OLIGONUCLEOTIDE COMPRISING AN INOSINE FOR TREATING DMD

Abstract

The invention provides an oligonucleotide comprising an inosine, and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-m RNA exon or at least part of a non-exon region of a dystrophin pre-m RNA said part being a contiguous stretch comprising at least 8 nucleotides. The invention further provides the use of said oligonucleotide for preventing or treating DMD or BMD.

Description

FIELD OF THE INVENTION

[0001] The invention relates to the fields of molecular biology and medicine.

BACKGROUND OF THE INVENTION

[0002] A muscle disorder is a disease that usually has a significant impact on the life of an individual. A muscle disorder can have either a genetic cause or a non-genetic cause. An important group of muscle diseases with a genetic cause are Becker Muscular Dystrophy (BMD) and Duchenne Muscular Dystrophy (DMD). These disorders are caused by defects in a gene for a muscle protein.

[0003] Becker Muscular Dystrophy and Duchenne Muscular Dystrophy are genetic muscular dystrophies with a relatively high incidence. In both Duchenne and Becker muscular dystrophy the muscle protein dystrophin is affected. In Duchenne dystrophin is absent, whereas in Becker some dystrophin is present but its production is most often not sufficient and/or the dystrophin present is abnormally formed. Both diseases are associated with recessive X-linked inheritance. DMD results from a frameshift mutation in the DMD gene. The frameshift in the DMD gene's transcript (mRNA) results in the production of a truncated non-functional dystrophin protein, resulting in progressive muscle wasting and weakness. BMD occurs as a mutation does not cause a frame-shift in the DMD transcript (mRNA). As in BMD some partly to largely functional dystrophin is present in contrast to DMD where dystrophin is absent, BMD has generally less severe symptoms then DMD. The onset of DMD is earlier than BMD. DMD usually manifests itself in early childhood, BMD in the teens or in early adulthood. The progression of BMD is slower and less predictable than DMD. Patients with BMD can survive into mid to late adulthood. Patients with DMD rarely survive beyond their thirties.

[0004] Dystrophin plays an important structural role in the muscle fiber, connecting the extracellular matrix and the cytoskeleton. The N-terminal region binds actin, whereas the C-terminal end is part of the dystrophin glycoprotein complex (DGC), which spans the sarcolemma. In the absence of dystrophin, mechanical stress leads to sarcolemmal ruptures, causing an uncontrolled influx of calcium into the muscle fiber interior, thereby triggering calcium-activated proteases and fiber necrosis.

[0005] For most genetic muscular dystrophies no clinically applicable and effective therapies are currently available. Exon skipping techniques are nowadays explored in order to combat genetic muscular dystrophies. Promising results have recently been reported by us and others on a genetic therapy aimed at restoring the reading frame of the dystrophin pre-mRNA in cells from the mdx mouse, the GRMD dog (reference 59) and DMD patients.sup.1-11. By the targeted skipping of a specific exon, a DMD phenotype (lacking dystrophin) is converted into a milder BMD phenotype (partly to largely functional dystrophin). The skipping of an exon is preferably induced by the binding of antisense oligoribonucleotides (AONs) targeting either one or both of the splice sites, or exon-internal sequences. Since an exon will only be included in the mRNA when both the splice sites are recognised by the spliceosome complex, splice sites have been considered obvious targets for AONs. More preferably, one or more AONs are used which are specific for at least part of one or more exonic sequences involved in correct splicing of the exon. Using exon-internal AONs specific for an exon 46 sequence, we were previously able to modulate the splicing pattern in cultured myotubes from two different DMD patients with an exon 45 deletion.sup.11. Following AON treatment, exon 46 was skipped, which resulted in a restored reading frame and the induction of dystrophin synthesis in at least 75% of the cells. We have recently shown that exon skipping can also efficiently be induced in human control and patient muscle cells for 39 different DMD exons using exon-internal AONs.sup.1, 2, 11-15.

[0006] Hence, exon skipping techniques applied on the dystrophin gene result in the generation of at least partially functional--albeit shorter--dystrophin protein in DMD patients. Since DMD is caused by a dysfunctional dystrophin protein, it would be expected that the symptoms of DMD are sufficiently alleviated once a DMD patient has been provided with functional dystrophin protein. However, the present invention provides the insight that, even though exon skipping techniques are capable of inducing dystrophin synthesis, the oligonucleotide used for exon skipping technique can be improved any further by incorporating an inosine and/or a nucleotide containing a base able to form a wobble base pair in said oligonucleotide.

DESCRIPTION OF THE INVENTION

[0007] Oligonucleotide

[0008] In a first aspect, there is provided an oligonucleotide comprising an inosine and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides.

[0009] The use of an inosine and/or a nucleotide containing a base able to form a wobble base pair in an oligonucleotide of the invention is very attractive as explained below. Inosine for example is a known modified base which can pair with three bases: uracil, adenine, and cytosine. Inosine is a nucleoside that is formed when hypoxanthine is attached to a ribose ring (also known as a ribofuranose) via a .beta.-N9-glycosidic bond. Inosine is commonly found in tRNAs and is essential for proper translation of the genetic code in wobble base pairs. A wobble base pair is a G-U and I-U/I-A/I-C pair fundamental in RNA secondary structure. Its thermodynamic stability is comparable to that of the Watson-Crick base pair. Wobble base pairs are critical for the proper translation of the genetic code. The genetic code makes up for disparities in the number of amino acids (20) for triplet codons (64), by using modified base pairs in the first base of the anti-codon. Similarly, when designing primers for polymerase chain reaction, inosine is useful in that it will indiscriminately pair with adenine, thymine, or cytosine. A first advantage of using such a base allows one to design a primer that spans a single nucleotide polymorphism (SNP), without worry that the polymorphism will disrupt the primer's annealing efficiency. Therefore in the invention, the use of such a base allows to design an oligonucleotide that may be used for an individual having a SNP within the dystrophin pre-mRNA stretch which is targeted by an oligonucleotide of the invention. A second advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is when said oligonucleotide would normally contain a CpG if one would have designed it as being complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides. The presence of a CpG in an oligonucleotide is usually associated with an increased immunogenicity of said oligonucleotide (reference 60). This increased immunogenicity is undesired since it may induce the breakdown of muscle fibers. Replacing one, two or more CpG by the corresponding inosine and/or a base able to form a wobble base pair in said oligonucleotide is expected to provide an oligonucleotide with a decreased and/or acceptable level of immunogenicity. Immunogenicity may be assessed in an animal model by assessing the presence of CD4.sup.+ and/or CD8+ cells and/or inflammatory mononucleocyte infiltration in muscle biopsy of said animal.

[0010] Immunogenicity may also be assessed in blood of an animal or of a human being treated with an oligonucleotide of the invention by detecting the presence of a neutralizing antibody and/or an antibody recognizing said oligonucleotide using a standard immunoassay known to the skilled person.

[0011] An increase in immunogenicity preferably corresponds to a detectable increase of at least one of these cell types by comparison to the amount of each cell type in a corresponding muscle biopsy of an animal before treatment or treated with a corresponding oligonucleotide having at least one inosine and/or a base able to form a wobble base pair. Alternatively, an increase in immunogenicity may be assessed by detecting the presence or an increasing amount of a neutralizing antibody or an antibody recognizing said oligonucleotide using a standard immunoassay.

[0012] A decrease in immunogenicity preferably corresponds to a detectable decrease of at least one of these cell types by comparison to the amount of corresponding cell type in a corresponding muscle biopsy of an animal before treatment or treated with a corresponding oligonucleotide having no inosine and/or a base able to form a wobble base pair. Alternatively a decrease in immunogenicity may be assessed by the absence of or a decreasing amount of said compound and/or neutralizing antibodies using a standard immunoassay.

[0013] A third advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is to avoid or decrease a potential multimerisation or aggregation of oligonucleotides. It is for example known that an oligonucleotide comprising a G-quartet motif has the tendency to form a quadruplex, a multimer or aggregate formed by the Hoogsteen base-pairing of four single-stranded oligonucleotides (reference 61), which is of course not desired: as a result the efficiency of the oligonucleotide is expected to be decreased. Multimerisation or aggregation is preferably assessed by standard polyacrylamid non-denaturing gel electrophoresis techniques known to the skilled person. In a preferred embodiment, less than 20% or 15%, 10%, 7%, 5% or less of a total amount of an oligonucleotide of the invention has the capacity to multimerise or aggregate assessed using the assay mentioned above.

[0014] A fourth advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is thus also to avoid quadruplex structures which have been associated with antithrombotic activity (reference 62) as well as with the binding to, and inhibition of, the macrophage scavenger receptor (reference 63).

