METHODS AND MEANS FOR EFFICIENT SKIPPING OF AT LEAST ONE OF THE FOLLOWING EXONS OF THE HUMAN DUCHENNE MUSCULAR DYSTROPHY GENE: 43, 46, 50-53

Abstract

The invention relates to a method wherein a molecule is used for inducing and/or promoting skipping of at least one of exon 43, exon 46, or exons 50-53 of the DMD pre-mRNA in a patient, the method comprising providing the patient with the molecule. The invention also relates to the molecule as such.

Claims

1. An antisense oligonucleotide whose base sequence consists of 5′-CUCUUGAUUGCUGGUCUUGUUUUUC-3′ (SEQ ID NO:246), wherein the oligonucleotide comprises a modification.

2. The antisense oligonucleotide of claim 1, wherein the modification comprises at least one nucleotide analogue, wherein the nucleotide analogue comprises a modified sugar moiety, a modified backbone, a modified internucleoside linkage, or a modified base, or a combination thereof.

3. The antisense oligonucleotide of claim 1, wherein the modification comprises a modified sugar moiety.

4. The antisense oligonucleotide of claim 3, wherein the modified sugar moiety is mono- or di-substituted at the 2′, 3′ and/or 5′ position.

5. The antisense oligonucleotide of claim 4, wherein the modified sugar moiety comprises a 2′-O-methyl ribose.

6. The antisense oligonucleotide of claim 1, wherein the modification comprises a modified backbone.

7. The antisense oligonucleotide of claim 6, wherein the modified backbone comprises 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 or an amide backbone, or a combination thereof.

8. The antisense oligonucleotide of claim 7, wherein the modified backbone comprises a morpholino backbone.

9. The antisense oligonucleotide of claim 1, wherein the modification comprises a modified internucleoside linkage.

10. The antisense oligonucleotide of claim 9, wherein the modified internucleoside linkage comprises a phosphorothioate linkage.

11. The antisense oligonucleotide of claim 1, wherein the modification comprises a modified base.

12. The antisense oligonucleotide of claim 1, wherein the oligonucleotide comprises a morpholino ring, a phosphorodiamidate internucleoside linkage, a peptide nucleic acid, a locked nucleic acid (LNA), or a combination thereof.

13. The antisense oligonucleotide of claim 1, wherein the oligonucleotide comprises a 2′-O-methyl phosphorothioate ribose.

14. The antisense oligonucleotide of claim 1, wherein the oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).

15. A pharmaceutical composition, comprising the antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.

16. A method of treating Duchenne muscular dystrophy or Becker muscular dystrophy in a subject, comprising administering to the subject the antisense oligonucleotide of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 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)

[0010] FIG. 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)

[0011] FIG. 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).

[0012] FIG. 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).

DETAILED DESCRIPTION

Method

[0013] In a first aspect, the present invention provides a method for inducing, and/or promoting skipping of at least one of exons 43, 46, 50-53 of the DMD pre-mRNA in a patient, preferably in an isolated cell of a patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 8 nucleotides within said exon. It is to be understood that said method encompasses an in vitro, in vivo or ex vivo method.

[0014] Accordingly, a method is provided for inducing and/or promoting skipping of at least one of exons 43, 46, 50-53 of DMD pre-mRNA in a patient, preferably in an isolated cell of said patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 8 nucleotides within said exon.

[0015] As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient.

[0016] 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.

[0017] 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 of splice acceptor sequence that 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.

[0018] 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.

[0019] Preferably, a method of the invention by inducing and/or promoting skipping of at least one of the following exons 43, 46, 50-53 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 is said patient.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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 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.

[0026] 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 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), exons 8-51, 51, 53, 53-55, 53-57, 53-59, 53-60, (correctable by exon 52 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).

[0027] 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 43, 46, 50-53 in the DMD gene, occurring in a total of 36% of all DMD patients with a deletion (Aartsma-Rus et al, Hum. Mut. 2009)

[0028] Furthermore, for some patients the simultaneous skipping of one of more exons in addition to exon 43, exon 46 and/or exon 50-53 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) 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.

[0029] In a preferred method, 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 52 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 50 and 51, or 52 and 53, or 30 43 and 51, or 43 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.

[0030] 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

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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 A Y 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.

[0036] A treatment in a method according to the invention 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.

[0037] 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.

[0038] 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”.

[0039] 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, 52, 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 multi-exon skipping (Aartsma-Rus A, Janson A A, Kaman W E, 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 W E, Weij R, den Dunnen J T, van Ommen G J, van Deutekom J C. 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. This multi-skipping was notably so for the combination of oligonucleotides derived from the DMD gene, wherein one oligonucleotide for exon 45 and one oligonucleotide for exon 51 was added to a cell transcribing the DMD gene. Such a set-up resulted in mRNA being produced that did not contain exons 45 to 51. Apparently, the structure of the pre-mRNA in the presence of the mentioned oligonucleotides was such that the splicing machinery was stimulated to connect exons 44 and 52 to each other.

[0040] 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.

[0041] 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

[0042] 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).

[0043] 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.

[0044] In a preferred embodiment, a molecule or an oligonucleotide of the invention 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.

[0045] A preferred molecule to be used in a method of the invention binds or is complementary to a continuous stretch of at least 8 nucleotides within one of the following nucleotide sequences selected from:

TABLE-US-00001 (SEQ ID NO: 2) 5′-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAUAGCA AGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3′ for skipping of exon 43; (SEQ ID NO: 3) 5′-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUGAACC UGGAAAAGAGCAGCAACUAAAAGAAAAGC-3′ for skipping of exon 46; (SEQ ID NO: 4) 5′-GGCGGUAAACCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUGACC UAGCUCCUGGACUGACCACUAUUGG-3′ for skipping of exon 50; (SEQ ID NO: 5) 5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACUAAG GAAACUGCCAUCUCCAAACUAGAAAUGCCAUCUUCCUUGAUGUUGGAGGU AC-3′ for skipping of exon 51; (SEQ ID NO: 6) 5′-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAU UACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3′ for skipping of exon 52; and (SEQ ID NO: 7) 5′-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAA GCUGAGCAGGUCUUAGGACAGGCCAGAG-3′ for skipping of exon 53.

[0046] 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 invention provides distinct molecules that can be used in a method for efficiently skipping of at least one of exon 43, exon 46 and/or exon 50-53. 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.

[0047] 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.

[0048] In a preferred embodiment for inducing and/or promoting the skipping of any of exon 43, exon 46 and/or exon 50-53, 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 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.

[0049] A preferred molecule of the invention comprises a nucleotide-based or nucleotide or an antisense oligonucleotide sequence of between 8 and 50 nucleotides or bases, more preferred between 10 and 50 nucleotides, more preferred between 20 and 40 nucleotides, more preferred between 20 and 30 nucleotides, such as 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides or 50 nucleotides. A most preferred molecule of the invention comprises a nucleotide-based sequence of 25 nucleotides.

[0050] 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.

[0051] 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 U.S. Pat. No. 6,875,736, which are herein incorporated by reference. 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) which is herein incorporated by reference.

[0052] 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.

[0053] In an even more preferred embodiment, the invention 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 embodiment, the invention 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.

[0054] Another preferred molecule of the invention 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 invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 70 to SEQ ID NO 122.

[0055] In an even more preferred embodiment, the invention 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 N0117.

[0056] In a most preferred embodiment, the invention 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.

[0057] Another preferred molecule of the invention 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 invention 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.

[0058] In an even more preferred embodiment, the invention 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.

[0059] In a most preferred embodiment, the invention 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.

[0060] Another preferred molecule of the invention 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 invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 168 to SEQ ID NO 241.

[0061] Another preferred molecule of the invention 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. More preferably, the invention 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 embodiment, the invention 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 embodiment, the invention 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.

