked muscle disorder mainly caused by nonsense or frame-shift mutations in the dystrophin gene resulting in a dystrophin deficiency in muscle cells. DMD has an incidence of about 1 :3500 newborn males. DMD patients suffer from severe, progressive muscle wasting and most will stop walking by the age of 12 years with about 90% not surviving beyond the age of 20. The milder allelic form of the disease, Becker muscular dystrophy (BMD), is usually caused by in-frame deletions resulting in expression of a shortened, but partially functional protein. BMD patients have milder symptoms and longer life expectancies when compared to DMD patients. The severity of the disease varies, and boys and men with Becker dystrophy have a longer life expectancy than those with Duchenne. The severity and rate of progression of Becker dystrophy depends on how much dystrophin is made and how well it functions in the muscles.

Myotonic dystrophy, also known as Steinert's disease, is the most common adult form of muscular dystrophy. Its name underscores an unusual symptom found only in this form of dystrophy — myotonia, which is similar to a spasm or stiffening of muscles after use. The disease causes muscle weakness and affects the central nervous system, heart, gastrointestinal tract, eyes (causing cataracts) and endocrine (hormone-producing) glands. Although muscle weakness progresses slowly, this symptom can vary greatly, even among members of a single family. Most often muscle weakness does not hamper daily living for many years after symptoms first occur.

Most often, the onset of Limb-Girdle muscular dystrophy (LGMD) is in adolescence or early adulthood, hi the most common forms, the disease causes progressive weakness that starts in the hips and moves to the shoulders. The weakness progresses to include the arms and legs. Within 20 years of onset, walking is difficult, if not impossible.

The word facioscapulohumeral refers to the muscles that move the face, scapula (shoulder blade) and humerus (upper arm bone). Common early signs of

Facioscapulohumeral muscular dystrophy (FSH) are a forward sloping of the shoulders as well as difficulty raising the arms over the head and closing the eyes. Progression is slow, with long periods of stability interspersed with shorter periods of rapid muscle deterioration and increased weakness. The muscles of the face and shoulder area are the first affected. The weakness spreads to the muscles of the abdomen, feet, upper arms, pelvic area and lower arms, usually in that order. The disease ranges in severity from very mild to considerably disabling, with impairment of walking, chewing, swallowing and speaking. About half of those with the disorder retain the ability to walk throughout their lives. Congenital muscular dystrophy (CMD) is a group of diseases in which symptoms can be noted from birth. One form that has been clearly described is Fukuyama congenital muscular dystrophy. This disorder involves severe weakness of the facial and limb muscles and a generalized lack of muscle tone, usually appearing before 9 months. Joint contractures are common. Brain abnormalities are also present, and most children have severe mental and speech problems. Seizures are often part of the disease, and medications are prescribed for these. Physical therapy is needed to minimize the contractures. Another form of congenital dystrophy seems to be related to a deficiency or malfunction of the protein merosin, which normally lies outside muscle cells and links them to the surrounding tissue. The disorder is similar to Fukuyama dystrophy, with muscle weakness evident at birth or in the first few months of life, severe and early contractures and often joint deformities. This disorder has been tentatively named congenital muscular dystrophy with merosin deficiency. Oculpharyngeal, meaning eye and throat, muscular dystrophy (OPMD) usually starts with drooping of the eyelids, most often in the 40s or 50s. This is followed by other signs of eye and facial muscle weakness, as well as by difficulty in swallowing. The later stages of this slowly progressive disease may include weakness in the pelvic and shoulder muscles. Swallowing problems can lead to choking and recurrent pneumonia.

Distal muscular dystrophy (DD) is actually a group of rare muscle diseases, which have in common weakness and wasting of the distal muscles of the forearms, hands, lower legs and feet. A type of distal dystrophy called Welander is inherited in an autosomal dominant pattern and affects the hands first. Another type, known as

Markesbery-Griggs, is autosomal dominant in its inheritance and affects the front of the lower legs first, as does Nonaka dystrophy. Miyoshi dystrophy, caused by a gene defect on chromosome 2, is autosomal recessive and affects the back of the lower legs first. Emery-Dreifuss dystrophy (EDMD) is a rare form of muscular dystrophy. Muscle weakness and wasting generally start in the shoulders, upper arms and lower legs. Weakness may later spread to involve the muscles of the chest and pelvic area. Contractures appear early in the disease, usually involving the ankle and elbow. Unlike other forms of muscular dystrophy, contractures in Emery-Dreifuss dystrophy often appear before the person experiences significant muscle weakness. Physical therapy is beneficial in minimizing the contractures. Life-threatening heart problems are a common part of this disorder. The heart problems are electrical and can be treated with a cardiac pacemaker. These problems can even occur in females who do not have the disease but are carriers, so sisters and mothers of boys with Emery-Dreifuss should be examined. The skeletal muscle weakness is less severe than it is in some other dystrophies, such as Duchenne. Emery-Dreifuss dystrophy is caused by a defect in the gene on the X chromosome that codes for the protein emerin.

Summary of the Invention

The present invention is based on the discovery that peptide nucleic acid (PNA) oligonucleotides are capable of inducing efficient exon skipping and thereby restoring the reading frame and expression of mutated mRNA, such as mutated dystrophin mRNA. The PNAs of the invention are therefore useful in antisense therapy of diseases that are caused by mutations affecting the expression of an mRNA/protein. In particular, the PNAs and the method of the invention are useful in antisense therapy of muscular diseases or dystrophies such as Duchenne muscular dystrophy (DMD) and myotonic dystrophy.

Accordingly, the invention provides a method of correcting expression of a gene in a human cell having a muscle disease or muscular dystrophy phenotype, wherein without correction the gene fails to express functional protein due to one or more mutations, said method comprising delivering to the cell a peptide nucleic acid (PNA) comprising a sequence capable of targeting a sequence responsible for exon skipping in the mutated pre-mRNA at an exon to be skipped or included, wherein expression is corrected by the PNA inducing exon skipping or inclusion and thereby correcting the expression of said mutated pre-mRNA

The present invention further provides a method of treating a muscular disease or dystrophy in a subject comprising administering a PNA as defined herein to said subject and thereby restoring the expression of mutated mRNA in said subject.

Brief Description of the Figures

Figure 1 demonstrates PNA-induced exon skipping H2K mdx cells. A. RT-PCR results for the cell transfection with neutral PNA. Total RNA from treated and untreated H2K mdx cells was amplified by nested RT-PCR across exon 20 to 26 of the dystrophin transcript. Full length transcript represented by 901bp product with the shorter product of 688bp (arrow), corresponding to the removal of exon 23, only present in cells transfected with neutral PNA. B. Sequence analysis confirming precise splicing of exon 22 to 24.

