HK1262582B - Methods and means for efficient skipping of at least exon 52 of the human duchenne muscular dystrophy gene - Google Patents

Description

Field

The invention relates to the field of genetics, more specifically human genetics. The invention in particular relates to modulation of splicing of the human Duchenne Muscular Dystrophy pre-mRNA.

Background of the invention

Myopathies are disorders that result in functional impairment of muscles. Muscular dystrophy (MD) refers to genetic diseases that are characterized by progressive weakness and degeneration of skeletal muscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. They are recessive disorders and because the gene responsible for DMD and BMD resides on the X-chromosome, mutations mainly affect males with an incidence of about 1 in 3500 boys.

DMD and BMD are caused by genetic defects in the DMD gene encoding dystrophin, a muscle protein that is required for interactions between the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction. DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and DMD patients often die before the age of thirty due to respiratory- or heart failure. In contrast, BMD patients often remain ambulatory until later in life, and have near normal life expectancies. DMD mutations in the DMD gene are characterized by frame shifting insertions or deletions or nonsense point mutations, resulting in the absence of functional dystrophin. BMD mutations in general keep the reading frame intact, allowing synthesis of a partly functional dystrophin.

During the last decade, specific modification of splicing in order to restore the disrupted reading frame of the dystrophin transcript has emerged as a promising therapy for Duchenne muscular dystrophy (DMD) (van Ommen, van Deutekom, Aartsma-Rus, Curr Opin Mol Ther. 2008;10(2):140-9, Yokota, Duddy, Partidge, Acta Myol. 2007;26(3): 179-84, van Deutekom et al., N Engl J Med. 2007;357(26):2677-86).

Using antisense oligonucleotides (AONs) interfering with splicing signals the skipping of specific exons can be induced in the DMD pre-mRNA, thus restoring the open reading frame and converting the severe DMD into a milder BMD phenotype (van Deutekom et al. Hum Mol Genet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet 2003; 12(8):907-14.). In vivo proof-of-concept was first obtained in the mdx mouse model, which is dystrophin-deficient due to a nonsense mutation in exon 23. Intramuscular and intravenous injections of AONs targeting the mutated exon 23 restored dystrophin expression for at least three months (Lu et al. Nat Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci US A. 2005;102(1):198-203). This was accompanied by restoration of dystrophin-associated proteins at the fiber membrane as well as functional improvement of the treated muscle. In vivo skipping of human exons has also been achieved in the hDMD mouse model, which contains a complete copy of the human DMD gene integrated in chromosome 5 of the mouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; 't Hoen et al. J Biol Chem. 2008; 283: 5899-907).

Recently, a first-in-man study was successfully completed where an AON inducing the skipping of exon 51 was injected into a small area of the tibialis anterior muscle of four DMD patients. Novel dystrophin expression was observed in the majority of muscle fibers in all four patients treated, and the AON was safe and well tolerated (van Deutekom et al. N Engl J Med. 2007; 357: 2677-86).

The invention is set out in the appended set of claims.

Description of the invention Method

In a first aspect, the present invention provides an in vitro or ex vivo method for inducing and/or promoting skipping of at least exon 52 (and optionally exon 43, 46, 50, 51 and/or 53) of the DMD pre-mRNA in an isolated cell of a patient, the method comprising providing said cell with an antisense oligonucleotide or an oligonucleotide equivalent as claimed herein or a viral-based vector as claimed herein. In a preferred embodiment, the molecule binds to a continuous stretch of at least 8 nucleotides within said exon.

Accordingly, an in vitro or ex vivo method is provided for inducing and/or promoting skipping of at least exon 52 and optionally exon 43, 46, 50, 51 and/or 53 of DMD pre-mRNA in an isolated cell of a patient, the method comprising providing said cell with a molecule that binds to a continuous stretch of at least 8 nucleotides within said exon.

As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient.

A patient is preferably intended to mean a patient having DMD or BMD as later defined herein or a patient susceptible to develop DMD or BMD due to his or her genetic background. In the case of a DMD patient, an oligonucleotide used will preferably correct one mutation as present in the DMD gene of said patient and therefore will preferably create a DMD protein that will look like a BMD protein: said protein will preferably be a functional dystrophin as later defined herein. In the case of a BMD patient, an oligonucleotide as used will preferably correct one mutation as present in the BMD gene of said patient and therefore will preferably create a dystrophin which will be more functional than the dystrophin which was originally present in said BMD patient.

Exon skipping refers to the induction in a cell of a mature mRNA that does not contain a particular exon that is normally present therein. Exon skipping is performed by providing a cell expressing the pre-mRNA of said mRNA with a molecule capable of interfering with essential sequences such as for example the splice donor or splice acceptor sequence that is required for splicing of said exon, or a molecule that is capable of interfering with an exon inclusion signal that is required for recognition of a stretch of nucleotides as an exon to be included in the mRNA. The term pre-mRNA refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template in the cell nucleus by transcription.

Within the context of the invention, inducing and/or promoting skipping of an exon as indicated herein means that at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more (muscle) cells of a treated patient will not contain said exon. This is preferably assessed by PCR as described in the examples.

Preferably, a method of the invention by inducing and/or promoting skipping of at least exon 52 and optionally one of the following exons 43, 46, 50, 51 and/or 53 of the DMD pre-mRNA in one or more (muscle) cells of a patient, provides said patient with a functional dystrophin protein and/or decreases the production of an aberrant dystrophin protein in said patient and/or increases the production of a functional dystrophin in said patient.

Providing a patient with a functional dystrophin protein and/or decreasing the production of an aberrant dystrophin protein in said patient is typically applied in a DMD patient. Increasing the production of a functional dystrophin is typically applied in a BMD patient.

Therefore a preferred method is a method, wherein a patient or one or more cells of said patient is provided with a functional dystrophin protein and/or wherein the production of an aberrant dystrophin protein in said patient is decreased and/or wherein the production of a functional dystrophin is increased in said patient, wherein the level of said aberrant or functional dystrophin is assessed by comparison to the level of said dystrophin in said patient at the onset of the method.

Decreasing the production of an aberrant dystrophin may be assessed at the mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional dystrophin mRNA or protein. A non-functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein.

Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional dystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional dystrophin mRNA. Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the protein level (by immunofluorescence and western blot analyses) and preferably means that a detectable amount of a functional dystrophin protein is detectable by immunofluorescence or western blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein.

As defined herein, a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 1. A functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a cysteine-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) each of these domains being present in a wild type dystrophin as known to the skilled person. The amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO:1. In other words, a functional dystrophin is a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. "At least to some extent" preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin. In this context, an activity of a functional dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC) (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immunofluorescence analysis of cross-sections, from a muscle biopsy, as known to the skilled person.

Individuals or patients suffering from Duchenne muscular dystrophy typically have a mutation in the gene encoding dystrophin that prevent synthesis of the complete protein, i.e. of a premature stop prevents the synthesis of the C-terminus. In Becker muscular dystrophy the DMD gene also comprises a mutation compared to the wild type gene but the mutation does typically not induce a premature stop and the C-terminus is typically synthesized. As a result, a functional dystrophin protein is synthesized that has at least the same activity in kind as the wild type protein, not although not necessarily the same amount of activity. The genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a cysteine-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) but its central rod shaped domain may be shorter than the one of a wild type dystrophin ( Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Exon skipping for the treatment of DMD is typically directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-shaped domain to correct the reading frame and allow synthesis of the remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain. In a preferred embodiment, an individual having DMD and being treated by a method as defined herein will be provided a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. More preferably, if said individual is a Duchenne patient or is suspected to be a Duchenne patient, a functional dystrophin is a dystrophin of an individual having BMD: typically said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). The central rod-shaped domain of wild type dystrophin comprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). For example, a central rod-shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC.

A method of the invention may alleviate one or more characteristics of a myogenic or muscle cell of a patient or alleviate one or more symptoms of a DMD patient having a deletion including but not limited to exons 8-51, 51, 53, 53-55, 53-57, 53-59, 53-60 (correctable by exon 52 skipping), and optionally exons 44, 44-46, 44-47, 44-48, 44-49, 44-51, 44-53 (correctable by exon 43 skipping), 19-45, 21-45, 43-45, 45, 47-54, 47-56 (correctable by exon 46 skipping), 51, 51-53, 51-55, 51-57 (correctable by exon 50 skipping), 13-50, 19-50, 29-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52 (correctable by exon 51 skipping), and exons 10-52, 42-52, 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, 52 (correctable by exon 53 skipping) in the DMD gene, occurring in a total of 68% of all DMD patients with a deletion (Aartsma-Rus et al., Hum. Mut. 2009).

Alternatively, a method of the invention may improve one or more characteristics of a muscle cell of a patient or alleviate one or more symptoms of a DMD patient having small mutations in, or single exon duplications of exon 52 and optionally exon 43, 46, 50, 51 and/or 53 in the DMD gene, occurring in a total of 36% of all DMD patients with a deletion (Aartsma-Rus et al, Hum. Mut. 2009)

Furthermore, for some patients the simultaneous skipping of one or more exons in addition to exon 52 is required to restore the open reading frame, including patients with specific deletions, small (point) mutations, or double or multiple exon duplications, such as (but not limited to) with a deletion of exons 44-50 requiring the co-skipping of exons 43 and 51, with a deletion of exons 46-50 requiring the co-skipping of exons 45 and 51, with a deletion of exons 44-52 requiring the co-skipping of exons 43 and 53, with a deletion of exons 46-52 requiring the co-skipping of exons 45 and 53, with a deletion of exons 51-54 requiring the co-skipping of exons 50 and 55, with a deletion of exons 53-54 requiring the co-skipping of exons 52 and 55, with a deletion of exons 53-56 requiring the co-skipping of exons 52 and 57, with a nonsense mutation in exon 43 or exon 44 requiring the co-skipping of exon 43 and 44, with a nonsense mutation in exon 45 or exon 46 requiring the co-skipping of exon 45 and 46, with a nonsense mutation in exon 50 or exon 51 requiring the co-skipping of exon 50 and 51, with a nonsense mutation in exon 51 or exon 52 requiring the co-skipping of exon 51 and 52, with a nonsense mutation in exon 52 or exon 53 requiring the co-skipping of exon 52 and 53, or with a double or multiple exon duplication involving exons 43, 46, 50, 51, 52, and/or 53.

In a preferred method, the skipping of exon 52 is induced and optionally the skipping of exon 43 is induced, or the skipping of exon 46 is induced, or the skipping of exon 50 is induced, or the skipping of exon 51 is induced, or the skipping of exon 53 is induced. An induction of the skipping of two of these exons is also encompassed by a method of the invention, for example, preferably skipping of exons 52 and 53, or 51 and 52. Depending on the type and the identity (the specific exons involved) of mutation identified in a patient, the skilled person will know which combination of exons needs to be skipped in said patient.