[0015] A fifth advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is to allow to design an oligonucleotide with improved RNA binding kinetics and/or thermodynamic properties. The RNA binding kinetics and/or thermodynamic properties are at least in part determined by the melting temperature of an oligonucleotide (Tm; calculated with the oligonucleotide properties calculator (http://www.unc.edu/.about.cail/biotool/oligo/index.html) for single stranded RNA using the basic Tm and the nearest neighbour model), and/or the free energy of the AON-target exon complex (using RNA structure version 4.5). If a Tm is too high, the oligonucleotide is expected to be less specific. An acceptable Tm and free energy depend on the sequence of the oligonucleotide. Therefore, it is difficult to give preferred ranges for each of these parameters. An acceptable Tm may be ranged between 35 and 65.degree. C. and an acceptable free energy may be ranged between 15 and 45 kcal/mol.

[0016] The skilled person may therefore first choose an oligonucleotide as a potential therapeutic compound. In a second step, he may use the invention to further optimise said oligonucleotide by decreasing its immunogenicity and/or avoiding aggregation and/or quadruplex formation and/or by optimizing its Tm and/or free energy of the AON-target complex. He may try to introduce at least one inosine and/or a base able to form a wobble base pair in said oligonucleotide at a suitable position and assess how the immunogenicity and/or aggregation and/or quadruplex formation and/or Tm and/or free energy of the AON-target complex have been altered by the presence of said inosine and/or a base able to form a wobble base pair. If the alteration does not provide the desired alteration or decrease of immunogenicity and/or aggregation and/or quadruplex formation and/or its Tm and/or free energy of the AON-target complex he may choose to introduce a further inosine and/or a base able to form a wobble base pair in said oligonucleotide and/or to introduce a given inosine and/or a base able to form a wobble base pair at a distinct suitable position within said oligonucleotide.

[0017] An oligonucleotide comprising an inosine and/or a base able to form a wobble base pair may be defined as an oligonucleotide wherein at least one nucleotide has been substituted with an inosine and/or a base able to form a wobble base pair. The skilled person knows how to test whether a nucleotide contains a base able to form a wobble base pair. Since for example inosine can form a base pair with uracil, adenine, and/or cytosine, it means that at least one nucleotide able to form a base pair with uracil, adenine and/or cytosine has been substituted with inosine. However, in order to safeguard specificity, the inosine containing oligonucleotide preferably comprises the substitution of at least one, two, three, four nucleotide(s) able to form a base pair with uracil or adenine or cytosine as long as an acceptable level of a functional activity of said oligonucleotide is retained as defined later herein.

[0018] An oligonucleotide comprising an inosine and/or a base able to form a wobble base pair is preferably an olignucleotide, which is still able to exhibit an acceptable level of a functional activity of a corresponding oligonucleotide not comprising an inosine and/or a base able to form a wobble base pair. A functional activity of said oligonucleotide is preferably to provide an individual with a functional dystrophin protein and/or mRNA and/or at least in part decreasing the production of an aberrant dystrophin protein and/or mRNA. Each of these features are later defined herein. An acceptable level of such a functional activity is preferably at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the functional activity of the corresponding oligonucleotide which does not comprise an inosine and/or a base able to form a wobble base pair. Such functional activity may be as measured in a muscular tissue or in a muscular cell of an individual or in vitro in a cell by comparison to the functional activity of a corresponding oligonucleotides not comprising an inosine and/or a base able to form a wobble base pair. The assessment of the functionality may be carried out at the mRNA level, preferably using RT-PCR. The assessment of the functionality may be carried out at the protein level, preferably using western blot analysis or immunofluorescence analysis of cross-sections.

[0019] Within the context of the invention, an inosine and/or a base able to form a wobble base pair as present in an oligonucleotide is/are present in a part of said oligonucleotide which is complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides. Therefore, in a preferred embodiment, an oligonucleotide comprising an inosine and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides and wherein said inosine and/or a nucleotide containing a base able is/are present within the oligonucleotide sequence which is complementary to at least part of a dystrophin pre-mRNA as defined in previous sentence.

[0020] However, as later defined herein such inosine and/or a base able to form a wobble base pair may also be present in a linking moiety present in an oligonucleotide of the invention. Preferred linking moieties are later defined herein.

[0021] In a preferred embodiment, such oligonucleotide is preferably a medicament. More preferably, said medicament is for preventing or treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual or a patient. 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 or a patient susceptible to develop DMD or BMD due to his or her genetic background.

[0022] In the case of a DMD patient, an oligonucleotide used will preferably correct at least one of the DMD mutations as present in the DMD gene of said patient and therefore will preferably create a dystrophin that will look like a BMD dystrophin: said dystropin will preferably be a functional dystrophin as later defined herein.

[0023] In the case of a BMD patient, an oligonucleotide as used will preferably correct at least one of the BMD mutations as present in the DMD gene of said patient and therefore will preferably create a, or more of a, dystrophin, which will be more functional than the dystrophin which was originally present in said BMD patient. Even more preferably, said medicament provides an individual with a functional or more (of a) functional dystrophin protein and/or mRNA and/or at least in part decreases the production of an aberrant dystrophin protein and/or mRNA.

[0024] Preferably, a method of the invention by inducing and/or promoting skipping of at least one exon of the DMD pre-mRNA as identified herein in one or more cells, preferably muscle cells of a patient, provides said patient with an increased production of a more (of a) functional dystrophin protein and/or mRNA and/or decreases the production of an aberrant or less functional dystrophin protein and/or mRNA in said patient.

[0025] Providing a patient with a more functional dystrophin protein and/or mRNA and/or decreasing the production of an aberrant dystrophin protein and/or mRNA in said patient is typically applied in a DMD patient. Increasing the production of a more functional or functional dystrophin and/or mRNA is typically applied in a BMD patient.

[0026] Therefore a preferred method is a method, wherein a patient or one or more cells of said patient is provided with an increased production of a more functional or functional dystrophin protein and/or mRNA and/or wherein the production of an aberrant dystrophin protein and/or mRNA in said patient is decreased, wherein the level of said aberrant or more functional dystrophin protein and/or mRNA is assessed by comparison to the level of said dystrophin and/or mRNA in said patient at the onset of the method.

[0027] 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 cystein-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 another embodiment, 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 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 wild type dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC).sup.5. 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 biopsy of a muscle suspected to be dystrophic, as known to the skilled person.

[0028] Individuals suffering from Duchenne muscular dystrophy typically have a mutation in the gene encoding dystrophin that prevents synthesis of the complete protein, i.e. a premature stop prevents the synthesis of the C-terminus of the protein. In Becker muscular dystrophy the dystrophin gene also comprises a mutation compared to the wild type but the mutation does typically not include a premature stop and the C-terminus of the protein is typically synthesized. As a result a functional dystrophin protein is synthesized that has at least the same activity in kind as a wild type protein, although not necessarily the same amount of activity. In a preferred embodiment, a functional dystrophin protein means an in frame dystrophin gene. 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 cystein-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.sup.56. Exon-skipping for the treatment of DMD is preferably but not exclusively directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-domain 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 using an oligonucleotide 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: preferably 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 et al (2006, ref 56). The central rod domain of wild type dystrophin comprises 24 spectrin-like repeats.sup.56. 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. Decreasing the production of an aberrant dystrophin in said patient or in a cell of said patient 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 or less to non-functional or semi-functional dystrophin mRNA or protein. A non-functional pre-mRNA dystrophin is preferably leads to an out of frame dystrophin protein, which means that no dystrophin protein will be produced and/or detected. 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.

[0029] 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 or in frame 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 or in frame dystrophin mRNA.

[0030] 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.

[0031] An increase or a decrease is preferably assessed in a muscular tissue or in a muscular cell of an individual or a patient by comparison to the amount present in said individual or patient before treatment with said molecule or composition of the invention. Alternatively, the comparison can be made with a muscular tissue or cell of said individual or patient, which has not yet been treated with said oligonucleotide or composition in case the treatment is local.

[0032] In a preferred method, one or more symptom(s) from a DMD or a BMD patient is/are alleviated and/or one or more characteristic(s) of a muscle cell or tissue from a DMD or a BMD patient is/are alleviated using a molecule or a composition of the invention. Such symptoms may be assessed on the patient self. Such characteristics may be assessed at the cellular, tissue level of a given patient. 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.

[0033] Alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual using a molecule or a composition of the invention may be assessed by any of the following assays: 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 at al (2008, ref 58) 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 molecule or composition of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (2006, ref 57). 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.

[0034] An oligonucleotide as used herein preferably comprises an antisense oligonucleotide or antisense oligoribonucleotide. In a preferred embodiment an exon skipping technique is applied. Exon skipping interferes with the natural splicing processes occurring within a eukaryotic cell. In higher eukaryotes the genetic information for proteins in the DNA of the cell is encoded in exons which are separated from each other by intronic sequences. These introns are in some cases very long. The transcription machinery of eukaryotes generates a pre-mRNA which contains both exons and introns, while the splicing machinery, often already during the production of the pre-mRNA, generates the actual coding region for the protein by splicing together the exons present in the pre-mRNA.