Figure 2 demonstrates restoration of dystrophin expression in tibialis anterior (TA) muscles of /nctc mice two weeks after intramuscular injection of PNA. A. Immunofluorescent staining for dystrophin in mdx TA muscles of different ages following 5μg injection of PNA. Nuclei were counterstained with 4', 6-diamidino-2- phenylindole. (a) normal C57BL6 control (b) saline injection (c, f, g) 3- week, 2-month and 6-month old respectively (d-e) 2-month old mdx mice injected with 2'0me PS AOs and PMOs, respectively (scale bar = 200μm). B. Quantitative evaluation of total dystrophin-positive fibres in TA muscles of various aged mdx mice, two weeks following a single intramuscular injection of 5μg PNA. All groups showed significant difference compared with age-matched mdx control (ANOVA * P<0.05, ** P<0.001). Figure 3 illustrates dystrophin expression in mdx TA following injection of increasing doses of PNA. A. Immunofluorescent dystrophin staining in TA muscles of 2-month old mdx mice two weeks after intramuscular injection, (a) untreated age- matched mdx mouse control; (b, c, d) 5μg lOμg and 20μg intramuscular injections of PNA respectively (scale bar = 200μm). B. Quantitative evaluation of total dystrophin- positive fibres in TA muscles 23 following various PNA dosages. 20μg injection showed significant increase compared with 5μg and lOμg injections of PNA (* P<0.05).

Figure 4 is a western blot analysis. Total protein was extracted from TA muscles of 2-month old mdx mice two weeks after a single intramuscular injection with 5μg PNA. No visible difference in the size of dystrophins between muscle treated with PNA and muscle from the normal C57BL6 mouse.

Figures 5 and 6 show PNA expression studies.

Figure 7 shows expression levels of different lengths of PNAs.

Description of Sequences

SEQ ID NO: 1 is the human dystrophin gene. The sequence was obtained from the following web link: http://vega.sanger.ac.uk/Homo_sapiens/transview?transcript=OTTHUMT00000056182

SEQ ED NO: 2 is a PNA sequence of the invention. SEQ ED NO: 3 to 125 are exon/intron boundary sequences that can be targeted by PNA sequences.

SEQ ED NO: 126 to 129 are RT-PCR primer sequences.

SEQ ID NO: 130 to 208 are mouse dystrophin gene exon sequence with partial flanking intron sequences. The full sequences can be accessed at the following web link: http : //vega. Sanger. ac.uk/Mus_musculus/transview?transcript=OTTM U ST00000043357

SEQ DD NO: 209 to 214 are sequences of oligos used in the Examples.

Detailed Description of the Invention The contents of the priority application (US Application No. 61/028,056 filed on

12 February 2008), including the sequences disclosed in the application, is incorporated by reference. DNA sequences are transcribed into pre-mRNAs which contain coding regions (exons) and generally also contain intervening non-coding regions (introns). Introns are removed from pre-mRNAs in a precise process called cis-splicing. Splicing takes place as a coordinated interaction of several small nuclear ribonucleoprotein particles (snRNPs) and many protein factors that assemble to form an enzymatic complex known as the spliceosome. Specific motifs in the pre-mRNA that are involved in the splicing process include splice site acceptors, splice site donors, exonic splicing enhancers (ESEs) and exon splicing silencers.

Pre-mRNA can be subject to various splicing events. Alternative splicing can result in several different mRNAs being capable of being produced from the same pre- mRNA. Alternative splicing can also occur through a mutation in the pre-mRNA, for instance generating an additional splice acceptor and/or splice donor sequence (cryptic sequences). Restructuring the exons in the pre-mRNA, by inducing exon skipping or inclusion, represents a means of correcting the expression from pre-mRNA exhibiting undesirable splicing or expression in an individual. Exon restructuring can be used to promote the production of a functional protein in a cell. Restructuring can lead to the generation of a coding region for a functional protein. This can be used to restore an open reading frame that was lost as a result of a mutation.

Antisense oligonucleotides (AOs) can be used to alter pre-mRNA processing via the targeted blockage of motifs involved in splicing. Hybridisation of antisense oligonucletides to splice site motifs prevents normal spliceosome assembly and results in the failure of the splicing machinery to recognize and include the target exon(s) in the mature gene transcript. This approach can be applied to diseases caused by aberrant splicing, or where alteration of normal splicing would abrogate the disease-causing mutation. This includes: (i) blockage of cryptic splice sites, (ii) exon removal or inclusion to alter isoform expression, and (iii) removal of exons to either eliminate a nonsense mutation or restore the reading frame around a genomic deletion.

An example of a gene in which the reading frame may be restored is the Duchenne muscular dystrophy (DMD) gene. The dystrophin protein is encoded by a plurality of exons over a range of at least 2.6 Mb. The human and mouse dystrophin gene sequences are represented in Figures 1 and 2 respectively. DMD is mainly caused by nonsense and frame-shift mutations in the dystrophin gene resulting in a deficiency in the expression of dystrophin protein. The dystrophin protein consists of two essential functional domains connected by a central rod domain. Dystrophin links the cytoskeleton to the extracellular matrix and is thought to be required to maintain muscle fibre stability during contraction. Mutations that disrupt the open reading frame result in prematurely truncated proteins unable to fulfill their bridge function. Ultimately this leads to muscle fibre damage and the continuous loss of muscle fibres, replacement of muscle tissue by fat and fibrotic tissue, impaired muscle function, and eventually the severe phenotype observed for DMD patients. In contrast, mutations that maintain the open reading frame allow for the generation of internally deleted, but partially functional, dystrophins. These mutations are associated with Becker muscular dystrophy (BMD), a much milder disease when compared with DMD. Patients generally remain ambulant until later in life and have near normal life expectancies.

The inventors have discovered that PNAs capable of targeting splice site motifs in mutated dystrophin mRNA can efficiently induce exon skipping. It is possible to target an exon which flanks an out-of frame deletion or duplication so that the reading frame can be restored and dystrophin production allowed. The removal of the mutated exon in this way allows shortened but functional (BMD-like) amounts of dystrophin protein to be produced. As a result, a severe DMD phenotype can be converted into a milder BMD phenotype.

Dystrophia myotonica (myotonic dystrophy) type 1 (DMl), the most common muscular dystrophy affecting adults, is caused by expansion of a CTG repeat in the 3' untranslated region of the gene encoding the DM protein kinase (DMPK). Evidence suggests that DMl is not caused by abnormal expression of DMPK protein, but rather that it involves a toxic gain of function by mutant DMPK transcripts that contain an expanded CUG repeat (CUGexp). The transcripts containing a CUGexp tract elicit abnormal regulation of alternative splicing, or spliceopathy. The splicing defect, which selectively affects a specific group of pre-mRNAs, is thought to result from reduced activity of splicing factors in the muscleblind (MBNL) family, increased levels of CUG- binding protein 1 , or both. Myotonia in mouse models of DM appears to result from abnormal inclusion of exon 7a in the ClC-I mRNA. Inclusion of exon 7a causes frame shift and introduction of a premature termination codon in the ClC-I mRNA. A therapeutic strategy for myotonic dystrophy is therefore to repress the inclusion of exon 7a in the mouse ClC-I mRNA, or the corresponding exon in human ClC-I mRNA. Just as targeted blockage of consensus splice sites and ESEs promotes exon exclusion, the blockage of exonic or intronic splicing silencers, or the introduction of splicing enhancer sequences, can enhance exon inclusion. This offers the ability to enhance expression of alternatively spliced 'weak' exons to induce the most functionally preferable isoform. In spinal muscular atrophy (SMA), mutations in the survival motor neuron (SMNl) gene are responsible for a degenerative disease that presents as childhood muscle weakness and, in the more serious forms, can cause fatal respiratory failure. The severity of the disease is modified by the production of SMN protein encoded by the paralogous gene, SMN2. Although SMN2 is nearly identical to SMNl, a silent C to T mutation in exon 7 abrogates an ESE site, weakening recognition of the upstream 3' splice site and resulting in the majority of SMN2 transcripts lacking exon 7. As this SMNΔ7 isoform is unstable, and at best, only partially functional, the level of full-length SMN protein is an important modifier of patient disease severity. Antisense technology can therefore be used to promote exon 7 inclusion in the SMN2 transcript.