In a preferred method, one or more symptom(s) of a DMD or a BMD patient is/are alleviated and/or one or more characteristic(s) of one or more muscle cells from a DMD or a BMD patient is/are improved. Such symptoms or characteristics may be assessed at the cellular, tissue level or on the patient self.

An alleviation of one or more characteristics may be assessed by any of the following assays on a myogenic cell or muscle cell from a patient: reduced calcium uptake by muscle cells, decreased collagen synthesis, altered morphology, altered lipid biosynthesis, decreased oxidative stress, and/or improved muscle fiber function, integrity, and/or survival. These parameters are usually assessed using immunofluorescence and/or histochemical analyses of cross sections of muscle biopsies.

The improvement of muscle fiber function, integrity and/or survival may be assessed using at least one of the following assays: a detectable decrease of creatine kinase in blood, a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic, and/or a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to the skilled person.

Creatine kinase may be detected in blood as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). A detectable decrease in creatine kinase may mean a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same DMD or BMD patient before treatment.

A detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in Hodgetts et al (Hodgetts S., et al (2006), Neuromuscular Disorders, 16: 591-602.2006) using biopsy cross-sections. A detectable decrease of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same DMD or BMD patient before treatment.

A detectable increase of the homogeneity of the diameter of a muscle fiber is preferably assessed in a muscle biopsy cross-section, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). The increase is measured by comparison to the homogeneity of the diameter of a muscle fiber in a same DMD or BMD patient before treatment.

An alleviation of one or more symptoms may be assessed by any of the following assays on the patient self: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur et al. (Manzur AY et al, (2008), Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review), Wiley publishers, The Cochrane collaboration.) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy has been alleviated in an individual using a method of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). Alternatively, the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.

A treatment in a method according to the disclosure may have a duration of at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more.

Each molecule or oligonucleotide or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of a molecule or an oligonucleotide or a composition of the invention may depend on several parameters such as the age of the patient, the mutation of the patient, the number of molecules (dose), the formulation of said molecule. The frequency may be ranged between at least once in a two weeks, or three weeks or four weeks or five weeks or a longer time period.

A molecule or oligonucleotide or equivalent thereof can be delivered as is to a cell. When administering said molecule, oligonucleotide or equivalent thereof to an individual, it is preferred that it is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred for a method of the invention is the use of an excipient that will further enhance delivery of said molecule, oligonucleotide or functional equivalent thereof as defined herein, to a cell and into a cell, preferably a muscle cell. Preferred excipients are defined in the section entitled "pharmaceutical composition".

In a preferred method of the invention, an additional molecule is used which is able to induce and/or promote skipping of another exon of the DMD pre-mRNA of a patient. Preferably, the second exon is selected from: exon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 53, 55, 57, 59, 62, 63, 65, 66, 69, or 75 of the DMD pre-mRNA of a patient. Molecules which can be used are depicted in any one of Table 1 to 7. This way, inclusion of two or more exons of a DMD pre-mRNA in mRNA produced from this pre-mRNA is prevented. This embodiment is further referred to as double- or multiexon skipping (Aartsma-Rus A, Janson AA, Kaman WE, et al. Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet 2004;74(1):83-92, Aartsma-Rus A, Kaman WE, Weij R, den Dunnen JT, van Ommen GJ, van Deutekom JC. Exploring the frontiers of therapeutic exon skipping for Duchenne muscular dystrophy by double targeting within one or multiple exons. Mol Ther 2006;14(3):401-7). In most cases double-exon skipping results in the exclusion of only the two targeted exons from the DMD pre-mRNA. However, in other cases it was found that the targeted exons and the entire region in between said exons in said pre-mRNA were not present in the produced mRNA even when other exons (intervening exons) were present in such region.

It is possible to specifically promote the skipping of also the intervening exons by providing a linkage between the two complementary oligonucleotides. Hence, in one embodiment stretches of nucleotides complementary to at least two dystrophin exons are separated by a linking moiety. The at least two stretches of nucleotides are thus linked in this embodiment so as to form a single molecule.

In case, more than one compounds or molecules are used in a method of the invention, said compounds can be administered to an individual in any order. In one embodiment, said compounds are administered simultaneously (meaning that said compounds are administered within 10 hours, preferably within one hour). This is however not necessary. In another embodiment, said compounds are administered sequentially.

Molecule

In a second aspect, there is provided a molecule for use in a method as described in the previous section entitled "Method". A molecule as defined herein is preferably an oligonucleotide or antisense oligonucleotide (AON).

It was found by the present investigators that any of exon 43, 46, 50-53 is specifically skipped at a high frequency using a molecule that preferably binds to a continuous stretch of at least 8 nucleotides within said exon. Although this effect can be associated with a higher binding affinity of said molecule, compared to a molecule that binds to a continuous stretch of less than 8 nucleotides, there could be other intracellular parameters involved that favor thermodynamic, kinetic, or structural characteristics of the hybrid duplex. In a preferred embodiment, a molecule that binds to a continuous stretch of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 nucleotides within said exon is used.

In a preferred embodiment, a molecule or an oligonucleotide of the disclosure which comprises a sequence that is complementary to a part of any of exon 43, 46, 50-53 of DMD pre-mRNA is such that the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, or even more preferably at least 95%, or even more preferably 98%, and most preferably up to 100%. "A part of said exon" preferably means a stretch of at least 8 nucleotides. In a most preferred embodiment, an oligonucleotide of the invention consists of a sequence that is complementary to part of said exon DMD pre-mRNA as defined herein. For example, an oligonucleotide may comprise a sequence that is complementary to part of said exon DMD pre-mRNA as defined herein and additional flanking sequences. In a more preferred embodiment, the length of said complementary part of said oligonucleotide is of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 ,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Preferably, additional flanking sequences are used to modify the binding of a protein to said molecule or oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity.

A preferred molecule to be used in a method of the disclosure binds or is complementary to a continuous stretch of at least 8 nucleotides within one of the following nucleotide sequences selected from:

• 5'-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAU AGCAAGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3' (SEQ ID NO: 2) for skipping of exon43;
• 5'-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUG AACCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3' (SEQ ID NO: 3) for skipping of exon 46;
• 5'-GGCGGUAAACCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUG ACCUAGC UCCUGGACUGACCACUAUUGG-3' (SEQ ID NO: 4) for skipping of exon 50;
• 5'-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACU AAGGAAACUGCCAUCUCCAAACUAGAAAUGCCAUCUUCCUUGAUG UUGGAGGUAC-3' (SEQ ID NO: 5) for skipping of exon 51;
• 5'-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAU UACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3' (SEQ ID NO:6) for skipping of exon 52, and
• 5'-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAA GCUGAGCAGGUCUUAGGACAGGCCAGAG-3' (SEQ ID NO:7) for skipping of exon 53.

Of the numerous molecules that theoretically can be prepared to bind to the continuous nucleotide stretches as defined by SEQ ID NO 2-7 within one of said exons, the disclosure provides distinct molecules that can be used in a method for efficiently skipping of at least exon 52 and optionally exon 43, exon 46, exon 50, exon 51 and/or exon 53. Although the skipping effect can be addressed to the relatively high density of putative SR protein binding sites within said stretches, there could be other parameters involved that favor uptake of the molecule or other, intracellular parameters such as thermodynamic, kinetic, or structural characteristics of the hybrid duplex.

It was found that a molecule that binds to a continuous stretch comprised within or consisting of any of SEQ ID NO 2-7 results in highly efficient skipping of exon 43, exon 46 and/or exon 50-53 respectively in a cell and/or in a patient provided with this molecule. Therefore, in a preferred embodiment, a method is provided wherein a molecule binds to a continuous stretch of at least 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50 nucleotides within SEQ ID NO 2-7.

In a preferred embodiment for inducing and/or promoting the skipping of exon 52 and optionally exon 43, exon 46, exon 50, exon 51 and/or exon 53, the invention provides a molecule comprising or consisting of an antisense nucleotide sequence selected from the antisense nucleotide sequences depicted in any of Tables 1 to 6. A molecule of the invention comprises or consist of the antisense nucleotide sequence of SEQ ID NO 287, and optionally a further molecule of the disclosure preferably comprises or consist of the antisense nucleotide sequence of SEQ ID NO 16, SEQ ID NO 65, SEQ ID NO 70, SEQ ID NO 91, SEQ ID NO 110, SEQ ID NO 117, SEQ ID NO 127, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 246, SEQ ID NO 299, SEQ ID NO 357.

A preferred molecule of the invention comprises a nucleotide-based or nucleotide or an antisense oligonucleotide sequence of between 22 and 30 nucleotides or bases, such as 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides. A most preferred molecule of the invention comprises a nucleotide-based sequence of 25 nucleotides.

Furthermore, none of the indicated sequences is derived from conserved parts of splice-junction sites. Therefore, said molecule is not likely to mediate differential splicing of other exons from the DMD pre-mRNA or exons from other genes.

In one embodiment, a molecule of the invention is a compound molecule that binds to the specified sequence, or a protein such as an RNA-binding protein or a non-natural zinc-finger protein that has been modified to be able to bind to the corresponding nucleotide sequence on a DMD pre-RNA molecule. Methods for screening compound molecules that bind specific nucleotide sequences are, for example, disclosed in PCT/NL01/00697 and US Patent 6,875,736 . Methods for designing RNA-binding Zinc-finger proteins that bind specific nucleotide sequences are disclosed by Friesen and Darby, Nature Structural Biology 5: 543- 546 (1998).

A preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 2: 5'-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAU AGCAAGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3' which is present in exon 43 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 8 to SEQ ID NO 69.

In an even more preferred embodiment, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 16 and/or SEQ ID NO 65.

In a most preferred aspect, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 65. It was found that this molecule is very efficient in modulating splicing of exon 43 of the DMD pre-mRNA in a muscle cell and/or in a patient.

Another preferred molecule of the disclosure binds to at least part of the sequence of SEQ ID NO 3: 5'-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUG AACCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3' which is present in exon 46 of the DMD gene. More preferably, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 70 to SEQ ID NO 122.

In an even more preferred aspect, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 70, SEQ ID NO 91, SEQ ID NO 110, and/or SEQ ID NO117.

In a most preferred aspect, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 117. It was found that this molecule is very efficient in modulating splicing of exon 46 of the DMD pre-mRNA in a muscle cell or in a patient.

Another preferred molecule of the disclosure binds to at least part of the sequence of SEQ ID NO 4: 5'-GGCGGUAAACCGUUUACUUCAAGAGCU GAGGGCAAAGCAGCCUG ACCUAGCUCCUGGACUGACCACUAUUGG-3' which is present in exon 50 of the DMD gene. More preferably, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 123 to SEQ ID NO 167 and/or SEQ ID NO 529 to SEQ ID NO 535.

In an even more preferred aspect, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 127, or SEQ ID NO 165, or SEQ ID NO 166 and/or SEQ ID NO 167.

In a most preferred aspect, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 127. It was found that this molecule is very efficient in modulating splicing of exon 50 of the DMD pre-mRNA in a muscle cell and/or in a patient.