[0062] Another preferred molecule of the invention 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 invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 311 to SEQ ID NO 358.

[0063] In a most preferred embodiment, the invention 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 one-carbon 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).

[0069] 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.

[0070] 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 alkylphosphonate including 3′-alkylene phosphonate, 5′-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3′-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.

[0071] 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′-0,4′-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.

[0072] 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.

[0073] 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.

[0074] A most preferred antisense oligonucleotide according to the invention comprises of 2′-O-methyl phosphorothioate ribose.

[0075] 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.

[0076] It will be understood by a skilled person that distinct antisense oligonucleotides can be combined for efficiently skipping any of exon 43, exon 46, exon 50, exon 51, exon 52 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, 43, 46, 50, 51, 52, 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:

−43 and 51, or
−43 and 53, or
−50 and 51, or
−51 and 52, or
−52 and 53.

[0077] 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.

[0078] 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.

[0079] A preferred antisense oligonucleotide comprises a peptide-linked PMO.

[0080] 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 43, 46, 50, 51, 52, or 53 of the human DMD pre-mRNA.

[0081] 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 agentia such as, for example, LipofectAMINE™ 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), or derivatives thereof.

[0082] One preferred antisense oligonucleotide expression system is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-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.

[0083] 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 Ul, a U6, or a U7 RNA promoter.

[0084] 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 43, exon 46, exon 50, exon 51, exon 52 or exon 53 of the human DMD pre-mRNA.

Pharmaceutical Composition

[0085] 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.

[0086] 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.

[0087] 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 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 52, 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 exon are identified in any one of Tables 1 to 7.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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) recognizing cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake into cells and/or the intracellular release of a compound as defined herein, preferably an oligonucleotide from vesicles, e.g. endosomes or lysosomes.

[0093] 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.

[0094] 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 optimized any further.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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 any of exon 43, 46, 50-53 as identified herein.

Use

[0099] 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. The splicing is preferably modulated in a human myogenic cell or muscle cell in vitro. More preferred is that splicing is modulated in a human muscle cell in vivo. Accordingly, the invention further relates to the use of the molecule as defined herein and/or the vector as defined herein and/or or the pharmaceutical composition as defined herein for modulating splicing of the DMD pre-mRNA or for the preparation of a medicament for the treatment of a DMD or BMD patient.

[0100] 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. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0101] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

EXAMPLES

Examples 1-4

Materials and Methods

[0102] AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by them-fold program, on (partly) overlapping putative SR— protein 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

[0103] 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), 200 nM and 500 nM (example 4) or 500 nM only (examples 1 and 3) of each AON. Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 μl UNIFectylin per 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.

[0104] 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 500 nM, as shown in FIG. 1. For comparison, also PS238 and PS240 are shown, inducing exon 43 skipping levels up to 13% and 36% respectively (FIG. 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.

[0105] 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 FIG. 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 (FIG. 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.

[0106] 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 FIG. 3. For comparison, also PS245, PS246, and PS247 are shown, inducing exon 50 skipping levels up to 14-16% at 500 nM (FIG. 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

[0107] 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.

[0108] 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 FIG. 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 (FIG. 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

[0109] 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).

TABLE-US-00002 SEQUENCE LISTING: DMD GENE AMINO ACID SEQUENCE SEO ID NO 1: MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKEGKQHIENLFSDLQDGRR LLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTD IVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQS TRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRL EHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSI EAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKP RFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSEGSSLMESEVNLDR YQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMMDLTAH QGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEK QSNLHRVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQV QQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR WANICRWTEDRWVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTT GFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVT QKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQPSLTQTTVMETVT TVTTREQILVKHAQEELPPPPPQKKRQITVDSEIRKRLDVDITELHSWI TRSEAVLQSPEFAIFRKEGNFSDLKEKVNAIEREKAEKERKLQDASRSA QALVEQMVNEGVNADSIKQASEQLNSRWIEFCQLLSERLNWLEYQNNII AFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKICKDEVNRLSG LQPQIERLKIQSIALKEKGQGPMFLDADFVAFTNHFKQVFSDVQAREKE LQTIFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLG ELQALQSSLQEQQSGLYYLSTTVKEMSKKAPSEISRKYQSEFEEIEGRW KKLSSQLVEHCQKLEEQMNKLRKIQNHIQTLKKWMAEVDVFLKEEWPAL GDSEILKKQLKQCRLLVSDIQTIQPSLNSVNEGGQKIKNEAEPEFASRL ETELKELNTQWDHMCQQVYARKEALKGGLEKTVSLQKDLSEMHEWMTQA EEEYLERDFEYKTPDELQKAVEEMKRAKEEAQQKEAKVKLLTESVNSVI AQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLEEVWACWHELLSY LEKANKWLNEVEFKLKTTENIPGGAEEISEVLDSLENLMRHSEDNPNQI RILAQTLTDGGVMDELINEELETFNSRWRELHEEAVRRQKLLEQSIQSA QETEKSLHLIQESLTFIDKQLAAYIADKVDAAQMPQEAQKIQSDLTSHE ISLEEMKKHNQGKEAAQRVLSQIDVAQKKLQDVSMKFRLFQKPANFEQR LQESKMILDEVKMHLPALETKSVEQEVVQSQLNHCVNLYKSLSEVKSEV EMVIKTGRQIVQKKQTENPKELDERVTALKLHYNELGAKVTERKQQLEK CLKLSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPSNLDSEVAWGKAT QKEIEKQKVHLKSITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSR AEEWLNLLLEYQKHMETFDQNVDHITKWIIQADTLLDESEKKKPQQKED VLKRLKAELNDIRPKVDSTRDQAANLMANRGDHCRKLVEPQISELNHRF AAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAEIQQGVNLKEEDFN KDMNEDNEGTVKELLQRGDNLQQRITDERKREEIKIKQQLLQTKHNALK DLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERK IKEIDRELQKKKEELNAVRRQAEGLSEDGAAMAVEPTQIQLSKRWREIE SKFAQFRRLNFAQIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQ ALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSK KTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWR RFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQ TVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKR LEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVK LLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWI KVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLE IYNQPNQEGPFDVQETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKR KLEDLSSEWKAVNRLLQELRAKQPDLAPGLTTIGASPTQTVTLVTQPVV TKETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQRVM VGDLEDINEMIIKQKATMQDLEQRRPQLEELITAAQNLKNKTSNQEART IITDRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLG QARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALK LLRDYSADDTRKVHMITENINASWRSIHKRVSEREAALEETHRLLQQFP LDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGE IEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKS LNIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQ KQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPREL PPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQ EATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLK ENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQL HEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPK MTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQH NLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNV YDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRL GLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLD WMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHF NYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFR TKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSH DDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSL NQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLK QQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQ ILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQP MLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNT PGKPMREDTM SEQ ID NO 2 (EXON 43): AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAUAGCAAG AAGACAG CAGCAUUGCAMGUGCAACGCCUGUGG SEQ ID NO 3 (EXON 46): UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUGAACCUG GAMAGAGCAGCAACUAAAAGAMAGC SEQ ID NO 4 (EXON 50): GGCGGUAMCCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUGACCUAG CUCCUGGACUGACCACUAUUGG SEQ ID NO 5 (EXON 51): CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACUAAGGA MCUGCCAUCUCCAAACUAGAAAUGCCAUCUUCCUUGAUGUUGGAGGUAC SEQ ID NO 6 (EXON 52): AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAUUACCG CUGCCCAAAAUUU GAAAAACAAGACCAGCAAUCAAGAGGCU SEQ ID NO 7 (EXON 53): AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAAGCUGA GCAGGUCUUAGGA CAGGCCAGAG