The current invention is directed to an antisense-based system, specifically peptide nucleic acids (PNAs), for inducing the skipping or inclusion of one or more exons in a pre-mRNA, thereby resulting in the expression of functional protein. Accordingly, the invention provides a method of correcting expression of a gene in a human cell having a muscle disease or muscular dystrophy phenotype, wherein without correction the gene fails to express functional protein due to one or more mutations, said method comprising delivering to the cell a peptide nucleic acid (PNA) comprising a sequence capable of targeting a sequence responsible for exon skipping in the mutated pre-mRNA at an exon to be skipped or included, wherein expression is corrected by the PNA inducing exon skipping or inclusion and thereby correcting the expression of said mutated pre-mRNA

The muscle disease or muscular dystrophy may be any muscular disease or dystrophy that is caused by the aberrant expression of a protein. The aberrant protein expression may be as a result of one or more nonsense or frame-shift mutations. The aberrant protein expression may be the result of a mutation that weakens a splice site resulting in the inclusion of an undesirable exon. Alternatively, the mutation may introduce a cryptic splice site resulting in the splicing of an exon that is desired to be included for protein function. Examples of muscle diseases include Duchenne muscular dystrophy (DMD), myotonic dystrophy, spinal muscular atrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculpharyngeal muscular dystrophy, distal muscular dystrophy and Emery-Dreifiiss dystrophy. Where the disease is DMD, the gene for which expression may be corrected is the dystrophin gene. Where the disease is myotonic dystrophy, the gene for which expression may be corrected is the muscle specific chloride channel (ClC-I) gene. Where is disease is spinal muscular atrophy, the gene for which expression may be corrected is the SMN2 gene. The human cell may be any human cell in which the gene for which expression is to be corrected has one or more mutations. The one or more mutations may be nonsense or frame-shift mutations. The one or more mutations may strengthen a cryptic splice site or may weaken a splice site. The cell has a muscle disease/dystrophy phenotype, i.e. does not produce a particular functional protein. The cell may be taken from a human patient that has a muscle disease/dystrophy. For example, the cell may be taken from a human patient that has DMD, myotonic dystrophy or spinal muscular atrophy.

In one aspect of the invention, the PNA is used for the purpose of inducing exon skipping. Alternatively, the PNA is used for inducing exon inclusion. In one aspect of the invention, more than one exon is induced to be skipped at a time. This is desirable because there are often numerous exons in a gene that could potentially be mutated resulting in muscle disease/dystrophy. By targeting the skipping of more than one exon it is possible to remove a larger region of potentially mutant mRNA resulting in the expression of a shortened but functional protein. Any number of exons may be skipped provided that the remaining exons are sufficient to result in the expression of suitably functional protein. Accordingly, 1, 2, 3, 4, 5, 6, 7, 8 or more exons may be skipped.

The method of the invention results in the induction of expression of functional protein. Typically, the amount of functional protein expressed in the cell is at least 10% of the amount of functional protein expressed in a cell in which the gene is not mutated. Preferably, the amount of functional protein expressed in a cell is at least 15%, 20%, 25%, 30%, or more preferably, at least 40% or 50% of the amount of functional protein expressed in a cell in which the gene is not mutated. A method for determining the relative amount of functional protein expressed may be any suitable method known in the art, for example Western blotting.

The functional protein that is expressed by the method of the invention is preferably capable of performing the function(s) of the corresponding protein expression from a non-mutated gene. The functional protein may not be 100% as effective as the normal protein but is preferably at least 50%, 60%, 70%, 80%, 90% or more preferably, at least 95% as effective as the normal protein. Functional activity may be determined by any method known in the art to the skilled person that is relevant to the protein concerned.

Therapeutic treatment

The ability of the PNAs of the invention to correct the expression of mutated mRNA results in the suitability of the PNAs of the invention for therapeutic treatment of muscle disease or muscular dystrophy in a subject having such a disease. Accordingly, the invention provides a method of treating a muscle disease or muscular dystrophy in a subject, comprising administering a PNA as described herein to said subject and thereby restoring the expression of mutated mRNA in said subject. As used herein, the term "treatment" is meant to encompass therapeutic, palliative and prophylactic uses. The method of treatment of the invention is suitable for any patient that has a muscle disease or muscular dystrophy. The disease may be caused by a nonsense or frameshift mutation. The aberrant protein expression may be the result of a mutation that weakens a splice site resulting in the inclusion of an unsuitable exon. Alternatively, the mutation may introduce a cryptic splice site resulting in the splicing of an exon that is important for protein function. The muscle disease or muscular dystrophy may be any muscle disease or dystrophy. Examples include Duchenne muscular dystrophy (DMD), myotonic dystrophy and spinal muscular atrophy.

Symptoms of DMD which may be used to determine whether a subject has DMD include progressive muscle wasting (loss of muscle mass), poor balance, frequent falls, walking difficulty, waddling gait, calf pain, limited range movement, muscle contractures, respiratory difficulty, drooping eyelids (ptosis), gonadal atrophy and scoliosis (curvature of the spine). Other symptoms can include cardiomyopathy and arrhythmias. Symptoms of myotonic dystrophy which may be used to determine whether a subject has myotonic dystrophy include abnormal stiffness of muscles and myotonia (difficulty or inability to relax muscles). Other symptoms of myotonic dystrophy include weakening and wasting of muscles (where the muscles shrink over time), cataracts, and heart problems. Myotonic dystrophy affects heart muscle, causing irregularities in the heartbeat. It also affects the muscles of the digestive system, causing constipation and other digestive problems. Myotonic dystrophy may cause cataracts, retinal degeneration, low IQ, frontal balding, skin disorders, atrophy of the testicles, insulin resistance and sleep apnea. A muscle disease of muscular dystrophy may be diagnosed on the basis of symptoms and characteristic traits such as those described above and/or on the results of a muscle biopsy, DNA or blood test. Blood tests work by determining the level of creatine phosphokinase (CPK). Other tests may include serum CPK, electromyography and electrocardiography. Muscular dystrophies can also alter the levels of myoglobin, LDH, creatine, AST and aldolase.

The method of treatment of the invention can be used to treat muscle disease or muscular dystrophy in a subject of any age. Preferably the individual to be treated is as young as possible and/or before symptoms of the condition develop. It is preferable to treat an individual before muscle damage occurs in order to preserve as much muscle as possible. The age of onset of DMD is usually between 2 and 5 years old. Without treatment, most DMD sufferers die by their early twenties, typically from respiratory disorders. Typically therefore, the age of the subject to be treated for DMD is from 2 to 20 years old. More preferably, the age of the subject to be treated is from 4 to 18, from 5 to 15 or from 8 to 12. Myotonic dystrophy generally affects adults with an age at onset of about 20 to about 40 years. Typically, the age of the subject to be treated for myotonic dystrophy is from 2 to 40 years old. More preferably, the age of the subject to be treated is from 4 to 35, from 8 to 30 or from 12 to 25. Preferably the individual to be treated is asymptomatic.