Another preferred molecule of the disclosure binds to at least part of the sequence of SEQ ID NO 5: 5'-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACU AAGGAAACUGCCAUCUCCAAACUAGAAAUGCCAUCUUCCUUGAUG UUGGAGGUAC-3' which is present in exon 51 of the DMD gene. More preferably, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 168 to SEQ ID NO 241.

Another preferred molecule of the disclosure binds to at least part of the sequence of SEQ ID NO 6: 5'-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAU UACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3' which is present in exon 52 of the DMD gene. The molecule of the invention comprises or consists of the antisense nucleotide sequence of SEQ ID NO 287. In a preferred aspect, the disclosure further provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 242 to SEQ ID NO 310. In an even more preferred aspect, the disclosure further provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 246 and/or SEQ ID NO 299. In a most preferred aspect, the disclosure further provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 299. It was found that this molecule is very efficient in modulating splicing of exon 52 of the DMD pre-mRNA in a muscle cell and/or in a patient.

Another preferred molecule of the disclosure binds to at least part of the sequence of SEQ ID NO 7: 5'-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAA GCUGAGCAGGUCUUAGGACAGGCCAGAG-3' which is present in exon 53 of the DMD gene. More preferably, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 311 to SEQ ID NO 358.

In a most preferred aspect, the disclosure provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 357. It was found that this molecule is very efficient in modulating splicing of exon 53 of the DMD pre-mRNA in a muscle cell and/or in a patient.

A nucleotide sequence of a molecule of the invention may contain RNA residues, or one or more DNA residues, and/or one or more nucleotide analogues or equivalents, as will be further detailed herein below.

It is preferred that a molecule of the invention comprises one or more residues that are modified to increase nuclease resistance, and/or to increase the affinity of the antisense nucleotide for the target sequence. Therefore, in a preferred embodiment, the antisense nucleotide sequence comprises at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.

In a preferred embodiment, the nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.

It is further preferred that that the linkage between the residues in a backbone do not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.

A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a onecarbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566-568).

A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.

In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3'-alkylene phosphonate, 5'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.

A further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2', 3' and/or 5' position such as a -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; -aminoxy; methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably a ribose or a derivative thereof, or a deoxyribose or a derivative thereof. Such preferred derivatized sugar moieties comprise Locked Nucleic Acid (LNA), in which the 2'-carbon atom is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2'-O,4'-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA.

It is understood by a skilled person that it is not necessary for all positions in an antisense oligonucleotide to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single antisense oligonucleotide or even at a single position within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents.

A preferred antisense oligonucleotide according to the invention comprises a 2'-O alkyl phosphorothioate antisense oligonucleotide, such as 2'-O-methyl modified ribose (RNA), 2'-O-ethyl modified ribose, 2'-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.

A most preferred antisense oligonucleotide according to the invention comprises of 2'-O-methyl phosphorothioate ribose.

A functional equivalent of a molecule of the invention may be defined as an oligonucleotide as defined herein wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is inducing exon 43, 46, 50, 51, 52, or 53 skipping and providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by detection of exon 43, 46, 50, 51, 52, or 53 skipping and by quantifying the amount of functional dystrophin protein. A functional dystrophin is herein preferably defined as being a dystrophin able to bind actin and members of the DGC protein complex. The assessment of said activity of an oligonucleotide is preferably done by RT-PCR or by immunofluorescence or Western blot analyses. Said activity is preferably retained to at least some extent when it represents at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% or more of corresponding activity of said oligonucleotide the functional equivalent derives from. Throughout this application, when the word oligonucleotide is used it may be replaced by a functional equivalent thereof as defined herein.

It will be understood by a skilled person that distinct antisense oligonucleotides can be combined for efficiently skipping exon 52 and optionally any of exon 43, exon 46, exon 50, exon 51 and/or exon 53 of the human DMD pre-mRNA. It is encompassed by the present invention to use one, two, three, four, five or more oligonucleotides for skipping one of said exons (i.e. exon 52 and optionally exon 43, 46, 50, 51, or 53). It is also encompassed to use at least two oligonucleotides for skipping at least two of said exons. Preferably two of said exons are skipped. More preferably, these two exons are:

• 51 and 52, or
• 52 and 53.

The skilled person will know which combination of exons is preferred to be skipped depending on the type, the number and the location of the mutation present in a DMD or BMD patient.

An antisense oligonucleotide can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells, preferably muscle cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.

A preferred antisense oligonucleotide comprises a peptide-linked PMO.

A preferred antisense oligonucleotide comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an antisense oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an antisense oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells. Therefore, in one embodiment it is preferred to use a combination of antisense oligonucleotides comprising different nucleotide analogs or equivalents for inducing skipping of exon 52 and optionally exon 43, 46, 50, 51, or 53 of the human DMD pre-mRNA.

A cell can be provided with a molecule capable of interfering with essential sequences that result in highly efficient skipping of exon 43, exon 46, exon 50, exon 51, exon 52 or exon 53 of the human DMD pre-mRNA by plasmid-derived antisense oligonucleotide expression or viral expression provided by adenovirus- or adeno-associated virus-based vectors. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette that drives expression of a molecule as identified herein. Expression is preferably driven by a polymerase III promoter, such as a U1, a U6, or a U7 RNA promoter. A muscle or myogenic cell can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution. Alternatively, a plasmid can be provided by transfection using known transfection agents such as, for example, LipofectAMINE^™ 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), or derivatives thereof.

One preferred antisense oligonucleotide expression system is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AA V-based vectors have been developed that can be used for prolonged expression of small antisense nucleotide sequences for highly efficient skipping of exon 43, 46, 50, 51, 52 or 53 of the DMD pre-mRNA.

A preferred AAV-based vector comprises an expression cassette that is driven by a polymerase III-promoter (Pol III). A preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA promoter.

The invention therefore also provides a viral-based vector, comprising a Pol III-promoter driven expression cassette for expression of one or more antisense sequences of the invention for inducing skipping of exon 52 and optionally exon 43, exon 46, exon 50, exon 51, or exon 53 of the human DMD pre-mRNA.

Pharmaceutical composition

If required, a molecule or a vector expressing an antisense oligonucleotide of the invention can be incorporated into a pharmaceutically active mixture or composition by adding a pharmaceutically acceptable carrier.

Therefore, in a further aspect, the invention provides a composition, preferably a pharmaceutical composition comprising a molecule comprising an antisense oligonucleotide according to the invention, and/or a viral-based vector expressing the antisense sequence(s) according to the invention and a pharmaceutically acceptable carrier.

A preferred pharmaceutical composition comprises a molecule as defined herein and/or a vector as defined herein, and a pharmaceutical acceptable carrier or excipient, optionally combined with a molecule and/or a vector as defined herein which is able to induce skipping of exon 52 and optionally exon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 53, 55, 57, 59, 62, 63, 65, 66, 69, or 75 of the DMD pre-mRNA. Preferred molecules able to induce skipping of any of these exons are identified in any one of Tables 1 to 7.

Preferred excipients include excipients capable of forming complexes, vesicles and/or liposomes that deliver such a molecule as defined herein, preferably an oligonucleotide complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine and derivatives, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, ExGen 500, synthetic amphiphils (SAINT-18), lipofectin^™, DOTAP and/or viral capsid proteins that are capable of self-assembly into particles that can deliver such molecule, preferably an oligonucleotide as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver (oligonucleotide such as antisense) nucleic acids to a wide variety of cultured cells, including muscle cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.

Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine(DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.

Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver a molecule or a compound as defined herein, preferably an oligonucleotide across cell membranes into cells.

In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate a compound as defined herein, preferably an oligonucleotide as colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of a compound as defined herein, preferably an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver a compound as defined herein, preferably an oligonucleotide for use in the current invention to deliver said compound for the treatment of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in humans.

In addition, a compound as defined herein, preferably an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake into the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an a compound as defined herein, preferably an oligonucleotide from vesicles, e.g. endosomes or lysosomes.

Therefore, in a preferred embodiment, a compound as defined herein, preferably an oligonucleotide are formulated in a medicament which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising a compound as defined herein, preferably an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. It is to be understood that a molecule or compound or oligonucleotide may not be formulated in one single composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each compound.

In a preferred embodiment, an in vitro concentration of a molecule or an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 µM is used. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM. A molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If several molecules or oligonucleotides are used, these concentrations may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.

More preferably, a compound preferably an oligonucleotide to be used in the invention to prevent, treat DMD or BMD are synthetically produced and administered directly to a cell, a tissue, an organ and/or patients in formulated form in a pharmaceutically acceptable composition or preparation. The delivery of a pharmaceutical composition to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body.

A preferred oligonucleotide as defined herein optionally comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells.

In this respect, systemic delivery of an oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting a different subset of muscle cells. Therefore, in this embodiment, it is preferred to use a combination of oligonucleotides comprising different nucleotide analogs or equivalents for modulating splicing of the DMD mRNA in at least one type of muscle cells.

In a preferred embodiment, there is provided a molecule or a viral-based vector for use as a medicament, preferably for modulating splicing of the DMD pre-mRNA, more preferably for promoting or inducing skipping of exon 52 and optionally any of exon 43, 46, 50, 51 and/or 53 as identified herein.

Use

In yet a further aspect, the invention provides the use of an antisense oligonucleotide or molecule according to the invention, and/or a viral-based vector that expresses one or more antisense sequences according to the invention and/or a pharmaceutical composition, for modulating splicing of the DMD pre-mRNA in a human myogenic cell or muscle cell in vitro. The invention further relates to the use of the molecule as defined herein and/or the vector as defined herein and/or the pharmaceutical composition as defined herein for the preparation of a medicament for the treatment of a DMD or BMD patient.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a molecule or a viral- based vector or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

Each embodiment as identified herein may be combined together unless otherwise indicated.

Examples Illustrative Examples 1-4 Materials and methods

AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program, on (partly) overlapping putative SRprotein binding sites as predicted by the ESE-finder software. AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2'-O-methyl RNA and full-length phosphorothioate (PS) backbones.

Tissue culturing, transfection and RT-PCR analysis

Myotube cultures derived from a healthy individual ("human control") (examples 1, 3, and 4; exon 43, 50, 52 skipping) or a DMD patient carrying an exon 45 deletion (example 2; exon 46 skipping) were processed as described previously (Aartsma-Rus et al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 50 nM and 150 nM (example 2), 200nM and 500 nM (example 4) or 500nM only (examples 1 and 3) of each AON. Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 □1 UNIFectylin per □g AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking the targeted exons (43, 46, 50, 51, 52, or 53). PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the DNA 1000 LabChips Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA).

Results DMD exon 43 skipping.

A series of AONs targeting sequences within exon 43 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 43 herein defined as SEQ ID NO 2, was indeed capable of inducing exon 43 skipping. PS237 (SEQ ID NO: 65) reproducibly induced highest levels of exon 43 skipping (up to 66%) at 500nM, as shown in Figure 1. For comparison, also PS238 and PS240 are shown, inducing exon 43 skipping levels up to 13% and 36% respectively (Figure 1). The precise skipping of exon 43 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 43 skipping was observed in non-treated cells (NT).