[0035] Exon-skipping results in mature mRNA that lacks at least one skipped exon. Thus, when said exon codes for amino acids, exon skipping leads to the expression of an altered product. Technology for exon-skipping is currently directed towards the use of antisense oligonucleotides (AONs). Much of this work is done in the mdx mouse model for Duchenne muscular dystrophy. The mdx mouse carries a nonsense mutation in exon 23. Despite the mdx mutation, which should preclude the synthesis of a functional dystrophin protein, rare, naturally occurring dystrophin positive fibers have been observed in mdx muscle tissue. These dystrophin-positive fibers are thought to have arisen from an apparently naturally occurring exon-skipping mechanism, either due to somatic mutations or through alternative splicing. AONs directed to, respectively, the 3' and/or 5' splice sites of introns 22 and 23 in dystrophin pre-mRNA, have been shown to interfere with factors normally involved in removal of intron 23 so that also exon 23 was removed from the mRNA.sup.3, 5, 6, 39, 40.

[0036] By the targeted skipping of a specific exon, a DMD phenotype is converted into a milder BMD phenotype. The skipping of an exon is preferably induced by the binding of AONs targeting either one or both of the splice sites, or exon-internal sequences. An oligonucleotide directed toward an exon internal sequence typically exhibits no overlap with non-exon sequences. It preferably does not overlap with the splice sites at least not insofar, as these are present in the intron. An oligonucleotide directed toward an exon internal sequence preferably does not contain a sequence complementary to an adjacent intron. Further provided is thus an oligonucleotide according to the invention, wherein said oligonucleotide, or a functional equivalent thereof, is for inhibiting inclusion of an exon of a dystrophin pre-mRNA into mRNA produced from splicing of said pre-mRNA. An exon skipping technique is preferably applied such that the absence of an exon from mRNA produced from dystrophin pre-mRNA generates a coding region for a more functional--albeit shorter--dystrophin protein. In this context, inhibiting inclusion of an exon preferably means that the detection of the original, aberrant dystrophin mRNA and/or protein is decreased as earlier defined herein.

[0037] Since an exon of a dystrophin pre-mRNA will only be included into the resulting mRNA when both the splice sites are recognised by the spliceosome complex, splice sites have been obvious targets for AONs. One embodiment therefore provides an oligonucleotide, or a functional equivalent thereof, comprising a sequence which is complementary to a non-exon region of a dystrophin pre mRNA. In one embodiment an AON is used which is solely complementary to a non-exon region of a dystrophin pre mRNA. This is however not necessary: it is also possible to use an AON which comprises an intron-specific sequence as well as exon-specific sequence. Such AON comprises a sequence which is complementary to a non-exon region of a dystrophin pre mRNA, as well as a sequence which is complementary to an exon region of a dystrophin pre mRNA. Of course, an AON is not necessarily complementary to the entire sequence of a dystrophin exon or intron. AONs, which are complementary to a part of such exon or intron are preferred. An AON is preferably complementary to at least part of a dystrohin exon and/or intron, said part having at least 8, 10, 13, 15, 20 nucleotides.

[0038] Splicing of a dystrophin pre-mRNA occurs via two sequential transesterification reactions. First, the 2'OH of a specific branch-point nucleotide within the intron that is defined during spliceosome assembly performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site forming the lariat intermediate. Second, the 3'OH of the released 5' exon then performs a nucleophilic attack at the last nucleotide of the intron at the 3' splice site thus joining the exons and releasing the intron lariat. The branch point and splice sites of an intron are thus involved in a splicing event. Hence, an oligonucleotide comprising a sequence, which is complementary to such branch point and/or splice site is preferably used for exon skipping. Further provided is therefore an oligonucleotide, or a functional equivalent thereof, which comprises a sequence which is complementary to a splice site and/or branch point of a dystrophin pre mRNA.

[0039] Since splice sites contain consensus sequences, the use of an oligonucleotide or a functional equivalent thereof (herein also called an AON) comprising a sequence which is complementary of a splice site involves the risk of promiscuous hybridization. Hybridization of AONs to other splice sites than the sites of the exon to be skipped could easily interfere with the accuracy of the splicing process. To overcome these and other potential problems related to the use of AONs which are complementary to an intron sequence, one preferred embodiment provides an oligonucleotide, or a functional equivalent thereof, comprising a sequence which is complementary to a dystrophin pre-mRNA exon. Preferably, said AON is capable of specifically inhibiting an exon inclusion signal of at least one exon in said dystrophin pre-mRNA. Interfering with an exon inclusion signal (EIS) has the advantage that such elements are located within the exon. By providing an AON for the interior of the exon to be skipped, it is possible to interfere with the exon inclusion signal thereby effectively masking the exon from the splicing apparatus. The failure of the splicing apparatus to recognize the exon to be skipped thus leads to exclusion of the exon from the final mRNA. This embodiment does not interfere directly with the enzymatic process of the splicing machinery (the joining of the exons). It is thought that this allows the method to be more specific and/or reliable. It is thought that an EIS is a particular structure of an exon that allows splice acceptor and donor to assume a particular spatial conformation. In this concept, it is the particular spatial conformation that enables the splicing machinery to recognize the exon. However, the invention is certainly not limited to this model. In a preferred embodiment, use is made of an oligonucleotide, which is capable of binding to an exon and is capable of inhibiting an EIS. An AON may specifically contact said exon at any point and still be able to specifically inhibit said EIS.

[0040] Within the context of the invention, a functional equivalent of an oligonucleotide preferably means an oligonucleotide as defined herein wherein one or more nucleotides have been substituted and wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by quantifying the amount of a functional dystrophin protein or by quantifying the amount of a functional dystrophin mRNA. A functional dystrophin protein (or a functional dystrophin mRNA) is herein preferably defined as being a dystrophin protein (or a dystrophin protein encoded by said mRNA) able to bind actin and members of the DGC protein. The assessment of said activity of an oligonucleotide is preferably done by RT-PCR (m-RNA) or by immunofluorescence or Western blot analyses (protein). 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. Such activity may be measured in a muscular tissue or in a muscular cell of an individual or in vitro in a cell by comparison to an activity of a corresponding oligonucleotide 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.

[0041] Hence, an oligonucleotide, or a functional equivalent thereof, comprising or consisting of a sequence which is complementary to a dystrophin pre-mRNA exon provides good DMD therapeutic results. In one preferred embodiment an oligonucleotide, or a functional equivalent thereof, is used which comprises or consists of a sequence which is complementary to at least part of either dystrophin pre-mRNA exons 2 to 75 said part having or comprising at least 13 nucleotides. However, said part may also have 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. A part of dystrophin pre-mRNA to which an oligonucleotide is complementary may also be called a contiguous stretch of dystrophin pre-mRNA.

[0042] Most preferably an AON is used which comprises or consists of a sequence which is complementary to at least part of dystrophin pre-mRNA exon 51, 45, 53, 44, 46, 52, 60, 43, 6, 7, 8, 55, 2, 2, 11, 1, 19, 21, 57, 59, 8263, 65, 66, 69, and/or 75 said part having or comprising at least 13 nucleotides. However, said part may also have 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, 36, 86, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. More preferred oligonucleotides are represented by a sequence that comprises or consists of each of the following sequences SEQ ID NO: 2 to SEQ ID NO:539 wherein at least one inosine and/or a base able to form a wobble base pair is present in said sequence. Preferably, an inosine has been introduced in one of these sequences to replace a guanosine, adenine, or a uracil. Accordingly, an even more preferred oligonucleotide as used herein is represented by a sequence that comprises or consists of SEQ ID NO:2 to SEQ ID NO:486 or SEQ ID NO:539, even more preferably SEQ ID NO:2 to NO 237 or SEQ ID NO:539, most preferably SEQ ID NO:76 wherein at least one inosine and/or a base able to form a wobble base pair is present in said sequence. Preferably, an inosine has been introduced in one of these sequences to replace a guanosine, adenine, or a uracil.

[0043] Accordingly, in another preferred embodiment, an oligonucleotide as used herein is represented by a sequence that comprises or consists of SEQ ID NO:540 to SEQ ID NO:576. More preferably, an oligonucleotide as used herein is represented by a sequence that comprises or consists of SEQ ID NO:557.

[0044] Said exons are listed in decreasing order of patient population applicability. Hence, the use of an AON comprising a sequence, which is complementary to at least part of dystrophin pre-mRNA exon 51 is suitable for use in a larger part of the DMD patient population as compared to an AON comprising a sequence which is complementary to dystrophin pre-mRNA exon 44, et cetera.