The method of the invention may be used to correct or alter splicing in any type of muscle tissue. The target muscle tissue may be skeletal muscle, cardiac muscle, or smooth muscle. Targeting the heart muscle may be preferable in patients with cardiac disease or early cardiac symptoms. Such patients may be preferable to treat because of the early mortality associated with this component of the disease. Current medications and treatments for muscular dystrophy are limited. Inactivity can worsen the disease. Physical therapy and orthopaedic instruments may be helpful. The cardiac problems that occur with myotonic dystrophy and Emery-Dreifuss muscular dystrophy may require a pacemaker. Conventional methods of coping with the disease include exercise, drugs that slow down or eliminate muscle wasting, anabolic steroids and dietary supplements such as creatine and glutamine. The antiinflammatory corticosteroid prednisone may be used to improve muscle strength and delay the progression of the disease. Other nutritional supplements and steroids that may be used in the treatment of DMD include deflazacort, albuterol, creatine, anabolic steroids, and calcium blockers. The myotonia occurring in myotonic dystrophy may be treated with medications such as quinine, phenytoin or mexiletine. All of the above treatments are aimed at slowing down the progression of the disease or reducing its symptoms. The treatment of the invention may be administered in combination with any such form of treating or alleviating the symptoms of muscle disease or muscular dystrophy.

Peptide nucleic acid oligonucleotides

In PNAs, the sugar phosphate backbone of DNA is replaced by an achiral polyamide backbone. PNAs have a high affinity for DNA and RNA and high sequence specificity. They are also highly resistant to degradation, being protease- and nuclease- resistant. PNAs are also stable over a wide pH range.

The PNAs used in the methods of the invention are typically at least 10 bases long, such as at least 12, 14, 15, 18, 20, 23 or 25 or more bases in length. Typically, the PNA is less than 35 bases in length. Such as less than 34, 32, 30 or 28 bases long. Preferably, the PNA will be in the range of 15 to 30 bases long, more preferably 15 to 25 or 20 to 30 bases long. The PNAs are preferably of length 18 or 25 bases and/or preferably have sequence which is homologous to SEQ ID NO: 213 or 214.

The PNAs of the invention are complementary to and selectively hybridise to one or more sequences that are responsible for or contribute to the promotion of exon splicing or inclusion. Such a sequence may be a splice site donor sequence, splice site acceptor sequence, splice site enhancer sequence or splice site silencer sequence. Splice site donor, acceptor and enhancer sequences are involved in the promotion of exon splicing and therefore can be targeting with one or more PNAs in order to inhibit exon splicing. Splice site silencers are involved in inhibiting splicing and can therefore be targeted with PNAs in order to promote exon splicing.

Splice site donor, acceptor, enhancer and silencer sequences may be located within the vicinity of the 5' or 3' end of the exon to be spliced from or, in the case of silencer sequences, included into the final mRNA. Splice site acceptor or donor sequences and splice site enhancer or silencer sequences are either known in the art or can be readily determined. Bioinformatic prediction programmes can be used to identify gene regions of relevance to splicing events as a first approximation. For example, software packages such as RESCUE-ESE, ESEfinder, and the PESX server predict putative ESE sites. Subsequent empirical experimental work, using splicing assays well known in the art, can then be carried out in order to validate or optimise the sequences involved in splicing for each exon that is being targeted.

Any exon in which there is a non-sense or frame-shift inducing mutation may be a potential target for deletion from the pre-mRNA by exon skipping. Any of the exons in the dystrophin gene can be targeted for deletion from the dystrophin pre-mRN A. Preferably, the exons that are targeted for deletion are any of the exons in the human dystrophin gene except for exons 65 to 69, which are essential for protein function. Preferably the exon(s) to be deleted are those that are commonly mutated in DMD, i.e. any of exons 2 to 20 or exons 45 to 53. Preferably, the patient is tested for which mutation they have in order to determine which exon is to be deleted or included. Preferably, the sequence of the PNA used for exon skipping comprises a sequence that is capable of selectively hybridising to a sequence that spans the exon/intron boundary of the exon to be deleted or included. The exon/intron boundary may be the 3' or 5' boundary of the exon to be included or deleted. The exon/intron boundary sequence information for a particular gene may be obtained from any source of sequence information, such as the ensemble database. Sequence information, including the exon/intron boundary locations, for the human and mouse dystrophin genes may be found at the following web links:

Human: http://vega.sanger.ac.uk/Homo_sapiens/transview?transcript=OTTHUMT00000056182

Mouse: http://vega.sanger.ac.uk/Mus_musculus/transview?transcript=OTTMUST00000043357 The exon/intron boundary sequence information for the human and mouse dystrophin genes is in SEQ ID NO: 1 and 130-208. The currently known mutations, including point mutations, deletions duplications in the entire human dystrophin gene may be accessed at the following web link: httρ://www. dmd.nl7DMD_deldup.html

More preferably, the PNA sequence is selected from sequences capable of selectively hybridising to the exon/intron boundary sequences provided in Table 1 or homologues thereof. The nomenclature in Table 1 is based upon target species (H, human, M, mouse), exon number, and annealing coordinates as described by Mann et al 2002 (Journal of Gene Medicine, 4: 644-654). The number of exonic nucleotides from the acceptor site is indicated as a positive number, whereas intronic bases are given a negative value. For example, H16A(-06+25) refers to an antisense oligonucleotide (AO) for human dystrophin exon 16 acceptor region, at coordinates 6 intronic bases from the splice site to 25 exonic bases into exon 16. The total length of this AO is 31 nucleotides and it covers the exon 16 acceptor site.

Table 1. Sequences of exon/intron boundaries in human and mouse dystrophin pre- mRNA (SEQ ID NO: 3 to 123).

Nomenclature Sequence (5'-3')

H2A(+12+41) CCA UUU UGU GAA UGU UUU CUU UUG AAC AUC

H3A(+20+40) GUA GGU CAC UGA AGA GGU UCU

H4A(+l l+40) UGU UCA GGG CAU GAA cue UUG UGG AUC CUU

H5A(+25+55) UCA GUU UAU GAU UUC CAU CUA CGA UGU CAG U

H6A(+69+91) UAC GAG UUG AUU GUC GGA CCC AG

H7A(+45+67) UGC AUG UUC CAG UCG UUG UGU GG

H9A(-06+23) CCC UGU GCU AGA CUG ACC GUG AUC UGC AG

H12A(+52+75) UCU UCU GUU UUU GUU AGC CAG UCA

H13A(+77+100) CAG CAG UUG CGU GAU cue CAC UAG

H14A(+32+61) GUA AAA GAA CCC AGC GGU CUU CUG UCC AUC

H15A(+48+71) UCU UUA AAG CCA GUU GUG UGA AUC

H16A(-12+19) CUA GAU CCG CUU UUA AAA ecu GUU AAA ACA A

H18A(+24+53) CAG CUU CUG AGC GAG UAA UCC AGC UGU GAA

HMl 9A(+35+65) GCC UGA GCU GAU CUG CUG GCA UCU UGC AGU U

H22A(+125+146) CUG CAA UUC CCC GAG UCU CUG C

H23A (+69+98) CGG CUA AUU UCA GAG GGC GCU UUC UUC GAC

H24A(+51+73) CAA GGG CAG GCC AUU ecu ecu UC

H25A(+95+119) UUG AGU UCU GUC UCA AGU cue GAA G

H27A(+82+106) UUA AGG ecu CUU GUG CUA CAG GUG G

H28A (+99+124) CAG AGA UUU ecu CAG cue CGC CAG GA

H29A(+57+81) UCC GCC AUC UGU UAG GGU CUG UGC C

H30A(+25+50) UCC UGG GCA GAC UGG AUG cue UGU UC Nomenclature Sequence (5-31)