DMD exon 46 skipping.

A series of AONs targeting sequences within exon 46 were designed and transfected in myotube cultures derived from a DMD patient carrying an exon 45 deletion in the DMD gene. For patients with such mutation antisense-induced exon 46 skipping would induce the synthesis of a novel, BMD-like dystrophin protein that may indeed alleviate one or more symptoms of the disease. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 46 herein defined as SEQ ID NO 3, was indeed capable of inducing exon 46 skipping, even at relatively low AON concentrations of 50 nM. PS182 (SEQ ID NO: 117) reproducibly induced highest levels of exon 46 skipping (up to 50% at 50 nM and 74% at 150 nM), as shown in Figure 2. For comparison, also PS177, PS179, and PS181 are shown, inducing exon 46 skipping levels up to 55%, 58% and 42% respectively at 150 nM (Figure 2). The precise skipping of exon 46 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 46 skipping was observed in non-treated cells (NT).

DMD exon 50 skipping.

A series of AONs targeting sequences within exon 50 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 50 herein defined as SEQ ID NO 4, was indeed capable of inducing exon 50 skipping. PS248 (SEQ ID NO: 127) reproducibly induced highest levels of exon 50 skipping (up to 35% at 500 nM), as shown in Figure 3. For comparison, also PS245, PS246, and PS247 are shown, inducing exon 50 skipping levels up to 14-16% at 500 nM (Figure 3). The precise skipping of exon 50 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 50 skipping was observed in non-treated cells (NT).

DMD exon 51 skipping.

A series of AONs targeting sequences within exon 51 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 51 herein defined as SEQ ID NO 5, was indeed capable of inducing exon 51 skipping. The AON with SEQ ID NO 180 reproducibly induced highest levels of exon 51 skipping (not shown).

DMD exon 52 skipping.

A series of AONs targeting sequences within exon 52 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 52 herein defined as SEQ ID NO 6, was indeed capable of inducing exon 52 skipping. PS236 (SEQ ID NO: 299) reproducibly induced highest levels of exon 52 skipping (up to 88% at 200 nM and 91% at 500 nM), as shown in Figure 4. For comparison, also PS232 and AON 52-1 (previously published by Aartsma-Rus et al. Oligonucleotides 2005) are shown, inducing exon 52 skipping at levels up to 59% and 10% respectively when applied at 500 nM (Figure 4). The precise skipping of exon 52 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 52 skipping was observed in non-treated cells (NT).

DMD exon 53 skipping.

A series of AONs targeting sequences within exon 53 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 53 herein defined as SEQ ID NO 7, was indeed capable of inducing exon 53 skipping. The AON with SEQ ID NO 328 reproducibly induced highest levels of exon 53 skipping (not shown).

Sequence listing: DMD gene amino acid sequence

• SEQ ID NO 1:
• SEQ ID NO 2 (exon 43):
• SEQ ID NO 3 (exon 46):
• SEQ ID NO 4 (exon 50):
• SEQ ID NO 5 (exon 51):
• SEQ ID NO 6 (exon 52):
• SEQ ID NO 7 (exon 53):

SEQ ID NO 8	CCACAGGCGUUGCACUUUGCAAUGC	SEQ ID NO 39	UCUUCUUGCUAUGAAUAAUGUCAAU
SEQ ID NO 9	CACAGGCGUUGCACUUUGCAAUGCU	SEQ ID NO 40	CUUCUUGCUAUGAAUAAUGUCAAUC
SEQ ID NO 10	ACAGGCGUUGCACUUUGCAAUGCUG	SEQ ID NO 41	UUCUUGCUAUGAAUAAUGUCAAUCC
SEQ ID NO 11	CAGGCGUUGCACUUUGCAAUGCUGC	SEQ ID NO 42	UCUUGCUAUGAAUAAUGUCAAUCCG
SEQ ID NO 12	AGGCGUUGCACUUUGCAAUGCUGCU	SEQ ID NO 43	CUUGCUAUGAAUAAUGUCAAUCCGA
SEQ ID NO 13	GGCGUUGCACUUUGCAAUGCUGCUG	SEQ ID NO 44	UUGCUAUGAAUAAUGUCAAUCCGAC
SEQ ID NO 14	GCGUUGCACUUUGCAAUGCUGCUGU	SEQ ID NO 45	UGCUAUGAAUAAUGUCAAUCCGACC
SEQ ID NO 15	CGUUGCACUUUGCAAUGCUGCUGUC	SEQ ID NO 46	GCUAUGAAUAAUGUCAAUCCGACCU
CGUUGCACUUUGCAAUGCUGCUG	SEQ ID NO 47	CUAUGAAUAAUGUCAAUCCGACCUG
SEQ ID NO 17	GUUGCACUUUGCAAUGCUGCUGUCU	SEQ ID NO 48	UAUGAAUAAUGUCAAUCCGACCUGA
SEQ ID NO 18	UUGCACUUUGCAAUGCUGCUGUCUU	SEQ ID NO 49	AUGAAUAAUGUCAAUCCGACCUGAG
SEQ ID NO 19	UGCACUUUGCAAUGCUGCUGUCUUC	SEQ ID NO 50	UGAAUAAUGUCAAUCCGACCUGAGC
SEQ ID NO 20	GCACUUUGCAAUGCUGCUGUCUUCU	SEQ ID NO 51	GAAUAAUGUCAAUCCGACCUGAGCU
SEQ ID NO 21	CACUUUGCAAUGCUGCUGUCUUCUU	SEQ ID NO 52	AAUAAUGUCAAUCCGACCUGAGCUU
SEQ ID NO 22	ACUUUGCAAUGCUGCUGUCUUCUUG	SEQ ID NO 53	AUAAUGUCAAUCCGACCUGAGCUUU
SEQ ID NO 23	CUUUGCAAUGCUGCUGUCUUCUUGC	SEQ ID NO 54	UAAUGUCAAUCCGACCUGAGCUUUG
SEQ ID NO 24	UUUGCAAUGCUGCUGUCUUCUUGCU	SEQ ID NO 55	AAUGUCAAUCCGACCUGAGCUUUGU
SEQ ID NO 25	UUGCAAUGCUGCUGUCUUCUUGCUA	SEQ ID NO 56	AUGUCAAUCCGACCUGAGCUUUGUU
SEQ ID NO 26	UGCAAUGCUGCUGUCUUCUUGCUAU	SEQ ID NO 57	UGUCAAUCCGACCUGAGCUUUGUUG
SEQ ID NO 27	GCAAUGCUGCUGUCUUCUUGCUAUG	SEQ ID NO 58	GUCAAUCCGACCUGAGCUUUGUUGU
SEQ ID NO 28	CAAUGCUGCUGUCUUCUUGCUAUGA	SEQ ID NO 59	UCAAUCCGACCUGAGCUUUGUUGUA
SEQ ID NO 29	AAUGCUGCUGUCUUCUUGCUAUGAA	SEQ ID NO 60	CAAUCCGACCUGAGCUUUGUUGUAG
SEQ ID NO 30	AUGCUGCUGUCUUCUUGCUAUGAAU	SEQ ID NO 61	AAUCCGACCUGAGCUUUGUUGUAGA
SEQ ID NO 31	UGCUGCUGUCUUCUUGCUAUGAAUA	SEQ ID NO 62	AUCCGACCUGAGCUUUGUUGUAGAC
SEQ ID NO 32	GCUGCUGUCUUCUUGCUAUGAAUAA	SEQ ID NO 63	UCCGACCUGAGCUUUGUUGUAGACU
SEQ ID NO 33	CUGCUGUCUUCUUGCUAUGAAUAAU	SEQ ID NO 64	CCGACCUGAGCUUUGUUGUAGACUA
SEQ ID NO 34	UGCUGUCUUCUUGCUAUGAAUAAUG	SEQ ID NO 65 PS237	CGACCUGAGCUUUGUUGUAG
SEQ ID NO 35	GCUGUCUUCUUGCUAUGAAUAAUGU	SEQ ID NO 66 PS238	CGACCUGAGCUUUGUUGUAGACUAU
SEQ ID NO 36	CUGUCUUCUUGCUAUGAAUAAUGUC	SEQ ID NO 67	GACCUGAGCUUUGUUGUAGACUAUC
SEQ ID NO 37	UGUCUUCUUGCUAUGAAUAAUGUCA	SEQ ID NO 68	ACCUGAGCUUUGUUGUAGACUAUCA
SEQ ID NO 38	GUCUUCUUGCUAUGAAUAAUGUCAA	SEQ ID NO 69	CCUGA GCUUU GUUGU AGACU AUC