TABLE-US-00003 TABLE 1 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 43 SEQ ID CCACAGGCGUUGCACUUUGCA NO 8 AUGC SEQ ID CACAGGCGUUGCACUUUGCAA NO 9 UGCU SEQ ID ACAGGCGUUGCACUUUGCAAU NO 10 GCUG SEQ ID CAGGCGUUGCACUUUGCAAUG NO 11 CUGC SEQ ID AGGCGUUGCACULTUGCAAUGC NO 12 UGCU SEQ ID GGCGUUGCACULTUGCAAUGCU NO 13 GCUG SEQ ID GCGUUGCACULTUGCAAUGCUG NO 14 CUGU SEQ ID CGUUGCACUUUGCAAUGCUGC NO 15 UGUC SEQ ID CGUUGCACULTUGCAAUGCUGC NO 16 UG PS240 SEQ ID GUUGCACUUUGCAAUGCUGCU NO 17 GUCU SEQ ID UUGCACUUUGCAAUGCUGCUG NO 18 UCUU SEQ ID UGCACUUUGCAAUGCUGCUGU NO 19 CUUC SEQ ID GCACUUUGCAAUGCUGCUGUC NO 20 UUCU SEQ ID CACUUUGCAAUGCUGCUGUCU NO 21 UCUU SEQ ID ACUUUGCAAUGCUGCUGUCUU NO 22 CUUG SEQ ID CUUUGCAAUGCUGCUGUCUUC NO 23 UUGC SEQ ID UUUGCAAUGCUGCUGUCUUCU NO 24 UGCU SEQ ID UUGCAAUGCUGCUGUCUUCUU NO 25 GCUA SEQ ID UGCAAUGCUGCUGUCUUCUUG NO 26 CUAU SEQ ID GCAAUGCUGCUGUCUUCUUGC NO 27 UAUG SEQ ID CAAUGCUGCUGUCUUCUUGCU NO 28 AUGA SEQ ID AAUGCUGCUGUCUUCUUGCUA NO 29 UGAA SEQ ID AUGCUGCUGUCUUCUUGCUAU NO 30 GAAU SEQ ID UGCUGCUGUCUUCUUGCUAUG NO 31 AAUA SEQ ID GCUGCUGUCUUCUUGCUAUGA NO 32 AUAA SEQ ID CUGCUGUCUUCUUGCUAUGAA NO 33 UAAU SEQ ID UGCUGUCUUCUUGCUAUGAAU NO 34 AAUG SEQ ID GCUGUCUUCUUGCUAUGAAUA NO 35 AUGU SEQ ID CUGUCUUCUUGCUAUGAAUAA NO 36 UGUC SEQ ID UGUCUUCUUGCUAUGAAUAAU NO 37 GUCA SEQ ID GUCUUCUUGCUAUGAAUAAUG NO 38 UCAA SEQ ID UCUUCUUGCUAUGAAUAAUGUC NO 39 AAU SEQ ID CUUCUUGCUAUGAAUAAUGUCA NO 40 AUC SEQ ID UUCUUGCUAUGAAUAAUGUCAA NO 41 UCC SEQ ID UCUUGCUAUGAAUAAUGUCAAU NO 42 CCG SEQ ID CUUGCUAUGAAUAAUGUCAAUC NO 43 CGA SEQ ID UUGCUAUGAAUAAUGUCAAUCC NO 44 GAC SEQ ID UGCUAUGAAUAAUGUCAAUCCG NO 45 ACC SEQ ID GCUAUGAAUAAUGUCAAUCCGA NO 46 CCU SEQ ID CUAUGAAUAAUGUCAAUCCGACC NO 47 UG SEQ ID UAUGAAUAAUGUCAAUCCGACC NO 48 UGA SEQ ID AUGAAUAAUGUCAAUCCGACCU NO 49 GAG SEQ ID UGAAUAAUGUCAAUCCGACCUG NO 50 AGC SEQ ID GAAUAAUGUCAAUCCGACCUGA NO 51 GCU SEQ ID AAUAAUGUCAAUCCGACCUGAGC NO 52 UU SEQ ID AUAAUGUCAAUCCGACCUGAGCU NO 53 UU SEQ ID UAAUGUCAAUCCGACCUGAGCUU NO 54 UG SEQ ID AAUGUCAAUCCGACCUGAGCUUU NO 55 GU SEQ ID AUGUCAAUCCGACCUGAGCUUUG NO 56 UU SEQ ID UGUCAAUCCGACCUGAGCUUUGU NO 57 UG SEQ ID GUCAAUCCGACCUGAGCUUUGUU NO 58 GU SEQ ID UCAAUCCGACCUGAGCUUUGUUG NO 59 UA SEQ ID CAAUCCGACCUGAGCUUUGUUGU NO 60 AG SEQ ID AAUCCGACCUGAGCUUUGUUGU NO 61 AGA SEQ ID AUCCGACCUGAGCUUUGUUGUA NO 62 GAC SEQ ID UCCGACCUGAGCUUUGUUGUAG NO 63 ACU SEQ ID CCGACCUGAGCUUUGUUGUAGAC NO 64 UA SEQ ID CGACCUGAGCUUUGUUGUAG NO 65 PS237 SEQ ID CGACCUGAGCUUUGUUGUAGAC NO 66 UAU PS238 SEQ ID GACCUGAGCUUUGUUGUAGACU NO 67 AUC SEQ ID ACCUGAGCUUUGUUGUAGACUA NO 68 UCA SEQ ID CCUGAGCUUUGUUGUAGACU NO 69 AUC

