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

Description

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/024,558, filed Jun. 29, 2018, which is a continuation of U.S. application Ser. No. 15/289,053, filed Oct. 7, 2016, which is a continuation of U.S. application Ser. No. 14/631,686, filed Feb. 25, 2015, now U.S. Pat. No. 9,499,818, which is a continuation of U.S. application Ser. No. 13/094,571, filed Apr. 26, 2011, which is a continuation of PCTNL2009/050113, filed Mar. 11, 2009, which is a continuation-in-part of PCT/NL2008/050673, filed Oct. 27, 2008. The disclosures of each of the above-referenced applications are incorporated by reference herein in their entirety.

SEQUENCE LISTING

This specification is being filed with a Sequence Listing in Computer Readable Form (CFR), which is entitled “0105_07 US1CN4_SL.txt” of 128885 bytes in size and was created on Dec. 14, 2020, the content of which is incorporated herein by reference in its entirety.

FIELD

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

BACKGROUND

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

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

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

Using antisense oligonucleotides (AONs) interfering with splicing signals the skipping of specific exons can be induced in the DMD pre-mRNA, thus restoring the open reading frame and converting the severe DMD into a milder BMD phenotype (van Deutekom et al. Hum Mol Genet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet 2003; 12(8):907-14.). In vivo proof-of-concept was first obtained in the mdx mouse model, which is dystrophin-deficient due to a nonsense mutation in exon 23. Intramuscular and intravenous injections of AONs targeting the mutated exon 23 restored dystrophin expression for at least three months (Lu et al. Nat Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci US A. 2005; 102(1):198-203). This was accompanied by restoration of dystrophin-associated proteins at the fiber membrane as well as functional improvement of the treated muscle. In vivo skipping of human exons has also been achieved in the hDMD mouse model, which contains a complete copy of the human DMD gene integrated in chromosome 5 of the mouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; ′t Hoen et al. J Biol Chem. 2008; 283: 5899-907).

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

BRIEF DESCRIPTION OF THE DRAWINGS

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)

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)

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).

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

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.

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.

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

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

Exon skipping refers to the induction in a cell of a mature mRNA that does not contain a particular exon that is normally present therein. Exon skipping is performed by providing a cell expressing the pre-mRNA of said mRNA with a molecule capable of interfering with essential sequences such as for example the splice donor 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.

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

Preferably, a method of the invention by inducing and/or promoting skipping of at least 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.

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

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

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

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

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

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

A method of the invention may alleviate one or more characteristics of a myogenic or muscle cell of a patient or alleviate one or more symptoms of a DMD patient having a deletion including but not limited to exons 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).

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)

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.

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.

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

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

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

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

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

A detectable increase of the homogeneity of the diameter of a muscle fiber is preferably assessed in a muscle biopsy cross-section, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). The increase is measured by comparison to the homogeneity of the diameter of a muscle fiber in a same DMD or BMD patient before treatment. An alleviation of one or more symptoms may be assessed by any of the following assays on the patient self: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur et al. (Manzur 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.

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.

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

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

In a preferred method of the invention, an additional molecule is used which is able to induce and/or promote skipping of another exon of the DMD pre-mRNA of a patient. Preferably, the second exon is selected from: exon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 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.

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

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

Molecule

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

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

In a preferred embodiment, a molecule or an oligonucleotide of the 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.

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:

(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.

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.

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

In a preferred embodiment for inducing and/or promoting the skipping of 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.

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.

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

In one embodiment, a molecule of the invention is a compound molecule that binds to the specified sequence, or a protein such as an RNA-binding protein or a non-natural zinc-finger protein that has been modified to be able to bind to the corresponding nucleotide sequence on a DMD pre-RNA molecule. Methods for screening compound molecules that bind specific nucleotide sequences are, for example, disclosed in PCT/NL01/00697 and 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.

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

In an even more preferred embodiment, the 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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

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

A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a 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).

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

In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other 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.

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.

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

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

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

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

It will be understood by a skilled person that distinct antisense oligonucleotides can be combined for efficiently skipping 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.

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

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

A preferred antisense oligonucleotide comprises a peptide-linked PMO.

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

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

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

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.

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

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

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

A preferred pharmaceutical composition comprises a molecule as defined herein and/or a vector as defined herein, and a pharmaceutical acceptable carrier or excipient, optionally combined with a molecule and/or a vector as defined herein which is able to induce skipping of exon 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.

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

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

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

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

In addition, a compound as defined herein, preferably an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake into the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) 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.

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

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

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

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

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

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

Use

In yet a further aspect, the invention provides the use of an antisense oligonucleotide or molecule according to the invention, and/or a viral-based vector that expresses one or more antisense sequences according to the invention and/or a pharmaceutical composition, for modulating splicing of the DMD pre-mRNA. 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.

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.

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

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

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.

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.

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.

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

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

DMD Exon 52 Skipping.

A series of AONs targeting sequences within exon 52 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 52 herein defined as SEQ ID NO 6, was indeed capable of inducing exon 52 skipping. PS236 (SEQ ID NO: 299) reproducibly induced highest levels of exon 52 skipping (up to 88% at 200 nM and 91% at 500 nM), as shown in 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

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

SEQUENCE LISTING:

DMD GENE AMINO ACID SEQUENCE

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 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 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 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 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 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 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 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