GCUUUUCUUUUAGUUGCUGCUCUUU	SEQ ID NO 51 NO 97	CCAGGUUCAAGUGGGAUACUAGCAA
SEQ ID NO 71	CUUUUCUUUUAGUUGCUGCUCUUUU	SEQ ID NO 51 NO 98	CAGGUUCAAGUGGGAUACUAGCAAU
SEQ ID NO 51 NO 72	UUUUCUUUUAGUUGCUGCUCUUUUC	SEQ ID NO 51 NO 99	AGGUUCAAGUGGGAUACUAGCAAUG
SEQ ID NO 51 NO 73	UUUCUUUUAGUUGCUGCUCUUUUCC	SEQ ID NO 51 NO 100	GGUUCAAGUGGGAUACUAGCAAUGU
SEQ ID NO 74	UUCUUUUAGUUGCUGCUCUUUUCCA	SEQ ID NO 51 NO 101	GUUCAAGUGGGAUACUAGCAAUGUU
SEQ ID NO 51 NO 75	UCUUUUAGUUGCUGCUCUUUUCCAG	SEQ ID NO 51 NO 102	UUCAAGUGGGAUACUAGCAAUGUUA
SEQ ID NO 76	CUUUUAGUUGCUGCUCUUUUCCAGG	SEQ ID NO 103	UCAAGUGGGAUACUAGCAAUGUUAU
SEQ ID NO 51 NO 77	UUUUAGUUGCUGCUCUUUUCCAGGU	SEQ ID NO 104	CAAGUGGGAUACUAGCAAUGUUAUC
SEQ ID NO 51 NO 78	UUUAGUUGCUGCUCUUUUCCAGGUU	SEQ ID NO 51 NO 105	AAGUGGGAUACUAGCAAUGUUAUCU
SEQ ID NO 51 NO 79	UUAGUUGCUGCUCUUUUCCAGGUUC	SEQ ID NO 106	AGUGGGAUACUAGCAAUGUUAUCUG
SEQ ID NO 51 NO 80	UAGUUGCUGCUCUUUUCCAGGUUCA	SEQ ID NO 107	GUGGGAUACUAGCAAUGUUAUCUGC
SEQ ID NO 51 NO 81	AGUUGCUGCUCUUUUCCAGGUUCAA	SEQ ID NO 108	UGGGAUACUAGCAAUGUUAUCUGCU
SEQ ID NO 51 NO 82	GUUGCUGCUCUUUUCCAGGUUCAAG	SEQID NO 109	GGGAUACUAGCAAUGUUAUCUGCUU
SEQ ID NO 83	UUGCUGCUCUUUUCCAGGUUCAAGU	GGAUACUAGCAAUGUUAUCUGCUUC
SEQ ID NO 51 NO 84	UGCUGCUCUUUUCCAGGUUCAAGUG	SEQ ID NO 51 NO 111	GAUACUAGCAAUGUUAUCUGCUUCC
SEQ ID NO 85	GCUGCUCUUUUCCAGGUUCAAGUGG	SEQ ID NO 51 NO 112	AUACUAGCAAUGUUAUCUGCUUCCU
SEQ ID NO 86	CUGCUCUUUUCCAGGUUCAAGUGGG	SEQ ID NO 113	UACUAGCAAUGUUAUCUGCUUCCUC
SEQ ID NO 87	UGCUCUUUUCCAGGUUCAAGUGGGA	SEQ ID NO 51 NO 114	ACUAGCAAUGUUAUCUGCUUCCUCC
SEQ ID NO 51 NO 88	GCUCUUUUCCAGGUUCAAGUGGGAC	SEQ ID NO 51 NO 115	CUAGCAAUGUUAUCUGCUUCCUCCA
SEQ ID NO 89	CUCUUUUCCAGGUUCAAGUGGGAUA	SEQ ID NO 116	UAGCAAUGUUAUCUGCUUCCUCCAA
SEQ ID NO 90	UCUUUUCCAGGUUCAAGUGGGAUAC	AGCAAUGUUAUCUGCUUCCUCCAAC
UCUUUUCCAGGUUCAAGUGG	SEQ ID NO 118	GCAAUGUUAUCUGCUUCCUCCAACC
SEQ ID NO 51 NO 92	CUUUUCCAGGUUCAAGUGGGAUACU	SEQ ID NO 51 NO 119	CAAUGUUAUCUGCUUCCUCCAACCA
SEQ ID NO 93	UUUUCCAGGUUCAAGUGGGAUACUA	SEQ ID NO 120	AAUGUUAUCUGCUUCCUCCAACCAU
SEQ ID NO 94	UUUCCAGGUUCAAGUGGGAUACUAG	SEQ ID NO 51 NO 121	AUGUUAUCUGCUUCCUCCAACCAUA
SEQ ID NO 51 NO 95	UUCCAGGUUCAAGUGGGAUACUAGC	SEQ ID NO 51 NO 122	UGUUAUCUGCUUCCUCCAACCAUAA
SEQ ID NO 96	UCCAGGUUCAAGUGGGAUACUAGCA

SEQ ID NO 123	CCAAUAGUGGUCAGUCCAGGAGCUA	SEQ ID NO 146	CUAGGUCAGGCUGCUUUGCCCUCAG
SEQ ID NO 124	CAAUAGUGGUCAGUCCAGGAGCUAG	SEQ ID NO 147	UAGGUCAGGCUGCUUUGCCCUCAGC
SEQ ID NO 125	AAUAGUGGUCAGUCCAGGAGCUAGG	SEQ ID NO 148	AGGUCAGGCUGCUUUGCCCUCAGCU
SEQ ID NO 126	AUAGUGGUCAGUCCAGGAGCUAGGU	SEQ ID NO 149	GGUCAGGCUGCUUUGCCCUCAGCUC
AUAGUGGUCAGUCCAGGAGCU	SEQ ID NO 150	GUCAGGCUGCUUUGCCCUCAGCUCU
SEQ ID NO 128	UAGUGGUCAGUCCAGGAGCUAGGUC	SEQ ID NO 151	UCAGGCUGCUUUGCCCUCAGCUCUU
SEQ ID NO 129	AGUGGUCAGUCCAGGAGCUAGGUCA	SEQ ID NO 152	CAGGCUGCUUUGCCCUCAGCUCUUG
SEQ ID NO 130	GUGGUCAGUCCAGGAGCUAGGUCAG	SEQ ID NO 153	AGGCUGCUUUGCCCUCAGCUCUUGA
SEQ ID NO 131	UGGUCAGUCCAGGAGCUAGGUCAGG	SEQ ID NO 154	GGCUGCUUUGCCCUCAGCUCUUGAA
SEQ ID NO 132	GGUCAGUCCAGGAGCUAGGUCAGGC	SEQ ID NO 155	GCUGCUUUGCCCUCAGCUCUUGAAG
SEQ ID NO 133	GUCAGUCCAGGAGCUAGGUCAGGCU	SEQ ID NO 156	CUGCUUUGCCCUCAGCUCUUGAAGU
SEQ ID NO 134	UCAGUCCAGGAGCUAGGUCAGGCUG	SEQ ID NO 157	UGCUUUGCCCUCAGCUCUUGAAGUA
SEQ ID NO 135	CAGUCCAGGAGCUAGGUCAGGCUGC	SEQ ID NO 158	GCUUUGCCCUCAGCUCUUGAAGUAA
SEQ ID NO 136	AGUCCAGGAGCUAGGUCAGGCUGCU	SEQ ID NO 159	CUUUGCCCUCAGCUCUUGAAGUAAA
SEQ ID NO 137	GUCCAGGAGCUAGGUCAGGCUGCUU	SEQ ID NO 160	UUUGCCCUCAGCUCUUGAAGUAAAC
SEQ ID NO 138	UCCAGGAGCUAGGUCAGGCUGCUUU	SEQ ID NO 161	UUGCCCUCAGCUCUUGAAGUAAACG
SEQ ID NO 139	CCAGGAGCUAGGUCAGGCUGCUUUG	SEQ ID NO 162	UGCCCUCAGCUCUUGAAGUAAACGG
SEQ ID NO 140	CAGGAGCUAGGUCAGGCUGCUUUGC	SEQ ID NO 163	GCCCUCAGCUCUUGAAGUAAACGGU
SEQ ID NO 141	AGGAGCUAGGUCAGGCUGCUUUGCC	SEQ ID NO 164	CCCUCAGCUCUUGAAGUAAACGGUU
SEQ ID NO 142	GGAGCUAGGUCAGGCUGCUUUGCCC	SEQ ID NO 165 PS246	CCUCAGCUCUUGAAGUAAAC
SEQ ID NO 143	GAGCUAGGUCAGGCUGCUUUGCCCU	CCUCAGCUCUUGAAGUAAACG
SEQ ID NO 144	AGCUAGGUCAGGCUGCUUUGCCCUC	CUCAGCUCUUGAAGUAAACG
SEQ ID NO 145	GCUAGGUCAGGCUGCUUUGCCCUCA	SEQ ID NO 529	CCUCAGCUCUUGAAGUAAACGGUUU
SEQ ID NO 530	CUCAGCUCUUGAAGUAAACGGUUUA	SEQ ID NO 531	UCAGCUCUUGAAGUAAACGGUUUAC
SEQ ID NO 532	CAGCUCUUGAAGUAAACGGUUUACC	SEQ ID NO 533	AGCUCUUGAAGUAAACGGUUUACCG
SEQ ID NO 534	GCUCUUGAAGUAAACGGUUUACCGC	SEQ ID NO 535	CUCUUGAAGUAAACGGUUUACCGCC

SEQ ID NO 168	GUACCUCCAACAUCAAGGAAGAUGG	SEQ ID NO 51 NO 205	GAGAUGGCAGUUUCCUUAGUAACCA
SEQ ID NO 169	UACCUCCAACAUCAAGGAAGAUGGC	SEQ ID NO 51 NO 206	AGAUGGCAGUUUCCUUAGUAACCAC
SEQ ID NO 170	ACCUCCAACAUCAAGGAAGAUGGCA	SEQ ID NO 51 NO 207	GAUGGCAGUUUCCUUAGUAACCACA
SEQ ID NO 171	CCUCCAACAUCAAGGAAGAUGGCAU	SEQ ID NO 208	AUGGCAGUUUCCUUAGUAACCACAG
SEQ ID NO 172	CUCCAACAUCAAGGAAGAUGGCAUU	SEQ ID NO 209	UGGCAGUUUCCUUAGUAACCACAGG
SEQ ID NO 51 NO 173	UCCAACAUCAAGGAAGAUGGCAUUU	SEQ ID NO 51 NO 210	GGCAGUUUCCUUAGUAACCACAGGU
SEQ ID NO 51 NO 174	CCAACAUCAAGGAAGAUGGCAUUUC	SEQ ID NO 51 NO 211	GCAGUUUCCUUAGUAACCACAGGUU
SEQ ID NO 51 NO 175	CAACAUCAAGGAAGAUGGCAUUUCU	SEQ ID NO 51 NO 212	CAGUUUCCUUAGUAACCACAGGUUG
SEQ ID NO 176	AACAUCAAGGAAGAUGGCAUUUCUA	SEQ ID NO 213	AGUUUCCUUAGUAACCACAGGUUGU
SEQ ID NO 177	ACAUCAAGGAAGAUGGCAUUUCUAG	SEQ ID NO 214	GUUUCCUUAGUAACCACAGGUUGUG
SEQ ID NO 178	CAUCAAGGAAGAUGGCAUUUCUAGU	SEQ ID NO 215	UUUCCUUAGUAACCACAGGUUGUGU
SEQ ID NO 179	AUCAAGGAAGAUGGCAUUUCUAGUU	SEQ ID NO 216	UUCCUUAGUAACCACAGGUUGUGUC
SEQ ID NO 180	UCAAGGAAGAUGGCAUUUCUAGUUU	SEQ ID NO 217	UCCUUAGUAACCACAGGUUGUGUCA
SEQ ID NO 51 NO 181	CAAGGAAGAUGGCAUUUCUAGUUUG	SEQ ID NO 218	CCUUAGUAACCACAGGUUGUGUCAC
SEQ ID NO 182	AAGGAAGAUGGCAUUUCUAGUUUGG	SEQ ID NO 51 NO 219	CUUAGUAACCACAGGUUGUGUCACC
SEQ ID NO 183	AGGAAGAUGGCAUUUCUAGUUUGGA	SEQ ID NO 220	UUAGUAACCACAGGUUGUGUCACCA
SEQ ID NO 51 NO 184	GGAAGAUGGCAUUUCUAGUUUGGAG	SEQ ID NO 221	UAGUAACCACAGGUUGUGUCACCAG
SEQ ID NO 185	GAAGAUGGCAUUUCUAGUUUGGAGA	SEQ ID NO 222	AGUAACCACAGGUUGUGUCACCAGA
SEQ ID NO 186	AAGAUGGCAUUUCUAGUUUGGAGAU	SEQ ID NO 51 NO 223	GUAACCACAGGUUGUGUCACCAGAG
SEQ ID NO 187	AGAUGGCAUUUCUAGUUUGGAGAUG	SEQ ID NO 224	UAACCACAGGUUGUGUCACCAGAGU
SEQ ID NO 188	GAUGGCAUUUCUAGUUUGGAGAUGG	SEQ ID NO 51 NO 225	AACCACAGGUUGUGUCACCAGAGUA
SEQ ID NO 51 NO 189	AUGGCAUUUCUAGUUUGGAGAUGGC	SEQ ID NO 51 NO 226	ACCACAGGUUGUGUCACCAGAGUAA
SEQ ID NO 190	UGGCAUUUCUAGUUUGGAGAUGGCA	SEQ ID NO 227	CCACAGGUUGUGUCACCAGAGUAAC
SEQ ID NO 191	GGCAUUUCUAGUUUGGAGAUGGCAG	SEQ ID NO 228	CACAGGUUGUGUCACCAGAGUAACA
SEQ ID NO 192	GCAUUUCUAGUUUGGAGAUGGCAGU	SEQ ID NO 229	ACAGGUUGUGUCACCAGAGUAACAG
SEQ ID NO 51 NO 193	CAUUUCUAGUUUGGAGAUGGCAGUU	SEQ ID NO 230	CAGGUUGUGUCACCAGAGUAACAGU
SEQ ID NO 194	AUUUCUAGUUUGGAGAUGGCAGUUU	SEQ ID NO 231	AGGUUGUGUCACCAGAGUAACAGUC
SEQ ID NO 195	UUUCUAGUUUGGAGAUGGCAGUUUC	SEQ ID NO 232	GGUUGUGUCACCAGAGUAACAGUCU
SEQ ID NO 196	UUCUAGUUUGGAGAUGGCAGUUUCC	SEQ ID NO 233	GUUGUGUCACCAGAGUAACAGUCUG
SEQ ID NO 197	UCUAGUUUGGAGAUGGCAGUUUCCU	SEQ ID NO 234	UUGUGUCACCAGAGUAACAGUCUGA
SEQ ID NO 198	CUAGUUUGGAGAUGGCAGUUUCCUU	SEQ ID NO 235	UGUGUCACCAGAGUAACAGUCUGAG
SEQ ID NO 199	UAGUUUGGAGAUGGCAGUUUCCUUA	SEQ ID NO 236	GUGUCACCAGAGUAACAGUCUGAGU
SEQ ID NO 200	AGUUUGGAGAUGGCAGUUUCCUUAG	SEQ ID NO 237	UGUCACCAGAGUAACAGUCUGAGUA
SEQ ID NO 201	GUUUGGAGAUGGCAGUUUCCUUAGU	SEQ ID NO 238	GUCACCAGAGUAACAGUCUGAGUAG
SEQ ID NO 202	UUUGGAGAUGGCAGUUUCCUUAGUA	SEQ ID NO 239	UCACCAGAGUAACAGUCUGAGUAGG
SEQ ID NO 203	UUGGAGAUGGCAGUUUCCUUAGUAA	SEQ ID NO 51 NO 240	CACCAGAGUAACAGUCUGAGUAGGA
SEQ ID NO 204	UGGAGAUGGCAGUUUCCUUAGUAAC	SEQ ID NO 241	ACCAGAGUAACAGUCUGAGUAGGAG