TABLE-US-00004 TABLE 2 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 46 SEQ ID GCUUUUCUUUUAGUUGCUGCUC NO 70 UUU PS179 SEQ ID CUUUUCUUUUAGUUGCUGCUCU NO 71 UUU SEQ ID UUUUCUUUUAGUUGCUGCUCU NO 72 UUUC SEQ ID UUUCUUUUAGUUGCUGCUCUU NO 73 UUCC SEQ ID UUCUUUUAGUUGCUGCUCUUU NO 74 UCCA SEQ ID UCUUUUAGUUGCUGCUCUUUUC NO 75 CAG SEQ ID CUUUUAGUUGCUGCUCUUUUCC NO 76 AGG SEQ ID UUUUAGUUGCUGCUCUUUUCCA NO 77 GGU SEQ ID UUUAGUUGCUGCUCUUUUCCAG NO 78 GUU SEQ ID UUAGUUGCUGCUCUUUUCCAGG NO 79 UUC SEQ ID UAGUUGCUGCUCUUUUCCAGGU NO 80 UCA SEQ ID AGUUGCUGCUCUUUUCCAGGUU NO 81  CAA SEQ ID GUUGCUGCUCUUUUCCAGGUUC NO 82 AAG SEQ ID UUGCUGCUCUUUUCCAGGUUCA NO 83 AGU SEQ ID UGCUGCUCUUUUCCAGGUUCAA NO 84 GUG SEQ ID GCUGCUCUUUUCCAGGUUCAAG NO 85 UGG SEQ ID CUGCUCUUUUCCAGGUUCAAGU NO 86 GGG SEQ ID UGCUCUUUUCCAGGUUCAAGUG NO 87 GGA SEQ ID GCUCUUUUCCAGGUUCAAGUGG NO 88 GAC SEQ ID CUCUUUUCCAGGUUCAAGUGGG NO 89 AUA SEQ ID UCUUUUCCAGGUUCAAGUGGG NO 90 AUAC SEQ ID UCUUUUCCAGGUUCAAGUGG NO 91 PS177 SEQ ID CUUUUCCAGGUUCAAGUGGGA NO 92 UACU SEQ ID UUUUCCAGGUUCAAGUGGGAU NO 93 ACUA SEQ ID UUUCCAGGUUCAAGUGGGAUA NO 94 CUAG SEQ ID UUCCAGGUUCAAGUGGGAUAC NO 95 UAGC SEQ ID UCCAGGUUCAAGUGGGAUACU NO 96 AGCA SEQ ID CCAGGUUCAAGUGGGAUACUA NO 97 GCAA SEQ ID CAGGUUCAAGUGGGAUACUAG NO 98 CAAU SEQ ID AGGUUCAAGUGGGAUACUAGC NO 99 AAUG SEQ ID GGUUCAAGUGGGAUACUAGCA NO 100 AUGU SEQ ID GUUCAAGUGGGAUACUAGCAA NO 101 UGUU SEQ ID UUCAAGUGGGAUACUAGCAAU NO 102 GUUA SEQ ID UCAAGUGGGAUACUAGCAAUG NO 103 UUAU SEQ ID CAAGUGGGAUACUAGCAAUGU NO 104 UAUC SEQ ID AAGUGGGAUACUAGCAAUGUU NO 105 AUCU SEQ ID AGUGGGAUACUAGCAAUGUUA NO 106 UCUG SEQ ID GUGGGAUACUAGCAAUGUUAU NO 107 CUGC SEQ ID UGGGAUACUAGCAAUGUUAUC NO 108 UGCU SEQ ID GGGAUACUAGCAAUGUUAUCU NO 109 GCUU SEQ ID GGAUACUAGCAAUGUUAUCUG NO 110 CUUC PS181 SEQ ID GAUACUAGCAAUGUUAUCUGC NO 111 UUCC SEQ ID AUACUAGCAAUGUUAUCUGCU NO 112 UCCU SEQ ID UACUAGCAAUGUUAUCUGCUU NO 113 CCUC SEQ ID ACUAGCAAUGUUAUCUGCUUCC NO 114 UCC SEQ ID CUAGCAAUGUUAUCUGCUUCCU NO 115 CCA SEQ ID UAGCAAUGUUAUCUGCUUCCUC NO 116 CAA SEQ ID AGCAAUGUUAUCUGCUUCCUCC NO 117 AAC PS182 SEQ ID GCAAUGUUAUCUGCUUCCUCCA NO 118 ACC SEQ ID CAAUGUUAUCUGCUUCCUCCAA NO 119 CCA SEQ ID AAUGUUAUCUGCUUCCUCCAAC NO 120 CAU SEQ ID AUGUUAUCUGCUUCCUCCAACC NO 121 AUA SEQ ID UGUUAUCUGCUUCCUCCAACCA NO 122 UAA

TABLE-US-00005 TABLE 3 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 50 SEQ ID CCAAUAGUGGUCAGUCCAGGA NO 123 GCUA SEQ ID CAAUAGUGGUCAGUCCAGGAG NO 124 CUAG SEQ ID AAUAGUGGUCAGUCCAGGAGC NO 125 UAGG SEQ ID AUAGUGGUCAGUCCAGGAGCU NO 126 AGGU SEQ ID AUAGUGGUCAGUCCAGGAGCU NO 127 PS248 SEQ ID UAGUGGUCAGUCCAGGAGCUA NO 128 GGUC SEQ ID AGUGGUCAGUCCAGGAGCUAG NO 129 GUCA SEQ ID GUGGUCAGUCCAGGAGCUAGG NO 130 UCAG SEQ ID UGGUCAGUCCAGGAGCUAGGU NO 131 CAGG SEQ ID GGUCAGUCCAGGAGCUAGGUC NO 132 AGGC SEQ ID GUCAGUCCAGGAGCUAGGUCA NO 133 GGCU SEQ ID UCAGUCCAGGAGCUAGGUCAG NO 134 GCUG SEQ ID CAGUCCAGGAGCUAGGUCAGG NO 135 CUGC SEQ ID AGUCCAGGAGCUAGGUCAGGC NO 136 UGCU SEQ ID GUCCAGGAGCUAGGUCAGGCU NO 137 GCUU SEQ ID UCCAGGAGCUAGGUCAGGCUG NO 138 CUUU SEQ ID CCAGGAGCUAGGUCAGGCUGC NO 139 UUUG SEQ ID  CAGGAGCUAGGUCAGGCUGCU NO 140 UUGC SEQ ID AGGAGCUAGGUCAGGCUGCUU NO 141 UGCC SEQ ID GGAGCUAGGUCAGGCUGCUUU NO 142 GCCC SEQ ID GAGCUAGGUCAGGCUGCUUUG NO 143 CCCU SEQ ID AGCUAGGUCAGGCUGCUUUGC NO 144 CCUC SEQ ID GCUAGGUCAGGCUGCUUUGCC NO 145 CUCA SEQ ID CUAGGUCAGGCUGCUUUGCCCU NO 146 CAG SEQ ID UAGGUCAGGCUGCUUUGCCCUC NO 147 AGC SEQ ID AGGUCAGGCUGCUUUGCCCUCA NO 148 GCU SEQ ID GGUCAGGCUGCUUUGCCCUCAG NO 149 CUC SEQ ID GUCAGGCUGCUUUGCCCUCAGC NO 150 UCU SEQ ID UCAGGCUGCUUUGCCCUCAGCU NO 151 CUU SEQ ID CAGGCUGCUUUGCCCUCAGCUC NO 152 UUG SEQ ID AGGCUGCUUUGCCCUCAGCUCU NO 153 UGA SEQ ID GGCUGCUUUGCCCUCAGCUCUU NO 154 GAA SEQ ID GCUGCUUUGCCCUCAGCUCUUG NO 155 AAG SEQ ID CUGCUUUGCCCUCAGCUCUUGA NO 156 AGU SEQ ID UGCUUUGCCCUCAGCUCUUGAA NO 157 GUA SEQ ID GCUUUGCCCUCAGCUCUUGAAG NO 158 UAA SEQ ID CUUUGCCCUCAGCUCUUGAAGU NO 159 AAA SEQ ID UUUGCCCUCAGCUCUUGAAGU NO 160 AAAC SEQ ID UUGCCCUCAGCUCUUGAAGUA NO 161 AACG SEQ ID UGCCCUCAGCUCUUGAAGUAA NO 162 ACGG SEQ ID GCCCUCAGCUCUUGAAGUAAAC NO 163 GGU SEQ ID CCCUCAGCUCUUGAAGUAAACG NO 164 GUU SEQ ID CCUCAGCUCUUGAAGUAAAC NO 165 PS246 SEQ ID CCUCAGCUCUUGAAGUAAACG NO 166 PS247 SEQ ID CUCAGCUCUUGAAGUAAACG NO 167 PS245 SEQ ID CCUCAGCUCUUGAAGUAAACG NO 529 GUUU SEQ ID CUCAGCUCUUGAAGUAAACGG NO 530 UUUA SEQ ID UCAGCUCUUGAAGUAAACGGU NO 531 UUAC SEQ ID CAGCUCUUGAAGUAAACGGUU NO 532 UACC SEQ ID AGCUCUUGAAGUAAACGGUUU NO 533 ACCG SEQ ID GCUCUUGAAGUAAACGGUUUA NO 534 CCGC SEQ ID CUCUUGAAGUAAACGGUUUAC NO 535 CGCC