[0045] In a preferred embodiment, an oligonucleotide of the invention, which comprises a sequence that is complementary to part of dystrophin pre-mRNA is such that the complementary part is at least 60% of the length of the oligonucleotide of the invention, more preferably at least 60%, 80 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% or even more preferably at least 99%, or even more preferably 100%. In a most preferred embodiment, the oligonucleotide of the invention consists of a sequence that is complementary to part of dystrophin pre-mRNA as defined herein. As an example, an oligonucleotide may comprise a sequence that is complementary to part of dystrophin 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 the oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity.

[0046] One preferred embodiment provides an oligonucleotide, or a functional equivalent thereof which comprises:

[0047] a sequence which is complementary to a region of a dystrophin pre-mRNA exon that is hybridized to another part of a dystrophin pre-mRNA exon (closed structure), and

[0048] a sequence which is complementary to a region of a dystrophin pre-mRNA exon that is not hybridized in said dystrophin pre-mRNA (open structure).

[0049] For this embodiment, reference is made to WO 2004/083432, which is incorporated by reference in its entirety. RNA molecules exhibit strong secondary structures, mostly due to base pairing of complementary or partly complementary stretches within the same RNA. It has long since been thought that structures in the RNA play a role in the function of the RNA. Without being bound by theory, it is believed that the secondary structure of the RNA of an exon plays a role in structuring the splicing process. The structure of an exon is one parameter which is believed to direct its inclusion into the mRNA. However, other parameters may also play a role therein. Herein this signalling function is referred to as an exon inclusion signal. A complementary oligonucleotide of this embodiment is capable of interfering with the structure of the exon and thereby capable of interfering with the exon inclusion signal of the exon. It has been found that many complementary oligonucleotides indeed comprise this capacity, some more efficient than others. Oligonucleotides of this preferred embodiment, i.e. those with the said overlap directed towards open and closed structures in the native exon RNA, are a selection from all possible oligonucleotides. The selection encompasses oligonucleotides that can efficiently interfere with an exon inclusion signal. Without being bound by theory it is thought that the overlap with an open structure improves the invasion efficiency of the oligonucleotide and prevents the binding of splicing factors (i.e. increases the efficiency with which the oligonucleotide can enter the structure), whereas the overlap with the closed structure subsequently increases the efficiency of interfering with the secondary structure of the RNA of the exon, and thereby interfere with the exon inclusion signal. It is found that the length of the partial complementarity to both the closed and the open structure is not extremely restricted. We have observed high efficiencies with oligonucleotides with variable lengths of complementarity in either structure. The term complementarity is used herein to refer to a stretch of nucleic acids that can hybridise to another stretch of nucleic acids under physiological conditions. It is thus not absolutely required that all the bases in the region of complementarity are capable of pairing with bases in the opposing strand. For instance, when designing the oligonucleotide one may want to incorporate for instance a residue that does not base pair with the base on the complementary strand. Mismatches may, to some extent, be allowed, if under the circumstances in the cell, the stretch of nucleotides is sufficiently capable of hybridising to the complementary part. In this context, "sufficiently" preferably means that using a gel mobility shift assay as described in example 1 of EP 1 619 249, binding of an oligonucleotide is detectable. Optionally, said oligonucleotide may further be tested by transfection into muscle cells of patients. Skipping of the targeted exon may be assessed by RT-PCR (as described in EP 1 619 249). The complementary regions are preferably designed such that, when combined, they are specific for the exon in the pre-mRNA. Such specificity may be created with various lengths of complementary regions as this depends on the actual sequences in other (pre-)mRNA in the system. The risk that also one or more other pre-mRNA will be able to hybridise to the oligonucleotide decreases with increasing size of the oligonucleotide. It is clear that oligonucleotides comprising mismatches in the region of complementarity but that retain the capacity to hybridise to the targeted region(s) in the pre-mRNA, can be used in the present invention. However, preferably at least the complementary parts do not comprise such mismatches as these typically have a higher efficiency and a higher specificity, than oligonucleotides having such mismatches in one or more complementary regions. It is thought, that higher hybridisation strengths, (i.e. increasing number of interactions with the opposing strand) are favourable in increasing the efficiency of the process of interfering with the splicing machinery of the system. Preferably, the complementarity is between 90 and 100%. In general this allows for approximately 1 or 2 mismatch(es) in an oligonucleotide of around 20 nucleotides

[0050] The secondary structure is best analysed in the context of the pre-mRNA wherein the exon resides. Such structure may be analysed in the actual RNA. However, it is currently possible to predict the secondary structure of an RNA molecule (at lowest energy costs) quite well using structure-modelling programs. A non-limiting example of a suitable program is RNA mfold version 3.1 server.sup.41. A person skilled in the art will be able to predict, with suitable reproducibility, a likely structure of the exon, given the nucleotide sequence. Best predictions are obtained when providing such modelling programs with both the exon and flanking intron sequences. It is typically not necessary to model the structure of the entire pre-mRNA.

[0051] The open and closed structure to which the oligonucleotide is directed, are preferably adjacent to one another. It is thought, that in this way the annealing of the oligonucleotide to the open structure induces opening of the closed structure whereupon annealing progresses into this closed structure. Through this action the previously closed structure assumes a different conformation. The different conformation results in the disruption of the exon inclusion signal. However, when potential (cryptic) splice acceptor and/or donor sequences are present within the targeted exon, occasionally a new exon inclusion signal is generated defining a different (neo) exon, i.e. with a different 5' end, a different 3' end, or both. This type of activity is within the scope of the present invention as the targeted exon is excluded from the mRNA. The presence of a new exon, containing part of the targeted exon, in the mRNA does not alter the fact that the targeted exon, as such, is excluded. The inclusion of a neo-exon can be seen as a side effect, which occurs only occasionally. There are two possibilities when exon skipping is used to restore (part of) an open reading frame of dystrophin that is disrupted as a result of a mutation. One is that the neo-exon is functional in the restoration of the reading frame, whereas in the other case the reading frame is not restored. When selecting oligonucleotides for restoring dystrophin reading frames by means of exon-skipping it is of course clear that under these conditions only those oligonucleotides are selected that indeed result in exon-skipping that restores the dystrophin open reading frame, with or without a neo-exon.

[0052] Further provided is an oligonucleotide, or a functional equivalent thereof, comprising a sequence that is complementary to a binding site for a serine-arginine (SR) protein in RNA of an exon of a dystrophin pre-mRNA. In WO 2006/112705 we have disclosed the presence of a correlation between the effectivity of an exon-internal antisense oligonucleotide (AON) in inducing exon skipping and the presence of a (for example by ESE finder) predicted SR binding site in the target pre-mRNA site of said AON. Therefore, in one embodiment an oligonucleotide is generated comprising determining a (putative) binding site for an SR (Ser-Arg) protein in RNA of a dystrophin exon and producing an oligonucleotide that is complementary to said RNA and that at least partly overlaps said (putative) binding site. The term "at least partly overlaps" is defined herein as to comprise an overlap of only a single nucleotide of an SR binding site as well as multiple nucleotides of said binding site as well as a complete overlap of said binding site. This embodiment preferably further comprises determining from a secondary structure of said RNA, a region that is hybridised to another part of said RNA (closed structure) and a region that is not hybridised in said structure (open structure), and subsequently generating an oligonucleotide that at least partly overlaps said (putative) binding site and that overlaps at least part of said closed structure and overlaps at least part of said open structure. In this way we increase the chance of obtaining an oligonucleotide that is capable of interfering with the exon inclusion from the pre-mRNA into mRNA. It is possible that a first selected SR-binding region does not have the requested open-closed structure in which case another (second) SR protein binding site is selected which is then subsequently tested for the presence of an open-closed structure. This process is continued until a sequence is identified which contains an SR protein binding site as well as a(n) (partly overlapping) open-closed structure. This sequence is then used to design an oligonucleotide which is complementary to said sequence.

[0053] Such a method, for generating an oligonucleotide, is also performed by reversing the described order, i.e. first generating an oligonucleotide comprising determining, from a secondary structure of RNA from a dystrophin exon, a region that assumes a structure that is hybridised to another part of said RNA (closed structure) and a region that is not hybridised in said structure (open structure), and subsequently generating an oligonucleotide, of which at least a part of said oligonucleotide is complementary to said closed structure and of which at least another part of said oligonucleotide is complementary to said open structure. This is then followed by determining whether an SR protein binding site at least overlaps with said open/closed structure. In this way the method of WO 2004/083432 is improved. In yet another embodiment the selections are performed simultaneously.

[0054] Without wishing to be bound by any theory it is currently thought that use of an oligonucleotide directed to an SR protein binding site results in (at least partly) impairing the binding of an SR protein to the binding site of an SR protein which results in disrupted or impaired splicing.

[0055] Preferably, an open/closed structure and an SR protein binding site partly overlap and even more preferred an open/closed structure completely overlaps an SR protein binding site or an SR protein binding site completely overlaps an open/closed structure. This allows for an improved disruption of exon inclusion.