SEQ ID NO 242	AGCCUCUUGAUUGCUGGUCUUGUUU	SEQ ID NO 51 NO 277	UUGGGCAGCGGUAAUGAGUUCUUCC
SEQ ID NO 51 NO 243	GCCUCUUGAUUGCUGGUCUUGUUUU	SEQ ID NO 278	UGGGCAGCGGUAAUGAGUUCUUCCA
SEQ ID NO 244	CCUCUUGAUUGCUGGUCUUGUUUUU	SEQ ID NO 279	GGGCAGCGGUAAUGAGUUCUUCCAA
SEQ ID NO 245	CCUCUUGAUUGCUGGUCUUG	SEQ ID NO 280	GGCAGCGGUAAUGAGUUCUUCCAAC
CUCUUGAUUGCUGGUCUUGUUUUUC	SEQ ID NO 281	GCAGCGGUAAUGAGUUCUUCCAACU
SEQ ID NO 247	UCUUGAUUGCUGGUCUUGUUUUUCA	SEQ ID NO 282	CAGCGGUAAUGAGUUCUUCCAACUG
SEQ ID NO 248	CUUGAUUGCUGGUCUUGUUUUUCAA	NO SEQ NO 283	AGCGGUAAUGAGUUCUUCCAACUGG
SEQ ID NO 249	UUGAUUGCUGGUCUUGUUUUUCAAA	SEQ ID NO 284	GCGGUAAUGAGUUCUUCCAACUGGG
SEQ ID NO 250	UGAUUGCUGGUCUUGUUUUUCAAAU	SEQ ID NO 285	CGGUAAUGAGUUCUUCCAACUGGGG
SEQ ID NO 251	GAUUGCUGGUCUUGUUUUUCAAAUU	SEQ ID NO 286	GGUAAUGAGUUCUUCCAACUGGGGA
SEQ ID NO 252	GAUUGCUGGUCUUGUUUUUC	SEQ ID NO 287	GGUAAUGAGUUCUUCCAACUGG
SEQ ID NO 51 NO 253	AUUGCUGGUCUUGUUUUUCAAAUUU	SEQ ID NO 288	GUAAUGAGUUCUUCCAACUGGGGAC
SEQ ID NO 254	UUGCUGGUCUUGUUUUUCAAAUUUU	SEQ ID NO 289	UAAUGAGUUCUUCCAACUGGGGACG
SEQ ID NO 255	UGCUGGUCUUGUUUUUCAAAUUUUG	SEQ ID NO 290	AAUGAGUUCUUCCAACUGGGGACGC
SEQ ID NO 256	GCUGGUCUUGUUUUUCAAAUUUUGG	SEQ ID NO 51 NO 291	AUGAGUUCUUCCAACUGGGGACGCC
SEQ ID	CUGGUCUUGUUUUUCAAAUUUUGGG	SEQ ID NO 51 NO 292	UGAGUUCUUCCAACUGGGGACGCCU
SEQ ID NO 258	UGGUCUUGUUUUUCAAAUUUUGGGC	SEQ ID NO 293	GAGUUCUUCCAACUGGGGACGCCUC
SEQ ID NO 259	GGUCUUGUUUUUCAAAUUUUGGGCA	SEQ ID NO 294	AGUUCUUCCAACUGGGGACGCCUCU
SEQ ID NO 260	GUCUUGUUUUUCAAAUUUUGGGCAG	SEQ ID NO 295	GUUCUUCCAACUGGGGACGCCUCUG
SEQ ID NO 261	UCUUGUUUUUCAAAUUUUGGGCAGC	SEQ ID	UUCUUCCAACUGGGGACGCCUCUGU
SEQ ID NO 262	CUUGUUUUUCAAAUUUUGGGCAGCG	SEQ ID NO 297	UCUUCCAACUGGGGACGCCUCUGUU
SEQ ID NO 263	UUGUUUUUCAAAUUUUGGGCAGCGG	SEQ ID NO 298	CUUCCAACUGGGGACGCCUCUGUUC
SEQ ID NO 264	UGUUUUUCAAAUUUUGGGCAGCGGU	UUCCAACUGGGGACGCCUCUGUUCC
SEQ ID NO 265	GUUUUUCAAAUUUUGGGCAGCGGUA	SEQ ID NO 300	UCCAACUGGGGACGCCUCUGUUCCA
NO SEQ ID 266	UUUUUCAAAUUUUGGGCAGCGGUAA	SEQ ID NO 301	CCAACUGGGGACGCCUCUGUUCCAA
SEQ ID NO 267	UUUUCAAAUUUUGGGCAGCGGUAAU	SEQ ID NO 302	CAACUGGGGACGCCUCUGUUCCAAA
SEQ ID NO 268	UUUCAAAUUUUGGGCAGCGGUAAUG	SEQ ID NO 51 NO 303	AACUGGGGACGCCUCUGUUCCAAAU
SEQ ID NO 269	UUCAAAUUUUGGGCAGCGGUAAUGA	SEQ ID NO 304	ACUGGGGACGCCUCUGUUCCAAAUC
SEQ ID NO 270	UCAAAUUUUGGGCAGCGGUAAUGAG	SEQ ID NO 305	CUGGGGACGCCUCUGUUCCAAAUCC
SEQ ID NO 271	CAAAUUUUGGGCAGCGGUAAUGAGU	SEQ ID NO 306	UGGGGACGCCUCUGUUCCAAAUCCU
SEQ ID NO 272	AAAUUUUGGGCAGCGGUAAUGAGUU	SEQ ID NO 307	GGGGACGCCUCUGUUCCAAAUCCUG
SEQ ID NO 273	AAUUUUGGGCAGCGGUAAUGAGUUC	SEQ ID NO 308	GGGACGCCUCUGUUCCAAAUCCUGC
SEQ ID NO 274	AUUUUGGGCAGCGGUAAUGAGUUCU	SEQ ID NO 309	GGACGCCUCUGUUCCAAAUCCUGCA
SEQ ID NO 275	UUUUGGGCAGCGGUAAUGAGUUCUU	SEQ ID NO 51 NO 310	GACGCCUCUGUUCCAAAUCCUGCAU
SEQ ID NO 276	UUUGGGCAGCGGUAAUGAGUUCUUC