TABLE-US-00006 TABLE 4 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 51 SEQ ID GUACCUCCAACAUCAAGGAAGA NO 168 UGG SEQ ID UACCUCCAACAUCAAGGAAGAU NO 169 GGC SEQ ID ACCUCCAACAUCAAGGAAGAUG NO 170 GCA SEQ ID CCUCCAACAUCAAGGAAGAUGG NO 171 CAU SEQ ID CUCCAACAUCAAGGAAGAUGGC NO 172 AUU SEQ ID UCCAACAUCAAGGAAGAUGGCA NO 173 UUU SEQ ID CCAACAUCAAGGAAGAUGGCAU NO 174 UUC SEQ ID CAACAUCAAGGAAGAUGGCAUU NO 175 UCU SEQ ID AACAUCAAGGAAGAUGGCAUUU NO 176 CUA SEQ ID ACAUCAAGGAAGAUGGCAUUUC NO 177 UAG SEQ ID CAUCAAGGAAGAUGGCAUUUCU NO 178 AGU SEQ ID AUCAAGGAAGAUGGCAUUUCUA NO 179 GUU SEQ ID UCAAGGAAGAUGGCAUUUCUAG NO 180 UUU SEQ ID CAAGGAAGAUGGCAUUUCUAGU NO 181 UUG SEQ ID AAGGAAGAUGGCAUUUCUAGUU NO 182 UGG SEQ ID AGGAAGAUGGCAUUUCUAGUUU NO 183 GGA SEQ ID GGAAGAUGGCAUUUCUAGUUUG NO 184 GAG SEQ ID GAAGAUGGCAUUUCUAGUUUGG NO 185 AGA SEQ ID AAGAUGGCAUUUCUAGUUUGGA NO 186 GAU SEQ ID AGAUGGCAUUUCUAGUUUGGAG NO 187 AUG SEQ ID GAUGGCAUUUCUAGUUUGGAGA NO 188 UGG SEQ ID AUGGCAUUUCUAGUUUGGAGAU NO 189 GGC SEQ ID UGGCAUUUCUAGUUUGGAGAUG NO 190 GCA SEQ ID GGCAUUUCUAGUUUGGAGAUGG NO 191 CAG SEQ ID GCAUUUCUAGUUUGGAGAUGGC NO 192 AGU SEQ ID CAUUUCUAGUUUGGAGAUGGCA NO 193 GUU SEQ ID AUUUCUAGUUUGGAGAUGGCAG NO 194 UUU SEQ ID UUUCUAGUUUGGAGAUGGCAGU NO 195 UUC SEQ ID UUCUAGUUUGGAGAUGGCAGUU NO 196 UCC SEQ ID UCUAGUUUGGAGAUGGCAGUUU NO 197 CCU SEQ ID CUAGUUUGGAGAUGGCAGUUUC NO 198 CUU SEQ ID UAGUUUGGAGAUGGCAGUUUCC NO 199 UUA SEQ ID AGUUUGGAGAUGGCAGUUUCCU NO 200 UAG SEQ ID GUUUGGAGAUGGCAGUUUCCUU NO 201 AGU SEQ ID UUUGGAGAUGGCAGUUUCCUUA NO 202 GUA SEQ ID UUGGAGAUGGCAGUUUCCUUAG NO 203 UAA SEQ ID UGGAGAUGGCAGUUUCCUUAGU NO 204 AAC SEQ ID GAGAUGGCAGUUUCCUUAGUAA NO 205 CCA SEQ ID AGAUGGCAGUUUCCUUAGUAAC NO 206 CAC SEQ ID GAUGGCAGUUUCCUUAGUAACC NO 207 ACA SEQ ID AUGGCAGUUUCCUUAGUAACCA NO 208 CAG SEQ ID UGGCAGUUUCCUUAGUAACCAC NO 209 AGG SEQ ID GGCAGUUUCCUUAGUAACCACA NO 210 GGU SEQ ID GCAGUUUCCUUAGUAACCACAG NO 211 GUU SEQ ID CAGUUUCCUUAGUAACCACAGG NO 212 UUG SEQ ID AGUUUCCUUAGUAACCACAGGU NO 213 UGU SEQ ID GUUUCCUUAGUAACCACAGGUU NO 214 GUG SEQ ID UUUCCUUAGUAACCACAGGUUG NO 215 UGU SEQ ID UUCCUUAGUAACCACAGGUUGU NO 216 GUC SEQ ID UCCUUAGUAACCACAGGUUGUG NO 217 UCA SEQ ID CCUUAGUAACCACAGGUUGUGU NO 218 CAC SEQ ID CUUAGUAACCACAGGUUGUGUC NO 219 ACC SEQ ID UUAGUAACCACAGGUUGUGUCA NO 220 CCA SEQ ID UAGUAACCACAGGUUGUGUCAC NO 221 CAG SEQ ID AGUAACCACAGGUUGUGUCACC NO 222 AGA SEQ ID GUAACCACAGGUUGUGUCACCA NO 223 GAG SEQ ID UAACCACAGGUUGUGUCACCAG NO 224 AGU SEQ ID AACCACAGGUUGUGUCACCAGA NO 225 GUA SEQ ID ACCACAGGUUGUGUCACCAGAG NO 226 UAA SEQ ID CCACAGGUUGUGUCACCAGAGU NO 227 AAC SEQ ID CACAGGUUGUGUCACCAGAGUA NO 228 ACA SEQ ID ACAGGUUGUGUCACCAGAGUAA NO 229 CAG SEQ ID CAGGUUGUGUCACCAGAGUAAC NO 230 AGU SEQ ID AGGUUGUGUCACCAGAGUAACA NO 231 GUC SEQ ID GGUUGUGUCACCAGAGUAACAG NO 232 UCU SEQ ID GUUGUGUCACCAGAGUAACAGU NO 233 CUG SEQ ID UUGUGUCACCAGAGUAACAGUC NO 234 UGA SEQ ID UGUGUCACCAGAGUAACAGUCU NO 235 GAG SEQ ID GUGUCACCAGAGUAACAGUCUG NO 236 AGU SEQ ID UGUCACCAGAGUAACAGUCUGA NO 237 GUA SEQ ID GUCACCAGAGUAACAGUCUGAG NO 238 UAG SEQ ID UCACCAGAGUAACAGUCUGAGU NO 239 AGG SEQ ID CACCAGAGUAACAGUCUGAGUA NO 240 GGA SEQ ID ACCAGAGUAACAGUCUGAGUA NO 241 GGAG