H31D(+03-22) UAG UUU CUG AAA UAA CAU AUA ecu G

H32A(44+73) CUU GUA GAC GCU GCU CAA AAU UGG CUG GUU

H33A(+64+88) CCG UCU GCU UUU UCU GUA CAA UCU G

H35A(+24+53) UCU GUG AUA cue UUC AGG UGC ACC UUC UGU

H37A(+134+157) UUC UGU GUG AAA UGG CUG CAA AUC

H38A(+88+112) UGA AGU CUU ecu CUU UCA GAU UCA C

H39A(+62+91) UUU ecu cue GCU UUC UCU CAU CUG UGA UUC

H41A(+44+69) CAA GCC cue AGC UUG ecu ACG CAC UG

H42A(-4+23) AUC GUU UCU UCA CGG ACA GUG UGC UGG

H47A(-06+24) CAG GGG CAA cue UUC CAC CAG UAA CUG AAA

H49A(-11+16) CUG CUA UUU CAG UUU ecu GGG GAA AAG

H51A(+66+90) ACA UCA AGG AAG AUG GCA UUU CUA G

H52A(+12+41) UCC AAC UGG GGA CGC cue UGU UCC AAA UCC

H53A(+39+69) CAU UCA ACU GUU GCC UCC GGU UCU GAA GGU G

H72A(+02+28) GUG UGA AAG CUG AGG GGA CGA GGC AGG

H74A(+48+72) CGA GGC UGG cue AGG GGG GAG UCC U

H75A(+34+58) GGA CAG GCC UUU AUG UUC GUG CUG C

H77A(+16+42) CUG UGC UUG UGU ecu GGG GAG GAC UGA

H78A(+04+29) UCU CAU UGG CUU UCC AGG GGU AUU UC

Hl lA(+75+97) CAU CUU CUG AUA AUU UUC CUG UU

H21A(+86+108) GUC UGC AUC CAG GAA CAU GGG UC

H36A(+22+51) UGU GAU GUG GUC CAC AUU CUG GUC AAA AGU

H40A(-5+17) CUU UGA GAC cue AAA UCC UGU U

H43A(+101+120) GGA GAG AGC UUC CUG UAG CU

H44A(+61+84) UGU UCA GCU UCU GUU AGC CAC UGA

H46A(+107+137) CAA GCU UUU CUU UUA GUU GCU GCU CUU UUC C

H48A(-07+23) UUC UCA GGU AAA GCU CUG GAA ACC UGA AAG

H57A (-12+18) CUG GCU UCC AAA UGG GAC CUG AAA AAG AAC

H60A(+37+66) CUG GCG AGC AAG GUC CUU GAC GUG GCU CAC

H61A(+10+40) GGG CUU CAU GCA GCU GCC UGA cue GGU CCU C

H68A(+22+48) CAU CCA GUC UAG GAA GAG GGC CGC UUC

H70A(+98+121) ecu CUA AGA CAG UCU GCA CUG GCA

H71A(-03+21) AAG UUG AUC AGA GUA ACG GGA CUG

H73A(+06+30) GAU CCA UUG CUG UUU UCC AUU UCU G

H26A(-07+19) ecu ecu UUC UGG CAU AGA ecu UCC AC

H45A(-06+20) CCA AUG CCA UCC UGG AGU UCC UGU AA

H50A(+02+30) CCA cue AGA GCU CAG AUC UUC UAA CUU CC

H55A(+141+160) CUU GGA GUC UUC UAG GAG CC

H56A(+102+126) GUU AUC CAA ACG UCU UUG UAA CAG G

H58A(+21+45) ACU CAU GAU UAC ACG UUC UUU AGU U

H59A(-06+16) UCC UCA GGA GGC AGC UCU AAA U

H62A (+8+34) GAG AUG GCU cue UCC CAG GGA CCC UGG

H63A(+l l+35) UGG GAU GGU CCC AGC AAG UUG UUU G

H64A(+47+74) GCA AAG GGC CUU CUG CAG UCU UCG GAG

H66A(-8+19) GAU ecu CCC UGU UCG UCC ecu AUU AUG

H67A(+22+47) GCG CUG GUC ACA AAA UCC UGU UGA AC Nomenclature Sequence (5-31)