[0056] Besides consensus splice sites sequences, many (if not all) exons contain splicing regulatory sequences such as exonic splicing enhancer (ESE) sequences to facilitate the recognition of genuine splice sites by the spliceosome.sup.42, 43. A subgroup of splicing factors, called the SR proteins, can bind to these ESEs and recruit other splicing factors, such as U1 and U2AF to (weakly defined) splice sites. The binding sites of the four most abundant SR proteins (SF2/ASF, SC35, SRp40 and SRp55) have been analyzed in detail and these results are implemented in ESE finder, a web source that predicts potential binding sites for these SR proteins.sup.42, 43. There is a correlation between the effectiveness of an AON and the presence/absence of an SF2/ASF, SC35 and SRp40 binding site. In a preferred embodiment, the invention thus provides a combination as described above, wherein said SR protein is SF2/ASF or SC35 or SRp40.

[0057] In one embodiment an oligonucleotide, or a functional equivalent thereof is capable of specifically binding a regulatory RNA sequence which is required for the correct splicing of a dystrophin exon in a transcript. Several cis-acting RNA sequences are required for the correct splicing of exons in a transcript. In particular, supplementary elements such as intronic or exonic splicing enhancers (ISEs and ESEs) or silencers (ISSs and ESEs) are identified to regulate specific and efficient splicing of constitutive and alternative exons. Using sequence-specific antisense oligonucleotides (AONs) that bind to the elements, their regulatory function is disturbed so that the exon is skipped, as shown for DMD. Hence, in one preferred embodiment an oligonucleotide or functional equivalent thereof is used which is complementary to an intronic splicing enhancer (ISE), an exonic splicing enhancer (ESE), an intronic splicing silencer (ISS) and/or an exonic splicing silencer (ESS). As already described herein before, a dystrophin exon is in one preferred embodiment skipped by an agent capable of specifically inhibiting an exon inclusion signal of said exon, so that said exon is not recognized by the splicing machinery as a part that needs to be included in the mRNA. As a result, a mRNA without said exon is formed.

[0058] An AON used herein is preferably complementary to a consecutive part or a contiguous stretch of between 8 and 50 nucleotides of dystrophin exon RNA or dystrophin intron RNA. In one embodiment an AON used herein is complementary to a consecutive part or a contiguous stretch of between 14 and 50 nucleotides of a dystrophin exon RNA or dystrophin intron RNA. Preferably, said AON is complementary to a consecutive part or contiguous stretch of between 14 and 26 nucleotides of said exon RNA. More preferably, an AON is used which comprises a sequence which is complementary to a consecutive part or a contiguous stretch of between 20 and 26 nucleotides of a dystrophin exon RNA or a dystrophin intron RNA.

[0059] Different types of nucleic acid may be used to generate an oligonucleotide. Preferably, said oligonucleotide comprises RNA, as RNA/RNA hybrids are very stable. Since one of the aims of the exon skipping technique is to direct splicing in subjects it is preferred that the oligonucleotide RNA comprises a modification providing the RNA with an additional property, for instance resistance to endonucleases, exonucleases, and RNaseH, additional hybridisation strength, increased stability (for instance in a bodily fluid), increased or decreased flexibility, reduced toxicity, increased intracellular transport, tissue-specificity, etc. Preferably, said modification comprises a 2'-O-methyl-phosphorothioate oligoribonucleotide modification. Preferably, said modification comprises a 2'-O-methyl-phosphorothioate oligodeoxyribonucleotide modification. One embodiment thus provides an oligonucleotide is used which comprises RNA which contains a modification, preferably a 2'-O-methyl modified ribose (RNA) or deoxyribose (DNA) modification.

[0060] In one embodiment the invention provides a hybrid oligonucleotide comprising an oligonucleotide comprising a 2'-O-methyl-phosphorothioate oligo(deoxy)ribonucleotide modification and locked nucleic acid. This particular oligonucleotide comprises better sequence specificity compared to an equivalent consisting of locked nucleic acid, and comprises improved effectivity when compared with an oligonucleotide consisting of 2'-O-methyl-phosphorothioate oligo(deoxy)ribonucleotide modification.

[0061] With the advent of nucleic acid mimicking technology it has become possible to generate molecules that have a similar, preferably the same hybridisation characteristics in kind not necessarily in amount as nucleic acid itself. Such functional equivalents are of course also suitable for use in the invention. Preferred examples of functional equivalents of an oligonucleotide are peptide nucleic acid and/or locked nucleic acid. Most preferably, a morpholino phosphorodiamidate is used. Suitable but non-limiting examples of equivalents of oligonucleotides of the invention can be found in.sup.44-50. Hybrids between one or more of the equivalents among each other and/or together with nucleic acid are of course also suitable. In a preferred embodiment locked nucleic acid is used as a functional equivalent of an oligonucleotide, as locked nucleic acid displays a higher target affinity and reduced toxicity and therefore shows a higher efficiency of exon skipping.

[0062] In one embodiment an oligonucleotide, or a functional equivalent thereof, which is capable of inhibiting inclusion of a dystrophin exon into dystrophin mRNA is combined with at least one other oligonucleotide, or functional equivalent thereof, that is capable of inhibiting inclusion of another dystrophin exon into dystrophin mRNA. This way, inclusion of two or more exons of a dystrophin pre-mRNA in mRNA produced from this pre-mRNA is prevented. This embodiment is further referred to as double- or multi-exon skipping.sup.2, 15. In most cases double-exon skipping results in the exclusion of only the two targeted exons from the dystrophin 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-exon 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. Other preferred examples of multi-exon skipping are:

[0063] the use of an oligonucleotide targeting exon 17, and a second one exon 48 which may result in the skipping of said both exons or of the entire region between exon 17 and exon 48.

[0064] the use of an oligonucleotide targeting exon 17, and a second one exon 51 which may result in the skipping of said both exons or of the entire region between exon 17 and exon 51.

[0065] the use of an oligonucleotide targeting exon 42, and a second one exon 55 which may result in the skipping of said both exons or of the entire region between exon 42 and exon 55.

[0066] the use of an oligonucleotide targeting exon 43, and a second one exon 51 which may result in the skipping of said both exons or of the entire region between exon 43 and exon 51.

[0067] the use of an oligonucleotide targeting exon 43, and a second one exon 55 which may result in the skipping of said both exons or of the entire region between exon 43 and exon 55.

[0068] the use of an oligonucleotide targeting exon 45, and a second one exon 55 which may result in the skipping of said both exons or of the entire region between exon 45 and exon 55.

[0069] the use of an oligonucleotide targeting exon 45, and a second one exon 59 which may result in the skipping of said both exons or of the entire region between exon 45 and exon 59.

[0070] the use of an oligonucleotide targeting exon 48, and a second one exon 59 which may result in the skipping of said both exons or of the entire region between exon 48 and exon 59.

[0071] the use of an oligonucleotide targeting exon 50, and a second one exon 51 which may result in the skipping of said both exons.

[0072] the use of an oligonucleotide targeting exon 51, and a second one exon 52 which may result in the skipping of said both exons.

[0073] Further provided is therefore an oligonucleotide which comprises at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a first exon of a dystrophin pre-mRNA and wherein a nucleotide sequence is used which comprises at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a second exon of said dystrophin pre-mRNA. Said first and said second exon may be the same.

[0074] In one preferred embodiment said first and said second exon are separated in said dystrophin pre-mRNA by at least one exon to which said oligonucleotide is not complementary. Alternatively, said first and said second exon are adjacent.

[0075] 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. Further provided is therefore an oligonucleotide, or functional equivalent thereof which is complementary to at least two exons in a dystrophin pre-mRNA, said oligonucleotide or functional equivalent comprising at least two parts wherein a first part comprises an oligonucleotide having at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a first of said at least two exons and wherein a second part comprises an oligonucleotide having at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a second exon in said dystrophin pre-mRNA. The linkage may be through any means, but is preferably accomplished through a nucleotide linkage. In the latter case, the number of nucleotides that do not contain an overlap between one or the other complementary exon can be zero, but is preferably between 4 to 40 nucleotides. The linking moiety can be any type of moiety capable of linking oligonucleotides. Preferably, said linking moiety comprises at least 4 uracil nucleotides. Currently, many different compounds are available that mimic hybridisation characteristics of oligonucleotides. Such a compound, called herein a functional equivalent of an oligonucleotide, is also suitable for the present invention if such equivalent comprises similar hybridisation characteristics in kind not necessarily in amount. Suitable functional equivalents are mentioned earlier in this description. As mentioned, oligonucleotides of the invention do not have to consist of only oligonucleotides that contribute to hybridisation to the targeted exon. There may be additional material and/or nucleotides added.