TABLE-US-00007 TABLE 5 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 52 SEQ ID AGCCUCUUGAUUGCUGGUCUUG NO 242 UUU SEQ ID GCCUCUUGAUUGCUGGUCUUGU NO 243 UUU SEQ ID CCUCUUGAUUGCUGGUCUUGUU NO 244 UUU SEQ ID CCUCUUGAUUGCUGGUCUUG NO 245 SEQ ID CUCUUGAUUGCUGGUCUUGUU NO 246 UUUC PS232 SEQ ID UCUUGAUUGCUGGUCUUGUUU NO 247 UUCA SEQ ID CUUGAUUGCUGGUCUUGUUUU NO 248 UCAA SEQ ID UUGAUUGCUGGUCUUGUUUUU NO 249 CAAA SEQ ID UGAUUGCUGGUCUUGUUUUUC NO 250 AAAU SEQ ID GAUUGCUGGUCUUGUUUUUCA NO 251 AAUU SEQ ID GAUUGCUGGUCUUGUUUUUC NO 252 SEQ ID AUUGCUGGUCUUGUUUUUCAA NO 253 AUUU SEQ ID UUGCUGGUCUUGUUUUUCAAA NO 254 UUUU SEQ ID UGCUGGUCUUGUUUUUCAAAU NO 255 UUUG SEQ ID GCUGGUCUUGUUUUUCAAAUU NO 256 UUGG SEQ ID CUGGUCUUGUUUUUCAAAUUU NO 257 UGGG SEQ ID UGGUCUUGUUUUUCAAAUUUU NO 258 GGGC SEQ ID GGUCUUGUUUUUCAAAUUUUG NO 259 GGCA SEQ ID GUCUUGUUUUUCAAAUUUUGG NO 260 GCAG SEQ ID UCUUGUUUUUCAAAUUUUGGG NO 261 CAGC SEQ ID CUUGUUUUUCAAAUUUUGGGC NO 262 AGCG SEQ ID UUGUUUUUCAAAUUUUGGGCA NO 263 GCGG SEQ ID UGUUUUUCAAAUUUUGGGCAG NO 264 CGGU SEQ ID GUUUUUCAAAUUUUGGGCAGC NO 265 GGUA SEQ ID UUUUUCAAAUUUUGGGCAGCG NO 266 GUAA SEQ ID UUUUCAAAUUUUGGGCAGCGG NO 267 UAAU SEQ ID UUUCAAAUUUUGGGCAGCGGU NO 268 AAUG SEQ ID UUCAAAUUUUGGGCAGCGGUA NO 269 AUGA SEQ ID UCAAAUUUUGGGCAGCGGUAA NO 270 UGAG SEQ ID CAAAUUUUGGGCAGCGGUAAU NO 271 GAGU SEQ ID AAAUUUUGGGCAGCGGUAAUG NO 272 AGUU SEQ ID AAUUUUGGGCAGCGGUAAUGA NO 273 GUUC SEQ ID AUUUUGGGCAGCGGUAAUGAG NO 274 UUCU SEQ ID UUUUGGGCAGCGGUAAUGAGU NO 275 UCUU SEQ ID UUUGGGCAGCGGUAAUGAGUU NO 276 CUUC SEQ ID UUGGGCAGCGGUAAUGAGUUCU NO 277 UCC SEQ ID UGGGCAGCGGUAAUGAGUUCUU NO 278 CCA SEQ ID GGGCAGCGGUAAUGAGUUCUUC NO 279 CAA SEQ ID GGCAGCGGUAAUGAGUUCUUCC NO 280 AAC SEQ ID GCAGCGGUAAUGAGUUCUUCCA NO 281 ACU SEQ ID CAGCGGUAAUGAGUUCUUCCAA NO 282 CUG SEQ ID AGCGGUAAUGAGUUCUUCCAAC NO 283 UGG SEQ ID GCGGUAAUGAGUUCUUCCAACU NO 284 GGG SEQ ID CGGUAAUGAGUUCUUCCAACUG NO 285 GGG SEQ ID GGUAAUGAGUUCUUCCAACUGG NO 286 GGA SEQ ID GGUAAUGAGUUCUUCCAACUGG NO 287 SEQ ID GUAAUGAGUUCUUCCAACUGGG NO 288 GAC SEQ ID UAAUGAGUUCUUCCAACUGGGG NO 289 ACG SEQ ID AAUGAGUUCUUCCAACUGGGGA NO 290 CGC SEQ ID AUGAGUUCUUCCAACUGGGGAC NO 291 GCC SEQ ID UGAGUUCUUCCAACUGGGGACG NO 292 CCU SEQ ID GAGUUCUUCCAACUGGGGACGC NO 293 CUC SEQ ID AGUUCUUCCAACUGGGGACGCC NO 294 UCU SEQ ID GUUCUUCCAACUGGGGACGCCU NO 295 CUG SEQ ID UUCUUCCAACUGGGGACGCCUC NO 296 UGU SEQ ID UCUUCCAACUGGGGACGCCUCU NO 297 GUU SEQ ID CUUCCAACUGGGGACGCCUCUG NO 298 UUC SEQ ID UUCCAACUGGGGACGCCUCUGU NO 299 UCC PS236 SEQ ID UCCAACUGGGGACGCCUCUGUU NO 300 CCA SEQ ID CCAACUGGGGACGCCUCUGUUC NO 301 CAA SEQ ID CAACUGGGGACGCCUCUGUUCC NO 302 AAA SEQ ID AACUGGGGACGCCUCUGUUCCA NO 303 AAU SEQ ID ACUGGGGACGCCUCUGUUCCAA NO 304 AUC SEQ ID CUGGGGACGCCUCUGUUCCAAA NO 305 UCC SEQ ID UGGGGACGCCUCUGUUCCAAAU NO 306 CCU SEQ ID GGGGACGCCUCUGUUCCAAAUC NO 307 CUG SEQ ID GGGACGCCUCUGUUCCAAAUCC NO 308 UGC SEQ ID GGACGCCUCUGUUCCAAAUCCU NO 309 GCA SEQ ID GACGCCUCUGUUCCAAAUCCUG NO 310 CAU

TABLE-US-00008 TABLE 6 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 53 SEQ ID CUCUGGCCUGUCCUAAGACCU NO 311 GCUC SEQ ID UCUGGCCUGUCCUAAGACCUG NO 312 CUCA SEQ ID CUGGCCUGUCCUAAGACCUGC NO 313 UCAG SEQ ID UGGCCUGUCCUAAGACCUGCU NO 314 CAGC SEQ ID GGCCUGUCCUAAGACCUGCUC NO 315 AGCU SEQ ID GCCUGUCCUAAGACCUGCUCA NO 316 GCUU SEQ ID CCUGUCCUAAGACCUGCUCAG NO 317 CUUC SEQ ID CUGUCCUAAGACCUGCUCAGC NO 318 UUCU SEQ ID UGUCCUAAGACCUGCUCAGCU NO 319 UCUU SEQ ID GUCCUAAGACCUGCUCAGCUU NO 320 CUUC SEQ ID UCCUAAGACCUGCUCAGCUUC NO 321 UUCC SEQ ID CCUAAGACCUGCUCAGCUUCU NO 322 UCCU SEQ ID CUAAGACCUGCUCAGCUUCUU NO 323 CCUU SEQ ID UAAGACCUGCUCAGCUUCUUC NO 324 CUUA SEQ ID AAGACCUGCUCAGCUUCUUCC NO 325 UUAG SEQ ID AGACCUGCUCAGCUUCUUCCU NO 326 UAGC SEQ ID GACCUGCUCAGCUUCUUCCUU NO 327 AGCU SEQ ID ACCUGCUCAGCUUCUUCCUUA NO 328 GCUU SEQ ID CCUGCUCAGCUUCUUCCUUAG NO 329 CUUC SEQ ID CUGCUCAGCUUCUUCCUUAGC NO 330 UUCC SEQ ID UGCUCAGCUUCUUCCUUAGCU NO 331 UCCA SEQ ID GCUCAGCUUCUUCCUUAGCUU NO 332 CCAG SEQ ID CUCAGCUUCUUCCUUAGCUUC NO 333 CAGC SEQ ID UCAGCUUCUUCCUUAGCUUCC NO 334 AGCC SEQ ID CAGCUUCUUCCUUAGCUUCCAG NO 335 CCA SEQ ID AGCUUCUUCCUUAGCUUCCAGC NO 336 CAU SEQ ID GCUUCUUCCUUAGCUUCCAGCC NO 337 AUU SEQ ID CUUCUUCCUUAGCUUCCAGCCA NO 338 UUG SEQ ID UUCUUCCUUAGCUUCCAGCCAU NO 339 UGU SEQ ID UCUUCCUUAGCUUCCAGCCAUU NO 340 GUG SEQ ID CUUCCUUAGCUUCCAGCCAUUG NO 341 UGU SEQ ID UUCCUUAGCUUCCAGCCAUUGU NO 342 GUU SEQ ID UCCUUAGCUUCCAGCCAUUGUG NO 343 UUG SEQ ID CCUUAGCUUCCAGCCAUUGUGU NO 344 UGA SEQ ID CUUAGCUUCCAGCCAUUGUGUU NO 345 GAA SEQ ID UUAGCUUCCAGCCAUUGUGUUG NO 346 AAU SEQ ID UAGCUUCCAGCCAUUGUGUUGA NO 347 AUC SEQ ID AGCUUCCAGCCAUUGUGUUGAA NO 348 UCC SEQ ID GCUUCCAGCCAUUGUGUUGAAU NO 349 CCU SEQ ID CUUCCAGCCAUUGUGUUGAAUC NO 350 CUU SEQ ID UUCCAGCCAUUGUGUUGAAUCC NO 351 UUU SEQ ID UCCAGCCAUUGUGUUGAAUCCU NO 352 UUA SEQ ID CCAGCCAUUGUGUUGAAUCCUU NO 353 UAA SEQ ID CAGCCAUUGUGUUGAAUCCUUU NO 354 AAC SEQ ID AGCCAUUGUGUUGAAUCCUUUA NO 355 ACA SEQ ID GCCAUUGUGUUGAAUCCUUUAA NO 356 CAU SEQ ID CCAUUGUGUUGAAUCCUUUAAC NO 357 AUU SEQ ID CAUUGUGUUGAAUCCUUUAACA NO 358 UUU