H69A(-06+18) UGC UUU AGA cue CUG UAC CUG AUA

H76A(+53+79) GCU GAC UGC UGU CGG ACC UCU GUA GAG

H8A(-06+18) GAU AGG UGG UAU CAA CAU CUG UAA

H10A(-05+16) CAG GAG CUU CCA AAU GCU GCA

H10A(+98+119) UCC UCA GCA GAA AGA AGC CAC G

H17A(-07+16) UGA CAG ecu GUG AAA UCU GUG AG

H20A(+44+71) CUG GCA GAA UUC GAU CCA CCG GCU GUU C

H20A(147+168) CAG CAG UAG UUG UCA UCU GCU C

H34A (+46+70) CAU UCA UUU ecu UUC GCA UCU UAC G

H34A(+95+120) AUC UCU UUG UCA AUU CCA UAU CUG UA

H54A(+67+89) UCU GCA GAA UAA UCC CGG AGA AG

H65A(-11+14) GCU CAA GAG AUC CAC UGC AAA AAA C

H65A(+63+87) UCU GCA GGA UAU CCA UGG GCU GGU C

H65D(+15-11) GCC AUA CGU ACG UAU CAU AAA CAU UC

H16A(-17+08) UUU AAA ACC UGU UAA AAC AAG AAA G

H16A(-12+19) CUA GAU CCG CUU UUA AAA ecu GUU AAA ACA A

H16A(-06+19) CUA GAU CCG CUU UUA AAA ecu GUU A

H16A(-06+25) UCU UUU CUA GAU CCG CUU UUA AAA ecu GUU A

H16A(-07+13) CCG CUU UUA AAA ecu GUU AA

H16A(+01+25) UCU UUU CUA GAU CCG CUU UUA AAA C

H16A(+06+30) CUU UUU CUU UUC UAG AUC CGC UUU U

H16A(+ll+35) GAU UGC UUU UUC UUU UCU AGA UCC G

H16A(+12+37) UGG AUU GCU UUU UCU UUU CUA GAU CC

H16A(+45+67) GAU CUU GUU UGA GUG AAU ACA GU

H16A(+87+109) CCG UCU UCU GGG UCA CUG ACU UA

H16A(+92+116) CAU GCU UCC GUC UUC UGG GUC ACU G

H16A(+105+126) GUU AUC CAG CCA UGC UUC CGU C

H16D(+11-11) GUA UCA CUA ACC UGU GCU GUA C

H16D(+05-20) UGA UAA UUG GUA UCA CUA ACC UGU G

H46A(+107+137) CAA' GCU UUU CUU UUA GUU GCU GCU CUU UUC C

H51A(-01+25) ACC AGA GUA ACA GUC UGA GUA GGA GC

H51A(+61+90) ACA UCA AGG AAG AUG GCA UUU CUA GUU UGG

H51A(+66+90) ACA UCA AGG AAG AUG GCA UUU CUA G

H51A(+66+95) cue CAA CAU CAA GGA AGA UGG CAU UUC UAG

H51A(+111+134) UUC UGU CCA AGC CCG GUU GAA AUC

H51A(+175+195) CAC CCA CCA UCA CCC UCU GUG

H51A(+199+220) AUC AUC UCG UUG AUA UCC UCA A

H51D(+08-17) AUC AUU UUU UCU CAU ACC UUC UGC U

H51D(+16-07) „ cue AUA ecu UCU GCU UGA UGA UC

H53A(-07+18) GAU UCU GAA UUC UUU CAA CUA GAA U

H53A(-12+10) AUU CUU UCA ACU AGA AUA AAA G

H53A(+23+47) CTG AAG GTG TTC TTG TAC TTC ATC C

H53A(+39+62) CUG UUG ecu CCG GUU CUG AAG GUG

H53A(+39+69) CAU UCA ACU GUU GCC UCC GGU UCU GAA GGU G

H53A(+45+69) CAU UCA ACU GUU GCC UCC GGU UCU G

H53A(+124+145) UUG GCU CUG GCC UGU ecu AAG A Nomenclature Sequence (5'-3')

H53A(+151+175) GUA UAG GGA CCC UCC UUC CAU GAC U

H53D(+09-18) GGU AUC UUU GAU ACU AAC CUU GGU UUC

H53D(+14-07) UAC UAA ecu UGG UUU CUG UGA

M23D(+07-18) GGC CAA ACC UCG GCU UAC CUG AAA U

M23D(+02-18) GGC CAA ACC UCG GCU UAC CU

M23D(+12-18) GGC CAA ACC UCG GCU UAC CUG AAA UUU UCG

M23D(+07-23) UUA AAG GCC AAA ecu CGG CUU ACC UGA AAU

Examples of preferred PNA sequences capable of inducing the splicing of exon 7a in the mouse ClC-I gene are sequences capable of selectively hybridising to the 3' or 5' splice sites of exon 7a. Such preferred PNA sequences may be capable of specifically hybridising to a sequence in Table 2 or a homologue thereof.

Table 2. Sequences of exon/intron boundaries in the mouse ClC-I pre-mRNA for mouse exon 7a (SEQ ID NO: 124 and 125).

Nomenclature Sequence (5-31)

M7a (-17+14) GUG CUU cue UGU UGC AGA CCG UGC CUG GGC A

M7a (+13-18) GCC CCT GAU GGA GGC AAG UUU CAC UUC cue C

Typically, only one PNA sequence is used to induce or inhibit exon skipping in a cell. However, more than one different PNA can be delivered to the sample of human cells or a patient, e.g. a cocktail of 2, 3, 4 or 5 or more different PNA sequences can be used to drive exon skipping or inhibit exon skipping in a cell. Such a combination of different PNA sequences can be delivered simultaneously, separately or sequentially. Selective hybridisation means that generally the polynucleotide can hybridize to the relevant polynucleotide, or portion thereof, at a level significantly above background. The signal level generated by the interaction between the polynucleotides is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides. The intensity of interaction may be measured, for example, by radiolabelling the polynucleotide, e.g. with 32P. Selective hybridisation is typically achieved using conditions of medium to high stringency (for example 0.03M sodium chloride and 0.003M sodium citrate at from about 5O0C to about 6O0C).

PNAs are produced synthetically using any known technique in the art. PNA is a DNA analog in which a polyamide backbone replaces the traditional phosphate ribose ring of DNA. Despite a radical change to the natural structure, PNA is capable of sequence-specific binding to DNA or RNA. Characteristics of PNA include a high binding affinity to complementary DNA or RNA, a destabilizing effect caused by single-base mismatch, resistance to nucleases and proteases, hybridization with DNA independent of salt concentration and triplex formation with homopurine DNA. Panagene™ has developed its proprietary Bts PNA monomers (Bts; benzothiazole-2-sulfonyl group) and proprietary oligomerisation process. The PNA oligomerisation using Bts PNA monomers is composed of repetitive cycles of deprotection, coupling and capping. Panagene's patents to this technology include US 6969766, US 7211668, US 7022851, US 7125994, US 7145006 and US 7179896. Homologues of polynucleotide sequences are referred to herein. Such homologues typically have at least 70% homology, preferably at least 80, 90%, 95%, 97% or 99% homology, for example over a region of at least 10, 15, 20, 25 or more contiguous nucleotides. The homology may be calculated on the basis of nucleotide identity (sometimes referred to as "hard homology"). For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J MoI Evol 36:290-300; Altschul, S, F et al (1990) J MoI Biol 215:403-10.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11 , the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. ScL USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. ScL USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

The homologous sequence typically differs by at least 1, 2, 5, 10, 20 or more mutations (which may be substitutions, deletions or insertions of nucleotides). These mutations may be measured across any of the regions mentioned above in relation to calculating homology.

Delivery/Administration

The PNA may be delivered to the cell by any suitable means. In the method of treating a subject having a muscle disease or muscular dystrophy, the method of delivery is any suitable means of delivering the PNA to the affected tissues i.e. muscle. The method of delivery may be selected from parenteral, intramuscular, intracerebral, intravenous, subcutaneous, or transdermal administration. Typically the method of delivery is by injection. Preferably the injection is intramuscular or intravascular, more preferably intramuscular. A physician will be able to determine the required route of administration for each particular patient. The PNA is preferably delivered as a composition. The composition may be formulated for parenteral, intramuscular, intracerebral, intravenous, subcutaneous, or transdermal administration. Uptake of nucleic acids by mammalian cells is enhanced by several known transfection techniques, for example, those that use transfection agents. The formulation that is administered may contain such agents. Examples of these agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam™ and transfectam™).

Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with a PNA oligonucleotide in conjunction with other muscle disease or muscular dystrophy therapeutic modalities (such as those described herein) in order to increase the efficacy of the treatment. The PNAs may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the PNA oligonucleotide. The composition may comprise other active agents that are used in DMD therapy such as anti-inflammatories. The PNAs may be used in combination with methods of gene therapy. For example, the PNAs may be delivered in combination (simultaneously, separately or sequentially) with a gene or partial gene encoding the protein which is mutated in the individual. For example, the gene may be the full-length or partial sequence of the dystrophin gene in cases of DMD. Gene delivery may be carried out by any means, but preferably via a viral vector such as AAV. Gene therapy targeting the myostatin gene or its receptor may also be used in conjunction with the PNA(s) in order to increase muscle mass and thereby restore strength in any remaining muscle.

The PNAs may be formulated in combination with other splice correction chemistries such as 2'-O-methyl phosphorothioate (2OMe) AOs, phosphorodiamidate morpholino oligomers (PMOs, but also referred to as morpholinos) and peptide-linked PMOs.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, liposomes, diluents and other suitable additives. Pharmaceutical compositions comprising the PNA oligonucleotides provided herein may include penetration enhancers in order to enhance the delivery of the oligonucleotides. Penetration enhancers may be classified as belonging to one of five broad categories, i.e. fatty acids, bile salts, chelating agents, surfactants and non- surfactants. One or more penetration enhancers from one or more of these broad categories may be included.