[0076] The DMD gene is a large gene, with many different exons. Considering that the gene is located on the X-chromosome, it is mostly boys that are affected, although girls can also be affected by the disease, as they may receive a bad copy of the gene from both parents, or are suffering from a particularly biased inactivation of the functional allele due to a particularly biased X chromosome inactivation in their muscle cells. The protein is encoded by a plurality of exons (79) over a range of at least 2.4 Mb. Defects may occur in any part of the DMD gene. Skipping of a particular exon or particular exons can, very often, result in a restructured mRNA that encodes a shorter than normal but at least partially functional dystrophin protein. A practical problem in the development of a medicament based on exon-skipping technology is the plurality of mutations that may result in a deficiency in functional dystrophin protein in the cell. Despite the fact that already multiple different mutations can be corrected for by the skipping of a single exon, this plurality of mutations, requires the generation of a series of different pharmaceuticals as for different mutations different exons need to be skipped. An advantage of an oligonucleotide or of a composition comprising at least two distinct oligonucleotide as later defined herein capable of inducing skipping of two or more exons, is that more than one exon can be skipped with a single pharmaceutical. This property is not only practically very useful in that only a limited number of pharmaceuticals need to be generated for treating many different DMD or particular, severe BMD mutations. Another option now open to the person skilled in the art is to select particularly functional restructured dystrophin proteins and produce compounds capable of generating these preferred dystrophin proteins. Such preferred end results are further referred to as mild phenotype dystrophins.

[0077] Dose ranges of oligonucleotide according to the invention are preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist. 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.

[0078] In a preferred embodiment, a concentration of an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 .mu.M is used. Preferably, this range is for in vitro use in a cellular model such as muscular cells or muscular tissue. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM. If several oligonucleotides are used, this concentration or dose may refer to the total concentration or dose of oligonucleotides or the concentration or dose of each oligonucleotide added.

[0079] The ranges of concentration or dose of oligonucleotide(s) as given above are preferred concentrations or doses 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 or dose of oligonucleotide(s) used may further vary and may need to be optimised any further.

[0080] An oligonucleotide as defined herein for use according to the invention may be suitable for 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 in vivo, ex vivo or in vitro. Said oligonucleotide may be directly or indirectly administrated to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing DMD or BMD, and may be administered directly or indirectly in vivo, ex vivo or in vitro. As Duchenne and Becker muscular dystrophy have a pronounced phenotype in muscle cells, it is preferred that said cells are muscle cells, it is further preferred that said tissue is a muscular tissue and/or it is further preferred that said organ comprises or consists of a muscular tissue. A preferred organ is the heart. Preferably, said cells comprise a gene encoding a mutant dystrophin protein. Preferably, said cells are cells of an individual suffering from DMD or BMD.

[0081] An oligonucleotide of the invention may be indirectly administrated using suitable means known in the art. An oligonucleotide may for example be provided to an individual or a cell, tissue or organ of said individual in the form of an expression vector wherein the expression vector encodes a transcript comprising said oligonucleotide. The expression vector is preferably introduced into a cell, tissue, organ or individual via a gene delivery vehicle. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette or a transcription cassette that drives expression or transcription of a molecule as identified herein. A preferred delivery vehicle is a viral vector such as an adeno-associated virus vector (AAV), or a retroviral vector such as a lentivirus vector.sup.4, 51, 52 and the like. Also, plasmids, artificial chromosomes, plasmids suitable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of an oligonucleotide as defined herein. Preferred for the current invention are those vectors wherein transcription is driven from PolII promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts. It is within the skill of the artisan to design suitable transcripts. Preferred are PolIII driven transcripts. Preferably, in the form of a fusion transcript with an U1 or U7 transcript.sup.4, 51, 52. Such fusions may be generated as described.sup.53, 54. The oligonucleotide may be delivered as is. However, the oligonucleotide may also be encoded by the viral vector. Typically, this is in the form of an RNA transcript that comprises the sequence of the oligonucleotide in a part of the transcript.

[0082] Improvements in means for providing an individual or a cell, tissue, organ of said individual with an oligonucleotide and/or an equivalent thereof, are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect on restructuring of mRNA using a method of the invention. An oligonucleotide and/or an equivalent thereof can be delivered as is to an individual, a cell, tissue or organ of said individual. When administering an oligonucleotide and/or an equivalent thereof, it is preferred that an oligonucleotide and/or an equivalent thereof 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 in the invention is the use of an excipient that will aid in delivery of each of the constituents as defined herein to a cell and/or into a cell, preferably a muscle cell. Preferred are excipients capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that deliver each constituent as defined herein, 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 (PEI), or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils (SAINT-18), Lipofectin.TM., DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver each constitutent as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver an 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.

[0083] 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.

[0084] 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 each constituent as defined herein, preferably an oligonucleotide across cell membranes into cells.

[0085] In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate an oligonucleotide with 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 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 an oligonucleotide for use in the current invention to deliver it for the treatment of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in humans.

[0086] In addition, an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes.

[0087] Therefore, in a preferred embodiment, an oligonucleotide is formulated in a composition or a medicament or a composition, which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device thereof to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said oligonucleotide to a cell and/or enhancing its intracellular delivery. It is to be understood that if a composition comprises an additional constituent such as an adjunct compound as later defined herein, each constituent of the composition may not be formulated in one single combination or composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each constituent as defined herein. In a preferred embodiment, the invention provides a composition or a preparation which is in the form of a kit of parts comprising an oligonucleotide and a further adjunct compound as later defined herein.

[0088] A preferred oligonucleotide is for preventing or treating Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) in an individual. An individual, which may be treated using an oligonucleotide of the invention may already have been diagnosed as having a DMD or a BMD. Alternatively, an individual which may be treated using an oligonucleotide of the invention may not have yet been diagnosed as having a DMD or a BMD but may be an individual having an increased risk of developing a DMD or a BMD in the future given his or her genetic background. A preferred individual is a human being.

[0089] Composition

[0090] In a further aspect, there is provided a composition comprising an oligonucleotide as defined herein. Preferably, said composition comprises at least two distinct oligonucleotide as defined herein. More preferably, these two distinct oligonucleotides are designed to skip distinct two or more exons as earlier defined herein for multi-exon skipping.

[0091] In a preferred embodiment, said composition being preferably a pharmaceutical composition said pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient. Such a pharmaceutical composition may comprise any pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient is also provided. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000. Each feature of said composition has earlier been defined herein.

[0092] If several oligonucleotides are used, concentration or dose already defined herein may refer to the total concentration or dose of all oligonucleotides used or the concentration or dose of each oligonucleotide used or added. Therefore in one embodiment, there is provided a composition wherein each or the total amount of oligonucleotide used is dosed in an amount ranged between 0.5 mg/kg and 10 mg/kg.

[0093] A preferred composition additionally comprises:

[0094] a) an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or

[0095] b) an adjunct compound for improving muscle fiber function, integrity and/or survival and/or

[0096] c) a compound exhibiting readthrough activity.

[0097] It has surprisingly been found that the skipping frequency of a dystrophin exon from a pre-MRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon, is enhanced if cells expressing said pre-mRNA are also provided with an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.

[0098] According to the present invention, even when a dystrophin protein deficiency has been restored in a DMD patient by administering an oligonucleotide of the invention, the presence of tissue inflammation and damaged muscle cells still continues to contribute to the symptoms of DMD. Hence, even though the cause of DMD--i.e. a dysfunctional dystrophin protein--is alleviated, treatment of DMD is still further improved by additionally using an adjunct therapy according to the present invention. Furthermore, the present invention provides the insight that a reduction of inflammation does not result in significant reduction of AON uptake by muscle cells. This is surprising because, in general, inflammation enhances the trafficking of cells, blood and other compounds. As a result, AON uptake/delivery is also enhanced during inflammation. Hence, before the present invention it would be expected that an adjunct therapy counteracting inflammation involves the risk of negatively influencing AON therapy. This, however, appears not to be the case.

[0099] An adjunct compound for reducing inflammation comprises any therapy which is capable of at least in part reducing inflammation, preferably inflammation caused by damaged muscle cells. Said adjunct compound is most preferably capable of reducing muscle tissue inflammation. Inflammation is preferably assessed by detecting an increase in the number of infiltrating immune cells such as neutrophils and/or mast cells and/or dendritic cells and/or lymphocytes in muscle tissue suspected to be dystrophic. This assessment is preferably carried out in cross-sections of a biopsy.sup.57 of muscle tissue suspected to be dystrophic after having specifically stained immune cells as identified above. The quantification is preferably carried out under the microscope. Reducing inflammation is therefore preferably assessed by detecting a decrease in the number of immune cells in a cross-section of muscle tissue suspected to be dystrophic. Detecting a decrease preferably means that the number of at least one sort of immune cells as identified above is decreased of at least 1%, 2%, 3%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the number of a corresponding immune cell in a same individual before treatment. Most preferably, no infiltrating immune cells are detected in cross-sections of said biopsy.

[0100] An adjunct compound for improving muscle fiber function, integrity and/or survival comprises any therapy, which is capable of measurably enhancing muscle fiber function, integrity and/or survival as compared to an otherwise similar situation wherein said adjunct compound is not present. 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.