TABLE-US-00009 TABLE 7 OLIGONUCLEOTIDES FOR SKIPPING OTHER EXONS OF THE DMD GENE AS IDENTIFIED DMD Gene Exon 6 SEQ ID CAUUUUUGACCUACAUGUGG NO 359 SEQ ID UUUGACCUACAUGUGGAAAG NO 360 SEQ ID UACAUUUUUGACCUACAUGUG NO 361 GAAA G SEQ ID GGUCUCCUUACCUAUGA NO 362 SEQ ID UCUUACCUAUGACUAUGGAUG NO 363 AGA SEQ ID AUUUUUGACCUACAUGGGAAA NO 364 G SEQ ID UACGAGUUGAUUGUCGGACCCA NO 365 G SEQ ID GUGGUCUCCUUACCUAUGACUG NO 366 UGG SEQ ID UGUCUCAGUAAUCUUCUUACCU NO 367 AU DMD Gene Exon 7 SEQ ID UGCAUGUUCCAGUCGUUGUGU NO 368 GG SEQ ID CACUAUUCCAGUCAAAUAGGU NO 369 CUGG SEQ ID AUUUACCAACCUUCAGGAUCGA NO 370 GUA SEQ ID GGCCUAAAACACAUACACAUA NO 371 DMD Gene Exon 11 SEQ ID CCCUGAGGCAUUCCCAUCUUG NO 372 AAU SEQ ID AGGACUUACUUGCUUUGUUU NO 373 SEQ ID CUUGAAUUUAGGAGAUUCAUCU NO 374 G SEQ ID CAUCUUCUGAUAAUUUUCCUGU NO 375 U DMD Gene Exon 17 SEQ ID CCAUUACAGUUGUCUGUGUU NO 376 SEQ ID UGACAGCCUGUGAAAUCUGUG NO 377 AG SEQ ID UAAUCUGCCUCUUCUUUUGG NO 378 DMD Gene Exon 19 SEQ ID CAGCAGUAGUUGUCAUCUGC NO 379 SEQ ID GCCUGAGCUGAUCUGCUGGCA NO 380 UCUUGC SEQ ID GCCUGAGCUGAUCUGCUGGCAU NO 381 CUUGCA GUU SEQ ID UCUGCUGGCAUCUUGC NO 382 DMD Gene Exon 21 SEQ ID GCCGGUUGACUUCAUCCUGUG NO 383 C SEQ ID GUCUGCAUCCAGGAACAUGGG NO 384 UC SEQ ID UACUUACUGUCUGUAGCUCUU NO 385 UCU SEQ ID CUGCAUCCAGGAACAUGGGUCC NO 386 SEQ ID GUUGAAGAUCUGAUAGCCGGUU NO 387 GA DMD Gene Exon 44 SEQ ID UCAGCUUCUGUUAGCCACUG NO 388 SEQ ID UUCAGCUUCUGUUAGCCACU NO 389 SEQ ID UUCAGCUUCUGUUAGCCACUG NO 390 SEQ ID UCAGCUUCUGUUAGCCACUGA NO 391 SEQ ID UUCAGCUUCUGUUAGCCACUG NO 392 A SEQ ID UCAGCUUCUGUUAGCCACUGA NO 393 SEQ ID UUCAGCUUCUGUUAGCCACUG NO 394 A SEQ ID UCAGCUUCUGUUAGCCACUGA NO 395 U SEQ ID UUCAGCUUCUGUUAGCCACUG NO 396 AU SEQ ID UCAGCUUCUGUUAGCCACUGA NO 397 UU SEQ ID UUCAGCUUCUGUUAGCCACUG NO 398 AUU SEQ ID UCAGCUUCUGUUAGCCACUGA NO 399 UUA SEQ ID UUCAGCUUCUGUUAGCCACUG NO 400 AUA SEQ ID UCAGCUUCUGUUAGCCACUGA NO 401 UUAA SEQ ID UUCAGCUUCUGUUAGCCACUG NO 402 AUUAA SEQ ID UCAGCUUCUGUUAGCCACUGA NO 403 UUAAA SEQ ID UUCAGCUUCUGUUAGCCACUG NO 404 AUUAAA SEQ ID CAGCUUCUGUUAGCCACUG NO 405 SEQ ID CAGCUUCUGUUAGCCACUGAU NO 406 SEQ ID AGCUUCUGUUAGCCACUGAUU NO 407 SEQ ID CAGCUUCUGUUAGCCACUGAU NO 408 U SEQ ID AGCUUCUGUUAGCCACUGAUU NO 409 A SEQ ID CAGCUUCUGUUAGCCACUGAU NO 410 UA SEQ ID AGCUUCUGUUAGCCACUGAUU NO 411 AA SEQ ID CAGCUUCUGUUAGCCACUGAU NO 412 UAA SEQ ID AGCUUCUGUUAGCCACUGAUUA NO 413 AA SEQ ID CAGCUUCUGUUAGCCACUGAUU NO 414 AAA SEQ ID AGCUUCUGUUAGCCACUGAUUA NO 415 AA SEQ ID AGCUUCUGUUAGCCACUGAU NO 416 SEQ ID GCUUCUGUUAGCCACUGAUU NO 417 SEQ ID AGCUUCUGUUAGCCACUGAUU NO 418 SEQ ID GCUUCUGUUAGCCACUGAUUA NO 419 SEQ ID AGCUUCUGUUAGCCACUGAUUA NO 420 SEQ ID GCUUCUGUUAGCCACUGAUUAA NO 421 SEQ ID AGCUUCUGUUAGCCACUGAUUA NO 422 A SEQ ID GCUUCUGUUAGCCACUGAUUAA NO 423 A SEQ ID AGCUUCUGUUAGCCACUGAUUA NO 424 AA SEQ ID GCUUCUGUUAGCCACUGAUUAA NO 425 A SEQ ID CCAUUUGUAUUUAGCAUGUUCC NO 426 C SEQ ID AGAUACCAUUUGUAUUUAGC NO 427 SEQ ID GCCAUUUCUCAACAGAUCU NO 428 SEQ ID GCCAUUUCUCAACAGAUCUGUC NO 429 A SEQ ID AUUCUCAGGAAUUUGUGUCUUU NO 430 C SEQ ID UCUCAGGAAUUUGUGUCUUUC NO 431 SEQ ID GUUCAGCUUCUGUUAGCC NO 432 SEQ ID CUGAUUAAAUAUCUUUAUAUC NO 433 SEQ ID GCCGCCAUUUCUCAACAG NO 434 SEQ ID GUAUUUAGCAUGUUCCCA NO 435 SEQ ID CAGGAAUUUGUGUCUUUC NO 436 DMD Gene Exon 45 SEQ ID UUUGCCGCUGCCCAAUGCCAU NO 437 CCUG SEQ ID AUUCAAUGUUCUGACAACAGU NO 438 UUGC SEQ ID CCAGUUGCAUUCAAUGUUCUG NO 439 ACAA SEQ ID CAGUUGCAUUCAAUGUUCUGA NO 440 C SEQ ID AGUUGCAUUCAAUGUUCUGA NO 441 SEQ ID GAUUGCUGAAUUAUUUCUUCC NO 442 SEQ ID GAUUGCUGAAUUAUUUCUUCC NO 443 CCAG SEQ ID AUUGCUGAAUUAUUUCUUCCC NO 444 CAGU SEQ ID