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (1- monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1- monocaprate, l-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono-and di-glycerides and physiologically acceptable salts thereof (i.e. oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.

Complex formulations comprising one or more penetration enhancers may be used. For example, bile salts may be used in combination with fatty acids to make complex formulations. Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g. sodium salicylate, 5- methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N- amino acyl derivatives of beta-diketones (enamines). Chelating agents have the added advantage of also serving as DNase inhibitors. Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9- lauryl ether and polyoxyethylene-20-cetyl ether and perfluorochemical emulsions, such as FC-43. Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl-andl- alkenylazacyclo-alkanone derivatives and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone. A "pharmaceutically acceptable carrier" (excipient) is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to a subject. The pharmaceutically acceptable carrier may be liquid or solid and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency etc when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e. g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc); fillers (e.g. lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc); lubricants (e.g. magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc); disintegrates (e.g. starch, sodium starch glycolate, etc); or wetting agents (e.g. sodium lauryl sulphate, etc).

The compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavouring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions provided herein.

Regardless of the method by which the oligonucleotides are introduced into a patient, colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the oligonucleotides and/or to target the oligonucleotides to a particular organ, tissue or cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterised structure. A preferred colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration.

A therapeutically effective amount of PNA is administered. The dose may be determined according to various parameters, especially according to the severity of the condition, age, and weight of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in vitro and in in vivo animal models. In general, dosage is from 0.01 mg/kg to 100 mg per kg of body weight. A typical daily dose is from about 0.1 to 50 mg per kg, preferably from about 0.1 mg/kg to lOmg/kg of body weight, according to the potency of the specific PNA, the age, weight and condition of the subject to be treated, the severity of the disease and the frequency and route of administration. Different dosages of PNA may be administered depending on whether administration is by intramuscular injection or systemic (intravenous or subcutaneous) injection. Preferably, the dose of a single intramuscular injection is in the range of about 5 to 20 ug. Preferably, the dose of single or multiple systemic injections is in the range of 10 to 100 mg/kg of body weight.

Due to PNA clearance and breakdown of the targeted mRNA transcript and protein, the patient may have to be treated repeatedly, for example once or more daily, weekly, monthly or yearly. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the PNA in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 mg/kg to 100 mg per kg of body weight, once or more daily, to once every 20 years. The invention is illustrated by the following Examples:

Example 1

Materials and methods

Animals

Three age groups of max mice were used: 20-21 days (referred to as 3 weeks; five mice for each test and control groups), 2-month old (five mice for test groups and control groups), and 5-6 months (referred to 6 months, six mice for each testing and control group). The experiments were carried out in the Animal unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK. Mice were killed by cervical dislocation at desired time points, and muscles were snap-frozen in liquid nitrogen-cooled isopentane and stored at -8O0C.

PNAs

AU PNAs were synthesized by EuroGentec (LIEGE Science Park, Belgium). The PNA antisense (AO) sequence against the boundary sequences of exon and intron 23 of the dystrophin gene was 5'-ggccaaacctcggcttacct-3' (SEQ ID NO: 2), and designated as PNA.

Cell culture and transfection The H2K mdx myoblasts were cultured at 33 0C under a 10% CO2 /90% air atmosphere in high-glucose DMEM supplemented with 20% fetal calf serum, 0.5% chicken embryo extract (PAA laboratories Ltd, Yeovil, UK), and 20 units/ml y- interferon (Roche applied science, Penzberg, Germany). Cells were then treated with trypsin and plated at 2x104 cells per well in 24- well plates coated with 200ug/ml gelatine (Sigma). H2K mdx cells were transfected 24h after trypsin treatment in a final volume of 0.5ml of antibiotic- and serum-free Opti-MEM (Life Technologies). Each well was treated with 25OnM of PNA complexed with corresponding amounts of lipofectin (weight ratio 1 :2=oligo:lipofectin) (Life Technologies) according to the supplier's instructions. After 4h of incubation, the transfection medium was replaced with DMEM supplemented medium.

RNA Extraction and Nested RT-PCR Analysis

Cells were transfected as triplicate wells with Lipofectin-Oligonucleotide complexes and total cellular RNA was then extracted 24h after transfection with RNAeasy mini kit (Qiagen) and 200ng of RNA template was used for 20μl RT-PCR with OneStep RT-PCR kit (Qiagen, West Sussex, UK). The primer sequences for the initial RT-PCR were Exon20Fo 5'-CAGAATTCTGCCAATTGCTGAG-S ' (SEQ ID NO: 126) and Ex26Ro 5'-TTCTTCAGCTTGTGTCATCC-S' (SEQ ID NO: 127) for amplification of mRNA from exons 20 to 26. The cycling conditions were 95 0C for 30sec, 55 0C for lmin, and 72 0C for 2min for 30 cycles. RT-PCR product (1 μl) was . then used as the template for secondary PCR performed in 25μl with 0.5unit TaqDNA polymerase (Invitrogen). The primer sequences for the second round were: Ex20Fi 5'-CCCAGTCTACCACCCTATCAGAGC-S' (SEQ ID NO: 128) and Ex2Ri 5'-CCTGCCTTTAAGGCTTCCTT-S' (SEQ ID NO: 129). The cycling conditions were 95 0C for lmin, 57 0C for lmin, and 72 0C for 2min for 25 cycles. Products were examined by electrophoresis on a 2% agarose gel. Intramuscular injection of PNA and PNA-peptide conjugates

One tibialis anterior (TA) muscle of each experimental mdx mouse was injected with a 40μl dose (3-week-old group injected with lOμl) of PNA with saline at a final concentration of 125μg/ml, and the contralateral muscle was injected with saline. The animals were sacrificed at various time points after injection, the muscles were removed and snap-frozen in liquid nitrogen-cooled isopentane and stored at -8O0C. To examine the dose-response profile, 2-month old mdx mice received the injections of lOμg and 20μg of PNA.

Immunohistochemistry and histology

Sections of 8μm were cut from at least two-thirds of muscle length of TA muscles at lOOμm intervals. The sections were then examined for dystrophin expression with a polyclonal antibody 2166 against the dystrophin carboxyl-terminal dystrophin. The maximum number of dystrophin-positive fibres in one section was counted using the Zeiss Axio Vision fluorescence microscope. The intervening muscle sections were collected either for Western blot or as serial sections for immunohistochemistry. Polyclonal antibodies were detected by goat-anti-rabbit IgGs Alexa Fluro 594 (Molecular probe). Routine H&E staining was used to examine overall muscle morphology and assess the level of infiltrating mononuclear cells.