[0101] Creatine kinase may be detected in blood as described in 57. 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 individual before treatment.

[0102] A detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in 57 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 individual before treatment.

[0103] 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 57.

[0104] In one embodiment, an adjunct compound for increasing turnover of damaged muscle cells is used. An adjunct compound for increasing turnover of damaged muscle cells comprises any therapy, which is capable of at least in part inducing and/or increasing turnover of damaged muscle cells. Damaged muscle cells are muscle cells, which have significantly less clinically measurable functionality than a healthy, intact muscle cell. In the absence of dystrophin, mechanical stress leads to sarcolemmal ruptures, causing an uncontrolled influx of calcium into the muscle fiber interior, thereby triggering calcium-activated proteases and fiber necrosis, resulting in damaged muscle cells. Increasing turnover of damaged muscle cells means that damaged muscle cells are more quickly broken down and/or removed as compared to a situation wherein turnover of damaged muscle cells is not increased. Turnover of damaged muscle cells is preferably assessed in a muscle biopsy, more preferably as described in 57 using a cross-section of a biopsy. A detectable increase of turnover may be an increase of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein turnover has been identified using a biopsy cross-section. The increase is measured by comparison to the turnover as assessed in a same individual before treatment.

[0105] Without wishing to be bound to theory, it is believed that increasing turnover of muscle cells is preferred because this reduces inflammatory responses.

[0106] According to the present invention, a composition of the invention further comprising an adjunct therapy for reducing inflammation, preferably for reducing muscle tissue inflammation in an individual, is particularly suitable for use as a medicament. Such composition is even better capable of alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy as compared to a combination not comprising said adjunct compound. This embodiment also enhances the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.

[0107] Further provided is therefore a composition further comprising an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation in said individual, for use as a medicament, preferably for treating or preventing counteracting DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein or altered or truncated dystrophin mRNA or protein is formed which is not sufficiently functional.

[0108] Preferred adjunct compound for reducing inflammation include a steroid, a TNF.alpha. inhibitor, a source of mIGF-1 and/or an antioxidant. However, any other compound able to reduce inflammation as defined herein is also encompassed within the present invention. Each of these compounds is later on extensively presented. Each of the compounds extensively presented may be used separately or in combination with each other and/or in combination with one or more of the adjunct compounds used for improving muscle fiber function, integrity and/or survival.

[0109] Furthermore, a composition comprising an adjunct therapy for improving muscle fiber function, integrity and/or survival in an individual is particularly suitable for use as a medicament, preferably for treating or preventing DMD. Such composition is even better capable of alleviating one or more symptom(s) of Duchenne Muscular Dystrophy as compared to a composition not comprising said adjunct compound.

[0110] Preferred adjunct compounds for improving muscle fiber function, integrity and/or survival include an ion channel inhibitor, a protease inhibitor, L-arginine and/or an angiotensin II type I receptor blocker. However, any other compound able to improving muscle fiber function, integrity and/or survival as defined herein is also encompassed within the present invention. Each of these compounds is later on extensively presented. Each of the compounds extensively presented may be used separately or in combination with each other and/or in combination with one or more of the adjunct compounds used for reducing inflammation.

[0111] In a particularly preferred embodiment, a composition further comprises a steroid. Such composition results in significant alleviation of DMD symptoms. This embodiment also enhances the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.

[0112] In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.

[0113] A steroid is a terpenoid lipid characterized by a carbon skeleton with four fused rings, generally arranged in a 6-6-6-5 fashion. Steroids vary by the functional groups attached to these rings and the oxidation state of the rings. Steroids include hormones and drugs, which are usually used to relieve swelling and inflammation, such as for instance prednisone, dexamethasone and vitamin D.

[0114] According to the present invention, supplemental effects of adjunct steroid therapy in DMD patients include reduction of tissue inflammation, suppression of cytotoxic cells, and improved calcium homeostasis. Most positive results are obtained in younger boys. Preferably, the steroid is a corticosteroid, more preferably, a glucocorticosteroid. Preferably, prednisone steroids such as prednisone, prednizolone or deflazacort are used in a combination according to the invention.sup.21. Dose ranges of steroid or of a glucocorticosteroid to be used in the therapeutic applications as described herein are designed on the basis of rising dose studies in clinical trials for which rigorous protocol requirements exist. The usual doses are 0.5-1.0 mg/kg/day, preferably 0.75 mg/kg/day for prednisone and prednisolone, and 0.4-1.4 mg/kg/day, preferably 0.9 mg/kg/day for deflazacort.

[0115] In one embodiment, a steroid is administered to said individual prior to administering a composition as earlier defined herein. In this embodiment, it is preferred that said steroid is administered at least one day, more preferred at least one week, more preferred at least two weeks, more preferred at least three weeks prior to administering said composition.

[0116] In another preferred embodiment, a combination further comprises a tumour necrosis factor-alpha (TNF.alpha.) inhibitor. Tumour necrosis factor-alpha (TNF.alpha.) is a pro-inflammatory cytokine that stimulates the inflammatory response. Pharmacological blockade of TNF.alpha. activity with the neutralising antibody infliximab (Remicade) is highly effective clinically at reducing symptoms of inflammatory diseases. In mdx mice, both infliximab and etanercept delay and reduce the necrosis of dystrophic muscle.sup.24, 25, with additional physiological benefits on muscle strength, chloride channel function and reduced CK levels being demonstrated in chronically treated exercised adult mdx mice.sup.26. Such highly specific anti-inflammatory drugs designed for use in other clinical conditions, are attractive alternatives to the use of steroids for DMD. In one embodiment, the use of a TNF.alpha. inhibitor is limited to periods of intensive muscle growth in boys when muscle damage and deterioration are especially pronounced.

[0117] A composition further comprising a TNF.alpha. inhibitor for use as a medicament is also provided. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. A preferred TNF.alpha. inhibitor is a dimeric fusion protein consisting of the extracellular ligand-binding domain of the human p75 receptor of TNF.alpha. linked to the Fc portion of human IgG1. A more preferred TNF.alpha. inhibitor is ethanercept (Amgen, America).sup.26. The usual doses of ethanercept is about 0.2 mg/kg, preferably about 0.5 mg/kg twice a week. The administration is preferably subcutaneous.

[0118] In another preferred embodiment, a composition of the invention further comprises a source of mIGF-1. As defined herein, a source of IGF-1 preferably encompasses mIGF-1 itself, a compound able of enhancing mIGF-1 expression and/or activity. Enhancing is herein synonymous with increasing. Expression of mIGF-1 is synonymous with amount of mIGF-1. mIGF-1 promotes regeneration of muscles through increase in satellite cell activity, and reduces inflammation and fibrosis.sup.27. Local injury of muscle results in increased mIGF-1 expression. In transgenic mice with extra IGF-1 genes, muscle hypertrophy and enlarged muscle fibers are observed.sup.27. Similarly, transgenic mdx mice show reduced muscle fiber degeneration.sup.28. Upregulation of the mIGF-1 gene and/or administration of extra amounts of mIGF-1 protein or a functional equivalent thereof (especially the mIGF-1 Ea isoform [as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577]) thus promotes the effect of other, preferably genetic, therapies for DMD, including antisense-induced exon skipping. The additional mIGF-1 levels in the above mentioned transgenic mice do not induce cardiac problems nor promote cancer, and have no pathological side effects. As stated before, the amount of mIGF-1 is for instance increased by enhancing expression of the mIGF-1 gene and/or by administration of mIGF-1 protein and/or a functional equivalent thereof (especially the mIGF-1 Ea isoform [as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577]). A composition of the invention further preferably comprises mIGF-1, a compound capable of enhancing mIGF-1 expression and/or an mIGF-1 activity, for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, such composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.

[0119] Within the context of the invention, an increased amount or activity of mIGF-1 may be reached by increasing the gene expression level of an IGF-1 gene, by increasing the amount of a corresponding IGF-1 protein and/or by increasing an activity of an IGF1-protein. A preferred mIGF-1 protein has been earlier defined herein. An increase of an activity of said protein is herein understood to mean any detectable change in a biological activity exerted by said protein or in the steady state level of said protein as compared to said activity or steady-state in a individual who has not been treated. Increased amount or activity of mIGF-1 is preferably assessed by detection of increased expression of muscle hypertrophy biomarker GATA-2 (as described in 27).

[0120] Gene expression level is preferably assessed using classical molecular biology techniques such as (real time) PCR, arrays or Northern analysis. A steady state level of a protein is determined directly by quantifying the amount of a protein. Quantifying a protein amount may be carried out by any known technique such as Western blotting or immunoassay using an antibody raised against a protein. The skilled person will understand that alternatively or in combination with the quantification of a gene expression level and/or a corresponding protein, the quantification of a substrate of a corresponding protein or of any compound known to be associated with a function or activity of a corresponding protein or the quantification of said function or activity of a corresponding protein using a specific assay may be used to assess the alteration of an activity or steady state level of a protein.