UUGCUGAAUUAUUUCUUCCCC NO 445 AGUU SEQ ID UGCUGAAUUAUUUCUUCCCCA NO 446 GUUG SEQ ID GCUGAAUUAUUUCUUCCCCAG NO 447 UUGC SEQ ID CUGAAUUAUUUCUUCCCCAGU NO 448 UGCA SEQ ID UGAAUUAUUUCUUCCCCAGUU NO 449 GCAU SEQ ID GAAUUAUUUCUUCCCCAGUUG NO 450 CAUU SEQ ID AAUUAUUUCUUCCCCAGUUGC NO 451 AUUC SEQ ID AUUAUUUCUUCCCCAGUUGCA NO 452 UUCA SEQ ID UUAUUUCUUCCCCAGUUGCAU NO 453 UCAA SEQ ID UAUUUCUUCCCCAGUUGCAUU NO 454 CAAU SEQ ID AUUUCUUCCCCAGUUGCAUUC NO 455 AAUG SEQ ID UUUCUUCCCCAGUUGCAUUCA NO 456 AUGU SEQ ID UUCUUCCCCAGUUGCAUUCAA NO 457 UGUU SEQ ID UCUUCCCCAGUUGCAUUCAAU NO 458 GUUC SEQ ID CUUCCCCAGUUGCAUUCAAUG NO 459 UUCU SEQ ID UUCCCCAGUUGCAUUCAAUGU NO 460 UCUG SEQ ID UCCCCAGUUGCAUUCAAUGUU NO 461 CUGA SEQ ID CCCCAGUUGCAUUCAAUGUUC NO 462 UGAC SEQ ID CCCAGUUGCAUUCAAUGUUCU NO 463 GACA SEQ ID CCAGUUGCAUUCAAUGUUCUG NO 464 ACAA SEQ ID CAGUUGCAUUCAAUGUUCUGA NO 465 CAAC SEQ ID AGUUGCAUUCAAUGUUCUGAC NO 466 AACA SEQ ID UCC UGU AGA AUA CUG GCA NO 467 UC SEQ ID UGCAGACCUCCUGCCACCGCAG NO 468 AUUCA SEQ ID UUGCAGACCUCCUGCCACCGCA NO 469 GAUUC AGGCUUC SEQ ID GUUGCAUUCAAUGUUCUGACAA NO 470 CAG SEQ ID UUGCAUUCAAUGUUCUGACAAC NO 471 AGU SEQ ID UGCAUUCAAUGUUCUGACAACA NO 472 GUU SEQ ID GCAUUCAAUGUUCUGACAACAG NO 473 UUU SEQ ID CAUUCAAUGUUCUGACAACAGU NO 474 UUG SEQ ID AUUCAAUGUUCUGACAACAGUU NO 475 UGC SEQ ID UCAAUGUUCUGACAACAGUUUG NO 476 CCG SEQ ID CAAUGUUCUGACAACAGUUUGC NO 477 CGC SEQ ID AAUGUUCUGACAACAGUUUGCC NO 478 GCU SEQ ID  AUGUUCUGACAACAGUUUGCCG NO 479 CUG SEQ ID UGUUCUGACAACAGUUUGCCGC NO 480 UGC SEQ ID GUUCUGACAACAGUUUGCCGCU NO 481 GCC SEQ ID UUCUGACAACAGUUUGCCGCUG NO 482 CCC SEQ ID UCUGACAACAGUUUGCCGCUGC NO 483 CCA SEQ ID CUGACAACAGUUUGCCGCUGCC NO 484 CAA SEQ ID UGACAACAGUUUGCCGCUGCCC NO 485 AAU SEQ ID GACAACAGUUUGCCGCUGCCCA NO 486 AUG SEQ ID ACAACAGUUUGCCGCUGCCCAA NO 487 UGC SEQ ID CAACAGUUUGCCGCUGCCCAAU NO 488 GCC SEQ ID AACAGUUUGCCGCUGCCCAAUG NO 489 CCA SEQ ID ACAGUUUGCCGCUGCCCAAUGC NO 490 CAU SEQ ID CAGUUUGCCGCUGCCCAAUGCC NO 491 AUC SEQ ID AGUUUGCCGCUGCCCAAUGCCA NO 492 UCC SEQ ID GUUUGCCGCUGCCCAAUGCCAU NO 493 CCU SEQ ID UUUGCCGCUGCCCAAUGCCAUC NO 494 CUG SEQ ID UUGCCGCUGCCCAAUGCCAUCC NO 495 UGG SEQ ID UGCCGCUGCCCAAUGCCAUCCU NO 496 GGA SEQ ID GCCGCUGCCCAAUGCCAUCCUG NO 497 GAG SEQ ID CCGCUGCCCAAUGCCAUCCUGG NO 498 AGU SEQ ID CGCUGCCCAAUGCCAUCCUGGA NO 499 GUU SEQ ID UGUUUUUGAGGAUUGCUGAA NO 500 SEQ ID UGUUCUGACAACAGUUUGCCGC NO 501 UGCCCA AUGCCAUCCUGG DMD Gene Exon 55 SEQ ID CUGUUGCAGUAAUCUAUGAG NO 502 SEQ ID UGCAGUAAUCUAUGAGUUUC NO 503 SEQ ID GAGUCUUCUAGGAGCCUU NO 504 SEQ ID UGCCAUUGUUUCAUCAGCUCUU NO 505 U SEQ ID UCCUGUAGGACAUUGGCAGU NO 506 SEQ ID CUUGGAGUCUUCUAGGAGCC NO 507 DMD Gene Exon 57 SEQ ID UAGGUGCCUGCCGGCUU NO 508 SEQ ID UUCAGCUGUAGCCACACC NO 509 SEQ ID CUGAACUGCUGGAAAGUCGCC NO 510 SEQ ID CUGGCUUCCAAAUGGGACCUGA NO 511 AAAAGA AC DMD Gene Exon 59 SEQ ID CAAUUUUUCCCACUCAGUAUU NO 512 SEQ ID UUGAAGUUCCUGGAGUCUU NO 513 SEQ ID UCCUCAGGAGGCAGCUCUAAAU NO 514 DMD Gene Exon 62 SEQ ID  UGGCUCUCUCCCAGGG NO 515 SEQ ID GAGAUGGCUCUCUCCCAGGGA NO 516 CCCUGG SEQ ID GGGCACUUUGUUUGGCG NO 517 DMD Gene Exon 63 SEQ ID GGUCCCAGCAAGUUGUUUG NO 518 SEQ ID  UGGGAUGGUCCCAGCAAGUUG NO 519 UUUG SEQ ID GUAGAGCUCUGUCAUUUUGGG NO 520 DMD Gene Exon 65 SEQ ID GCUCAAGAGAUCCACUGCAAA NO 521 AAAC SEQ ID  GCCAUACGUACGUAUCAUAAA NO 522 CAUUC SEQ ID UCUGCAGGAUAUCCAUGGGCUG NO 523 GUC DMD Gene Exon 66 SEQ ID GAUCCUCCCUGUUCGUCCCCUA NO 524 UUAUG DMD Gene Exon 69 SEQ ID UGCUUUAGACUCCUGUACCUG NO 525 AUA DMD Gene Exon 75 SEQ ID GGCGGCCUUUGUGUUGAC NO 526 SEQ ID GGACAGGCCUUUAUGUUCGUG NO 527 CUGC SEQ ID CCUUUAUGUUCGUGCUGCU NO 528