SEQ ID NO 311	CUCUGGCCUGUCCUAAGACCUGCUC	SEQ ID NO 335	CAGCUUCUUCCUUAGCUUCCAGCCA
SEQ ID NO 312	UCUGGCCUGUCCUAAGACCUGCUCA	SEQ ID NO 336	AGCUUCUUCCUUAGCUUCCAGCCAU
SEQ ID NO 313	CUGGCCUGUCCUAAGACCUGCUCAG	SEQ ID NO 337	GCUUCUUCCUUAGCUUCCAGCCAUU
SEQ ID NO 314	UGGCCUGUCCUAAGACCUGCUCAGC	SEQ ID NO 338	CUUCUUCCUUAGCUUCCAGCCAUUG
SEQ ID NO 315	GGCCUGUCCUAAGACCUGCUCAGCU	SEQ ID NO 51 NO 339	UUCUUCCUUAGCUUCCAGCCAUUGU
SEQ ID NO 316	GCCUGUCCUAAGACCUGCUCAGCUU	SEQ ID NO 340	UCUUCCUUAGCUUCCAGCCAUUGUG
SEQ ID NO 317	CCUGUCCUAAGACCUGCUCAGCUUC	SEQ ID NO 341	CUUCCUUAGCUUCCAGCCAUUGUGU
SEQ ID NO 318	CUGUCCUAAGACCUGCUCAGCUUCU	SEQ ID NO 342	UUCCUUAGCUUCCAGCCAUUGUGUU
SEQ ID NO 319	UGUCCUAAGACCUGCUCAGCUUCUU	SEQ ID NO 343	UCCUUAGCUUCCAGCCAUUGUGUUG
SEQ ID NO 320	GUCCUAAGACCUGCUCAGCUUCUUC	SEQ ID NO 344	CCUUAGCUUCCAGCCAUUGUGUUGA
SEQ ID NO 51 NO 321	UCCUAAGACCUGCUCAGCUUCUUCC	SEQ ID NO 345	CUUAGCUUCCAGCCAUUGUGUUGAA
SEQ ID NO 322	CCUAAGACCUGCUCAGCUUCUUCCU	SEQ ID NO 346	UUAGCUUCCAGCCAUUGUGUUGAAU
SEQ ID NO 51 NO 323	CUAAGACCUGCUCAGCUUCUUCCUU	SEQ ID NO 347	UAGCUUCCAGCCAUUGUGUUGAAUC
SEQ ID NO 324	UAAGACCUGCUCAGCUUCUUCCUUA	SEQ ID NO 348	AGCUUCCAGCCAUUGUGUUGAAUCC
SEQ ID NO 325	AAGACCUGCUCAGCUUCUUCCUUAG	SEQ ID NO 349	GCUUCCAGCCAUUGUGUUGAAUCCU
SEQ ID NO 326	AGACCUGCUCAGCUUCUUCCUUAGC	SEQ ID NO 350	CUUCCAGCCAUUGUGUUGAAUCCUU
SEQ ID NO 327	GACCUGCUCAGCUUCUUCCUUAGCU	SEQ ID NO 351	UUCCAGCCAUUGUGUUGAAUCCUUU
SEQ ID NO 328	ACCUGCUCAGCUUCUUCCUUAGCUU	SEQ ID NO 352	UCCAGCCAUUGUGUUGAAUCCUUUA
SEQ ID NO 329	CCUGCUCAGCUUCUUCCUUAGCUUC	SEQ ID NO 353	CCAGCCAUUGUGUUGAAUCCUUUAA
SEQ ID NO 330	CUGCUCAGCUUCUUCCUUAGCUUCC	SEQ ID NO 354	CAGCCAUUGUGUUGAAUCCUUUAAC
SEQ ID NO 331	UGCUCAGCUUCUUCCUUAGCUUCCA	SEQ ID NO 355	AGCCAUUGUGUUGAAUCCUUUAACA
SEQ ID NO 332	GCUCAGCUUCUUCCUUAGCUUCCAG	SEQ ID NO 356	GCCAUUGUGUUGAAUCCUUUAACAU
SEQ ID NO 333	CUCAGCUUCUUCCUUAGCUUCCAGC	SEQ ID NO 357	CCAUUGUGUUGAAUCCUUUAACAUU
SEQ ID NO 51 NO 334	UCAGCUUCUUCCUUAGCUUCCAGCC	SEQ ID NO 358	CAUUGUGUUGAAUCCUUUAACAUUU

SEQ ID NO 359	CAUUUUUGACCUACAUGUGG	SEQ ID NO 364	AUUUUUGACCUACAUGGGAAA G
SEQ ID NO 360	UUUGACCUACAUGUGGAAAG	SEQ ID NO 365	UACGAGUUGAUUGUCGGACCCAG
SEQ ID NO 51 NO 361	UACAUUUUUGACCUACAUGUGGAAA G	SEQ ID NO 366	GUGGUCUCCUUACCUAUGACUGUGG
SEQ ID NO 362	GGUCUCCUUACCUAUGA	SEQ ID NO 367	UGUCUCAGUAAUCUUCUUACCUAU
SEQ ID NO 363	UCUUACCUAUGACUAUGGAUGAGA

SEQ ID NO368	UGCAUGUUCCAGUCGUUGUGUGG	SEQ ID NO 370	AUUUACCAACCUUCAGGAUCGAGUA
SEQ ID NO 369	CACUAUUCCAGUCAAAUAGGUCUGG	SEQ ID NO 371	GGCCUAAAACACAUACACAUA

SEQ ID NO 51 NO 372	CCCUGAGGCAUUCCCAUCUUGAAU	SEQ ID NO 374	CUUGAAUUUAGGAGAUUCAUCUG
SEQ ID NO 373	AGGACUUACUUGCUUUGUUU	SEQ ID NO 375	CAUCUUCUGAUAAUUUUCCUGUU

SEQ ID NO 376	CCAUUACAGUUGUCUGUGUU	SEQ ID NO 378	UAAUCUGCCUCUUCUUUUGG
SEQ ID NO 377	UGACAGCCUGUGAAAUCUGUGAG

SEQ ID NO 379	CAGCAGUAGUUGUCAUCUGC	SEQ ID NO 381	GCCUGAGCUGAUCUGCUGGCAUCUUGCA GUU
SEQ ID NO 380	GCCUGAGCUGAUCUGCUGGCAUCUUGC	SEQ ID NO 382	UCUGCUGGCAUCUUGC

SEQID NO 383	GCCGGUUGACUUCAUCCUGUGC	SEQ ID NO 51 NO 386	CUGCAUCCAGGAACAUGGGUCC
SEQ ID NO 384	GUCUGCAUCCAGGAACAUGGGUC	SEQ ID NO 387	GUUGAAGAUCUGAUAGCCGGUUGA
SEQ ID NO 385	UACUUACUGUCUGUAGCUCUUUCU

SEQ ID NO 388	UCAGCUUCUGUUAGCCACUG	SEQ IDNO 413	AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 389	UUCAGCUUCUGUUAGCCACU	SEQ ID NO 414	CAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 390	UUCAGCUUCUGUUAGCCACUG	SEQ ID NO 415	AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 391	UCAGCUUCUGUUAGCCACUGA	SEQ ID NO 416	AGCUUCUGUUAGCCACUGAU
SEQ ID NO 392	UUCAGCUUCUGUUAGCCACUGA	SEQ ID NO 417	GCUUCUGUUAGCCACUGAUU
SEQ ID NO 393	UCAGCUUCUGUUAGCCACUGA	SEQ ID NO 418	AGCUUCUGUUAGCCACUGAUU
SEQ ID NO 394	UUCAGCUUCUGUUAGCCACUGA	SEQ ID NO 419	GCUUCUGUUAGCCACUGAUUA
SEQ ID NO 395	UCAGCUUCUGUUAGCCACUGAU	SEQ ID NO 51 NO 420	AGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 396	UUCAGCUUCUGUUAGCCACUGAU	SEQ ID NO 421	GCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 397	UCAGCUUCUGUUAGCCACUGAUU	SEQ ID NO 422	AGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 398	UUCAGCUUCUGUUAGCCACUGAUU	SEQ ID NO 423	GCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 399	UCAGCUUCUGUUAGCCACUGAUUA	SEQ ID NO 424	AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 400	UUCAGCUUCUGUUAGCCACUGAUA	SEQ ID NO 425	GCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 401	UCAGCUUCUGUUAGCCACUGAUUAA	SEQ ID NO 426	CCAUUUGUAUUUAGCAUGUUCCC
SEQ ID NO 402	UUCAGCUUCUGUUAGCCACUGAUUAA	SEQ ID NO 427	AGAUACCAUUUGUAUUUAGC
SEQ ID NO 403	UCAGCUUCUGUUAGCCACUGAUUAAA	SEQ ID NO 428	GCCAUUUCUCAACAGAUCU
SEQ ID NO 404	UUCAGCUUCUGUUAGCCACUGAUUAAA	SEQ ID NO 429	GCCAUUUCUCAACAGAUCUGUCA
SEQ ID NO 405	CAGCUUCUGUUAGCCACUG	SEQ ID NO 430	AUUCUCAGGAAUUUGUGUCUUUC
SEQ ID NO 406	CAGCUUCUGUUAGCCACUGAU	SEQ ID NO 431	UCUCAGGAAUUUGUGUCUUUC
SEQ ID NO 407	AGCUUCUGUUAGCCACUGAUU	SEQ ID NO 432	GUUCAGCUUCUGUUAGCC
SEQ ID NO 408	CAGCUUCUGUUAGCCACUGAUU	SEQ ID NO 433	CUGAUUAAAUAUCUUUAUAU C
SEQ ID NO 409	AGCUUCUGUUAGCCACUGAUUA	SEQ ID NO 434	GCCGCCAUUUCUCAACAG
SEQ ID NO 410	CAGCUUCUGUUAGCCACUGAUUA	SEQ ID NO 435	GUAUUUAGCAUGUUCCCA
SEQ IDNO 411	AGCUUCUGUUAGCCACUGAUUAA	SEQ ID NO 436	CAGGAAUUUGUGUCUUUC
SEQ ID NO 412	CAGCUUCUGUUAGCCACUGAUUAA