Protein extraction and Western blot

The collected sections were placed in a 1.5ml polypropylene eppendorf tube (Anachem, Bedfordshire,UK) on dry ice. The tissue sections were lysed with 150μl protein extraction buffer containing 125mM TrisΗCl (pH6.8), 10%SDS, 2M urea, 20% glycerol and 5% 2-mercaptoeethanol. The mixture was boiled for 5 min and centrifuged. The supernatant was collected and the protein concentration was quantified by BCA assay (Sigma). Protein (5μg) from normal C57BL6 mice as a positive control and 50μg of protein from muscles of treated or untreated mdx mice were loaded onto SDS-PAGE gels (4% stacking, 6% resolving). Samples were electrophoresed for 4h at 8OmA and transferred to nitrocellulose overnight at 50V at 40C. The membrane was then washed and blocked with 5% skimmed milk and probed with DYSl (monoclonal antibody against dystrophin R8 repeat, 1 :200, NovoCastra) overnight. The bound primary antibody was detected by horseradish peroxidase-conjugated rabbit anti-mouse IgGs and ECL Western Blotting Analysis system (Amersham Pharmacia Biosciences). The intensity of the bands obtained from treated mdx muscles was measured by Image J and compared with that from normal muscle of C57BL6 mice.

Statistical analysis

All data are reported as mean value ±SEM. Statistical differences between treatment groups were evaluated by SigmaStat (Systat Software Inc, UK).

Results

Neutral PNAs induce dystrophin exon skipping in cell culture

To evaluate the ability of PNAs to induce exon-skipping in the dystrophin gene, we transfected H2K myoblasts from an mdx mouse with a 20-mer PNA directed at the boundary sequences of exon 23 and intron 23 of the dystrophin gene (Figure IA). An in-frame transcript arising from the removal of exon 23 was clearly demonstrated in the cells treated with the PNA AO at the concentration of 250 nM. No other shorter RT- PCR products were generated. Sequence analysis confirmed the precise truncation from exon 22 to exon 24 (Figure IB).

Exon skipping and dystrophin expression in muscles of mdx mouse

We next examined the effects of PNA in mdx mice using intramuscular injections. Initial experiments were performed in 2-month old mdx mice with a single dose of 5μg PNA in saline into the TA muscle. Two weeks after injection, dystrophin- positive fibres were identified by immunohistochemistry and the number of dystrophin- positive fibres in TA muscles of treated mice was significantly increased compared with that in the age-matched control mdx mice (Figure 2A). Whole muscle transverse sections showed a uniform distribution of dystrophin-positive fibres within the injected region of the muscles, with average counts of dystrophin-positive fibres of 329(±51) (PO.001). The expression of dystrophin was further demonstrated by Western blot in the

PNA treated muscles. As expected, the size of the induced dystrophin was indistinguishable from that of normal dystrophin, since there is only 71 amino acids (encoded by exon 23) difference between the two proteins; and there was about 5% of normal level induced when compared with normal C57BL6. No dystrophin was detected in the TA muscle extracts from untreated mdx control muscle (Figure 4). To determine whether different stages of muscular dystrophy affected the efficiency of exon skipping by neutral PNA, we injected 5μg of PNA into TA muscles of 3 -week old mdx mice and examined dystrophin induction 2 weeks later.

Immunohistochemical evaluation revealed uniformly distributed dystrophin-positive fibres (235± 53) in the injected region. Similarly, the same amount of PNA induced wide-spread dystrophin expression in 6-month old mdx mice with the average number of dystrophin positive fibre at 172 (±49) (Figure 2B). Although statistically not significant, the relatively higher number of fibres expressing dystrophin in the 2-month old mdx mice than in the 6 month-old mice suggests that increased extracellular matrix associated with the progress of muscle degeneration might have negative effect on the delivery of AO. This would be consistent with age-related diminution in efficiency of gene delivery into skeletal muscle with increasing levels of muscle degeneration. To examine the dose-response of PNA, we quantified the dystrophin-positive fibres in TA muscles of 2-month old mdx mice injected with 5μg, lOμg and 20μg PNA. Immunohistochemical results showed that the number of dystrophin-positive fibres increased with the increase in the amount of PNA injected (Figure 3A), with an average of 427(±46) dystrophin-positive fibres in muscles injected with lOμg PNA and an average of 686 (±100) in muscles injected with 20μg PNA (P<0.05) (Figure 3B). Also important is that the number of dystrophin-positive fibres reached more than 1000, about 50% of whole TA muscle in some samples. This number of dystrophin-positive fibres was very close to that reportedly achieved by morpholino AO of the same dosage.

Further work

Results from further work are shown in Figures 5 to 7. Figures 5 and 6 show expression to persist over 20 weeks. A single IM injection was given to mice and at about 8 weeks about 20% normal expression is reached. Figure 7 shows expression results using the oligos below. As can be seen 18mer and 20mer oligos show high expression levels.

M23D(-1-18): 5'-ggccaaacctcggcttac-3'— M23D-1 (18) M23D(-2-18): 5'-ggccaaacctcggctta-3'~- M23D-2 (17) M23D(+2-14): 5'-aaacctcggcttacct-3'— M23D-3 (16) M23D(+2-13): 5'-aacctcggcttacct-3'~- M23D-4 (15)

PNA18 (+2-16): 5'-ccaaacctcggcttacct-3'- (18)

M23D (07-18) : 5'-GGCCAAACCTCG GCTTACCTGAAAT-3' (25)

Claims

1. A method of correcting expression of a gene in a human cell having a muscle disease or muscular dystrophy phenotype, wherein without correction the gene fails to express functional protein due to one or more mutations, said method comprising delivering to the cell a peptide nucleic acid (PNA) comprising a sequence capable of targeting a sequence responsible for exon skipping in the mutated pre-mRNA at an exon to be skipped or included, wherein expression is corrected by the PNA inducing exon skipping or inclusion and thereby correcting the expression of said mutated pre-mRNA.
2. The method according to claim 1, wherein the sequence responsible for exon skipping is a splice site acceptor, splice site donor, splice site enhancer or splice site silencer and the PNA comprises a sequence which is 15 to 30 nucleotides in length.
3. The method according to claim 1 or 2, wherein the gene is selected from dystrophin and CIC-I.
4. The method according to claim 3, wherein the gene is dystrophin.
5. The method according to claim 4, wherein the cell is in a human subject that has Duchenne muscular dystrophy (DMD).
6. The method according to claim 4 or 5, wherein the PNA comprises a sequence which is 15 to 30 nucleotides in length said sequence being hybridisable to any one of the sequences identified in Table 1.
7. The method according to any one of claims 4 to 6, wherein more than one PNA is used and the PNA(s) target the removal of exon 51 and 53 separately or by the removal of exons 51 to 53 simultaneously.
8. The method according to any one of the preceding claims wherein said cell is in a human subject, said human being the age of about 2 to 20 and optionally asymptomatic.
9. A method of treating DMD in a human subject comprising administering a PNA as defined in any one of the previous claims to the subject.
10. The method according to claim 9, wherein the PNA is administered with a pharmaceutically acceptable carrier.
11. The method according to claim 9 or 10, wherein the administration of PNA is by injection.
12. The method according to claim 11 , wherein the injection is intramuscular or systemic.
13. The method according to claim 12, wherein the dose of PNA is 5 to 20 ug per intramuscular injection or 10 to 100 mg/kg body weight per single or multiple systemic injection.
14. The method according to any one of the preceding claims wherein the PNA has a length of 18 or 25 bases and/or has sequence the same as or homologous to SEQ ID NO: 213 or 214.
PCT/GB2009/000379 2008-02-12 2009-02-12 Treatment of muscular dystrophy using peptide nucleic acid ( pna) WO2009101399A1 (en)

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