SEQ ID NO 437	UUUGCCGCUGCCCAAUGCCAUCCUG	SEQ ID NO 470	GUUGCAUUCAAUGUUCUGACAACAG
SEQ IDNO 438	AUUCAAUGUUCUGACAACAGUUUGC	SEQ ID NO 471	UUGCAUUCAAUGUUCUGACAACAGU
SEQ IDNO 439	CCAGUUGCAUUCAAUGUUCUGACAA	SEQ ID NO 472	UGCAUUCAAUGUUCUGACAACAGUU
SEQ IDNO 440	CAGUUGCAUUCAAUGUUCUGAC	SEQ ID NO 473	GCAUUCAAUGUUCUGACAACAGUUU
SEQ ID NO 441	AGUUGCAUUCAAUGUUCUGA	SEQ ID NO 474	CAUUCAAUGUUCUGACAACAGUUUG
SEQ ID	GAUUGCUGAAUUAUUUCUUCC	SEQ ID	AUUCAAUGUUCUGACAACAGUUUGC
NO 442	NO 475
SEQ ID NO 443	GAUUGCUGAAUUAUUUCUUCCCCAG	SEQ ID NO 476	UCAAUGUUCUGACAACAGUUUGCCG
SEQ ID NO 444	AUUGCUGAAUUAUUUCUUCCCCAGU	SEQ ID NO 477	CAAUGUUCUGACAACAGUUUGCCGC
SEQ ID NO 445	UUGCUGAAUUAUUUCUUCCCCAGUU NO	SEQ ID 478	AAUGUUCUGACAACAGUUUGCCGCU
SEQ ID NO 446	UGCUGAAUUAUUUCUUCCCCAGUUG	SEQ ID NO 479	AUGUUCUGACAACAGUUUGCCGCUG
SEQ ID NO 447	GCUGAAUUAUUUCUUCCCCAGUUGC	SEQ ID NO 480	UGUUCUGACAACAGUUUGCCGCUGC
SEQ ID NO 448	CUGAAUUAUUUCUUCCCCAGUUGCA	SEQ ID NO 481	GUUCUGACAACAGUUUGCCGCUGCC
SEQ ID NO 449	UGAAUUAUUUCUUCCCCAGUUGCAU	SEQ ID NO 482	UUCUGACAACAGUUUGCCGCUGCCC
SEQ ID NO 450	GAAUUAUUUCUUCCCCAGUUGCAUU	SEQ ID NO 483	UCUGACAACAGUUUGCCGCUGCCCA
SEQ IDNO 451	AAUUAUUUCUUCCCCAGUUGCAUUC	SEQ ID NO 484	CUGACAACAGUUUGCCGCUGCCCAA
SEQ ID No 452	AUUAUUUCUUCCCCAGUUGCAUUCA	SEQ ID NO 485	UGACAACAGUUUGCCGCUGCCCAAU
SEQ ID NO 453	UUAUUUCUUCCCCAGUUGCAUUCAA	SEQ ID NO 486	GACAACAGUUUGCCGCUGCCCAAUG
SEQ ID NO 454	UAUUUCUUCCCCAGUUGCAUUCAAU	SEQ ID NO 487	ACAACAGUUUGCCGCUGCCCAAUGC
SEQ ID NO 455	AUUUCUUCCCCAGUUGCAUUCAAUG	SEQ ID NO 488	CAACAGUUUGCCGCUGCCCAAUGCC
SEQ ID NO 456	UUUCUUCCCCAGUUGCAUUCAAUGU	SEQ ID NO 489	AACAGUUUGCCGCUGCCCAAUGCCA
SEQ ID NO 457	UUCUUCCCCAGUUGCAUUCAAUGUU	SEQ ID NO 490	ACAGUUUGCCGCUGCCCAAUGCCAU
SEQ ID NO 458	UCUUCCCCAGUUGCAUUCAAUGUUC	SEQ ID NO 491	CAGUUUGCCGCUGCCCAAUGCCAUC
SEQ ID NO 51 NO 459	CUUCCCCAGUUGCAUUCAAUGUUCU	SEQ ID NO 492	AGUUUGCCGCUGCCCAAUGCCAUCC
SEQ ID NO 460	UUCCCCAGUUGCAUUCAAUGUUCUG	SEQ ID NO 493	GUUUGCCGCUGCCCAAUGCCAUCCU
SEQ ID NO 461	UCCCCAGUUGCAUUCAAUGUUCUGA	SEQ ID NO 494	UUUGCCGCUGCCCAAUGCCAUCCUG
SEQ ID NO 462	CCCCAGUUGCAUUCAAUGUUCUGAC	SEQ ID NO 495	UUGCCGCUGCCCAAUGCCAUCCUGG
SEQ ID NO 463	CCCAGUUGCAUUCAAUGUUCUGACA	SEQ ID NO 496	UGCCGCUGCCCAAUGCCAUCCUGGA
SEQ ID NO 464	CCAGUUGCAUUCAAUGUUCUGACAA	SEQ ID NO 51 NO 497	GCCGCUGCCCAAUGCCAUCCUGGAG
SEQ ID NO 465	CAGUUGCAUUCAAUGUUCUGACAAC	SEQ ID NO 498	CCGCUGCCCAAUGCCAUCCUGGAGU
SEQ ID NO 466	AGUUGCAUUCAAUGUUCUGACAACA	SEQ ID NO 499	CGCUGCCCAAUGCCAUCCUGGAGUU
SEQ ID NO 467	UCC UGU AGA AUA CUG GCA UC	SEQ ID NO 500	UGUUUUUGAGGAUUGCUGAA
SEQ ID NO 468	UGCAGACCUCCUGCCACCGCAGAUUCA	SEQ ID NO 501	UGUUCUGACAACAGUUUGCCUCUUCCCA AUGCCAUCCUGG
SEQ ID NO 469	UUGCAGACCUCCUGCCACCGCAGAUUC AGGCUUC

SEQ ID NO 51	CUGUUGCAGUAAUCUAUGAG	SEQ ID NO 505	UGCCAUUGUUUCAUCAGCUCUUU
SEQ ID NO 503	UGCAGUAAUCUAUGAGUUUC	SEQ ID NO 506	UCCUGUAGGACAUUGGCAGU
SEQ ID NO 504	GAGUCUUCUAGGAGCCUU	SEQ ID NO 507	CUUGGAGUCUUCUAGGAGCC

SEQ ID No 508	UAGGUGCCUGCCGGCUU	SEQ ID NO 510	CUGAACUGCUGGAAAGUCGCC
SEQ ID NO 509	UUCAGCUGUAGCCACACC	SEQ ID NO 511	CUGGCUUCCAAAUGGGACCUGAAAAAGA AC

SEQ ID NO 512	CAAUUUUUCCCACUCAGUAUU	SEQ ID NO 514	UCCUCAGGAGGCAGCUCUAAAU
SEQ ID NO 513	UUGAAGUUCCUGGAGUCUU

SEQ ID NO 515	UGGCUCUCUCCCAGGG	SEQ ID NO 517	GGGCACUUUGUUUGGCG
NO 515 SEQ ID NO 516	GAGAUGGCUCUCUCCCAGGGACCCUGG

SEQ ID NO 518	GGUCCCAGCAAGUUGUUUG	SEQ ID NO 520	GUAGAGCUCUGUCAUUUUGGG
SEQ ID NO 519	UGGGAUGGUCCCAGCAAGUUGUUUG

SEQ ID NO 521	GCUCAAGAGAUCCACUGCAAAAAAC	SEQ ID NO 523	UCUGCAGGAUAUCCAUGGGCUGGUC
NO 521 SEQ ID NO 522	GCCAUACGUACGUAUCAUAAACAUUC

SEQ ID NO 524	GAUCCUCCCUGUUCGUCCCCUAUUAUG

SEQ ID NO 525	UGCUUUAGACUCCUGUACCUGAUA

SEQ ID NO 526	GGCGGCCUUUGUGUUGAC	SEQ ID NO 528	CCUUUAUGUUCGUGCUGCU
SEQ ID NO 527	GGACAGGCCUUUAUGUUCGUGCUGC

Figure legends

• Figure 1. In human control myotubes, a series of AONs (PS237, PS238, and PS240; SEQ ID NO 65, 66, 16 respectively) targeting exon 43 was tested at 500 nM. PS237 (SEQ ID NO 65) reproducibly induced highest levels of exon 43 skipping. (M: DNA size marker; NT: non-treated cells)
• Figure 2. In myotubes from a DMD patient with an exon 45 deletion, a series of AONs (PS177, PS179, PS181, and PS182; SEQ ID NO 91, 70, 110, and 117 respectively) targeting exon 46 was tested at two different concentrations (50 and 150 nM). PS182 (SEQ ID NO 117) reproducibly induced highest levels of exon 46 skipping. (M: DNA size marker)
• Figure 3. In human control myotubes, a series of AONs (PS245, PS246, PS247, and PS248; SEQ ID NO 167, 165, 166, and 127 respectively) targeting exon 50 was tested at 500 nM. PS248 (SEQ ID NO 127) reproducibly induced highest levels of exon 50 skipping. (M: DNA size marker; NT: non-treated cells).
• Figure 4. In human control myotubes, two novel AONs (PS232 and PS236; SEQ ID NO 246 and 299 respectively) targeting exon 52 were tested at two different concentrations (200 and 500 nM) and directly compared to a previously described AON (52-1). PS236 (SEQ ID NO 299) reproducibly induced highest levels of exon 52 skipping. (M: DNA size marker; NT: non-treated cells).

Claims (17)

1. An antisense oligonucleotide or an oligonucleotide equivalent comprising a nucleotide sequence of between 22 and 30 nucleotides, wherein said nucleotide sequence comprises or consists of the antisense nucleotide sequence 5'-GGUAAUGAGUUCUUCCAACUGG-3' (SEQ ID NO:287).
2. An oligonucleotide equivalent according to claim 1, wherein at least one of the nucleotides is a nucleotide analogue.
3. An antisense oligonucleotide or an oligonucleotide equivalent according to claim 1 or 2 comprising one or more RNA residues, and/or one or more DNA residues.
4. An oligonucleotide equivalent according to claim 2, comprising at least one nucleotide analogue, wherein said nucleotide analogue has a modified base, and/or a non-natural internucleoside linkage, or a combination of these modifications.
5. An oligonucleotide equivalent according to claim 4, wherein the nucleotide analogue has a modified base.
6. An oligonucleotide equivalent according to claim 4, wherein the oligonucleotide comprises a modified backbone.
7. An oligonucleotide equivalent according to claim 2, wherein the nucleotide analogue comprises one or more sugar moieties that are mono- or disubstituted at the 2', 3' and/or 5' position, preferably comprising a 2'-O-alkyl phosphorothioate antisense oligonucleotide, more preferably comprising a 2'-O methyl phosphorothioate ribose.
8. An oligonucleotide equivalent according to claim 7, which is a phosphorothioate antisense oligonucleotide wherein the sugar moieties are 2'-O-methyl substituted.
9. An oligonucleotide equivalent according to claim 6, wherein the modified backbone is selected from the group consisting of a morpholino backbone, a carbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl backbone, a thioformacetyl backbone, a methyleneformacetyl backbone, a riboacetyl backbone, an alkene containing backbone, a sulfamate backbone, a sulfonate backbone, a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazino backbone and an amide backbone.
10. An oligonucleotide equivalent according to any of claims 1-6 or 9 wherein said oligonucleotide comprises a morpholino ring and/or a phosphorodiamidate internucleoside linkage, and/or a peptide nucleic acid, and/or a locked nucleic acid.
11. An oligonucleotide equivalent according to claim 10, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO), preferably comprising at least one modified base.
12. A viral-based vector, comprising an expression cassette that drives expression of an oligonucleotide as defined in claim 1.
13. An antisense oligonucleotide or an oligonucleotide equivalent according to any one of claims 1 to 11 or the viral-based vector according to claim 12 for use as a medicament, preferably for modulating splicing of the dystrophin pre-mRNA of a DMD or BMD patient or for the treatment of a DMD or BMD patient.
14. A pharmaceutical composition comprising an antisense oligonucleotide or an oligonucleotide equivalent as defined in any one of claims 1 to 11 and/or the viral-based vector of claim 12, a pharmaceutical acceptable carrier, and optionally combined with an antisense oligonucleotide which is able to induce or promote skipping of at least one of exon 51, 53, 55, 57, 59 or 69 of the dystrophin pre-mRNA of a patient.
15. An in vitro or ex vivo method for inducing and/or promoting skipping of at least exon 52 of the dystrophin pre-mRNA in an isolated cell of a patient, the method comprising providing said cell with an antisense oligonucleotide or an oligonucleotide equivalent as defined in any one of claims 1 to 11 or the viral-based vector of claim 12.
16. A method according to claim 15, wherein an additional antisense oligonucleotide is used which is able to induce or promote skipping of at least one of exon 51, 53, 55, 57, 59 or 69 of the dystrophin pre-mRNA of a patient.
17. Use of the antisense oligonucleotide or oligonucleotide equivalent as defined in any one of claims 1-11, the viral-based vector of claim 12, or the pharmaceutical composition of claim 14 for modulating splicing of the dystrophin pre-mRNA in a human myogenic cell or muscle cell in vitro, or for the preparation of a medicament for the treatment of a DMD or BMD patient.