[0121] In the invention, an activity or steady-state level of a said protein may be altered at the level of the protein itself, e.g. by providing a protein to a cell from an exogenous source.

[0122] Preferably, an increase or an up-regulation of the expression level of a said gene means an increase of at least 5% of the expression level of said gene using arrays. More preferably, an increase of the expression level of said gene means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more. In another preferred embodiment, an increase of the expression level of said protein means an increase of at least 5% of the expression level of said protein using Western blotting and/or using ELISA or a suitable assay. More preferably, an increase of the expression level of a protein means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.

[0123] In another preferred embodiment, an increase of a polypeptide activity means an increase of at least 5% of a polypeptide activity using a suitable assay. More preferably, an increase of a polypeptide activity means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more. The increase is preferably assessed by comparison to corresponding activity in the individual before treatment.

[0124] A preferred way of providing a source of mIGF1 is to introduce a transgene encoding mIGF1, preferably an mIGF-1 Ea isoform (as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577), more preferably in an AAV vector as later defined herein. Such source of mIGF1 is specifically expressed in muscle tissue as described in mice in 27.

[0125] In another preferred embodiment, a composition further comprises an antioxidant. Oxidative stress is an important factor in the progression of DMD and promotes chronic inflammation and fibrosis.sup.2. The most prevalent products of oxidative stress, the peroxidized lipids, are increased by an average of 35% in Duchenne boys. Increased levels of the enzymes superoxide dismutase and catalase reduce the excessive amount of free radicals causing these effects. In fact, a dietary supplement Protandim.RTM. (LifeVantage) was clinically tested and found to increase levels of superoxide dismutase (up to 30%) and catalase (up to 54%), which indeed significantly inhibited the peroxidation of lipids in 29 healthy persons.sup.30. Such effective management of oxidative stress thus preserves muscle quality and so promotes the positive effect of DMD therapy. Idebenone is another potent antioxidant with a chemical structure derived from natural coenzyme Q10. It protects mitochondria where adenosine triphosphate, ATP, is generated by oxidative phosphorylation. The absence of dystrophin in DMD negatively affects this process in the heart, and probably also in skeletal muscle. Idebenone was recently applied in clinical trials in the US and Europe demonstrating efficacy on neurological aspects of Friedreich's Ataxia.sup.31. A phase-Ha double-blind, placebo-controlled randomized clinical trial with Idebenone has recently been started in Belgium, including 21 Duchenne boys at 8 to 16 years of age. The primary objective of this study is to determine the effect of Idebenone on heart muscle function. In addition, several different tests will be performed to detect the possible functional benefit on muscle strength in the patients. When effective, Idebenone is a preferred adjunct compound for use in a combination according to the present invention in order to enhance the therapeutic effect of DMD therapy, especially in the heart. A composition further comprising an antioxidant for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the antioxidant, the skilled person will know which quantities are preferably used. An antioxidant may include bacoside, silymarin, curcumin and/or a polyphenol. Preferably, a polyphenol is or comprises epigallocatechin-3-gallate (EGCG). Preferably, an antioxidant is a mixture of antioxidants as the dietary supplement Protandim.RTM. (LifeVantage). A daily capsule of 675 mg of Protandim.RTM. comprises 150 mg of B. monniera (45% bacosides), 225 mg of S. marianum (70-80% silymarin), 150 mg of W. somnifera powder, 75 mg green tea (98% polyphenols wherein 45% EGCG) and 75 mg turmeric (95% curcumin).

[0126] In another preferred embodiment, a composition further comprises an ion channel inhibitor. The presence of damaged muscle membranes in DMD disturbs the passage of calcium ions into the myofibers, and the consequently disrupted calcium homeostasis activates many enzymes, e.g. proteases, that cause additional damage and muscle necrosis. Ion channels that directly contribute to the pathological accumulation of calcium in dystrophic muscle are potential targets for adjunct compounds to treat DMD. There is evidence that some drugs, such as pentoxifylline, block exercise-sensitive calcium channels.sup.32 and antibiotics that block stretch activated channels reduce myofibre necrosis in mdx mice and CK levels in DMD boys.sup.33. A composition further comprising an ion channel inhibitor for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.

[0127] Preferably, an ion channel inhibitor of the class of xanthines is used. More preferably, said xanthines are derivatives of methylxanthines, and most preferably, said methylxanthine derivates are chosen from the group consisting of pentoxifylline, furafylline, lisofylline, propentofylline, pentifylline, theophylline, torbafylline, albifylline, enprofylline and derivatives thereof. Most preferred is the use of pentoxifylline. Ion channel inhibitors of the class of xanthines enhance the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.

[0128] Depending on the identity of the ion channel inhibitor, the skilled person will know which quantities are preferably used. Suitable dosages of pentoxifylline are between 1 mg/kg/day to 100 mg/kg/day, preferred dosages are between 10 mg/kg/day to 50 mg/kg/day. Typical dosages used in humans are 20 mg/kg/day.

[0129] In one embodiment, an ion channel inhibitor is administered to said individual prior to administering a composition comprising an oligonucleotide. In this embodiment, it is preferred that said ion channel inhibitor is administered at least one day, more preferred at least one week, more preferred at least two weeks, more preferred at least three weeks prior to administering a composition comprising an oligonucleotide.

[0130] In another preferred embodiment, a composition further comprises a protease inhibitor. Calpains are calcium-activated proteases that are increased in dystrophic muscle and account for myofiber degeneration. Calpain inhibitors such as calpastatin, leupeptin.sup.34, calpeptin, calpain inhibitor III, or PD150606 are therefore applied to reduce the degeneration process. A new compound, BN 82270 (Ipsen) that has dual action as both a calpain inhibitor and an antioxidant increased muscle strength, decreased serum CK and reduced fibrosis of the mdx diaphragm, indicating a therapeutic effect with this new compound.sup.35. Another compound of Leupeptin/Carnitine (Myodur) has recently been proposed for clinical trials in DMD patients.

[0131] MG132 is another proteasomal inhibitor that has shown to reduce muscle membrane damage, and to ameliorate the histopathological signs of muscular dystrophy.sup.36. MG-132 (CBZ-leucyl-leucyl-leucinal) is a cell-permeable, proteasomal inhibitor (Ki=4 nM), which inhibits NFkappaB activation by preventing IkappaB degradation (IC50=3 .mu.M). In addition, it is a peptide aldehyde that inhibits ubiquitin-mediated proteolysis by binding to and inactivating 20S and 26S proteasomes. MG-132 has shown to inhibit the proteasomal degradation of dystrophin-associated proteins in the dystrophic mdx mouse model.sup.36. This compound is thus also suitable for use as an adjunct pharmacological compound for DMD. A composition further comprising a protease inhibitor for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the protease inhibitor, the skilled person will know which quantities are preferably used.

[0132] In another preferred embodiment, a composition further comprises L-arginine. Dystrophin-deficiency is associated with the loss of the DGC-complex at the fiber membranes, including neuronal nitric oxide synthase (nNOS). Expression of a nNOS transgene in mdx mice greatly reduced muscle membrane damage. Similarly, administration of L-arginine (the substrate for nitric oxide synthase) increased NO production and upregulated utrophin expression in mdx mice. Six weeks of L-arginine treatment improved muscle pathology and decreased serum CK in mdx mice.sup.37. The use of L-arginine as a further constituent in a composition of the invention has not been disclosed.

[0133] A composition further comprising L-arginine for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.

[0134] In another preferred embodiment, a composition further comprises angiotensin II type 1 receptor blocker Losartan, which normalizes muscle architecture, repair and function, as shown in the dystrophin-deficient mdx mouse model.sup.23. A composition further comprising angiotensin II type 1 receptor blocker Losartan for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the angiotensin II type 1 receptor blocker, the skilled person will know which quantities are preferably used.

[0135] In another preferred embodiment, a composition further comprises an angiotensin-converting enzyme (ACE) inhibitor, preferably perindopril. ACE inhibitors are capable of lowering blood pressure. Early initiation of treatment with perindopril is associated with a lower mortality in DMD patients.sup.22. A composition further comprising an ACE inhibitor, preferably perindopril for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. The usual doses of an ACE inhibitor, preferably perindopril are about 2 to 4 mg/day.sup.22. In a more preferred embodiment, an ACE inhibitor is combined with at least one of the previously identified adjunct compounds.

[0136] In another preferred embodiment, a composition further comprises a compound exhibiting a readthrough activity. A compound exhibiting a readthrough activity may be any compound, which is able to suppress a stop codon. For 20% of DMD patients, the mutation in the dystrophin gene is comprising a point mutation, of which 13% is a nonsense mutation. A compound exhibiting a readthrough activity or which is able to suppress a stop codon is a compound which is able to provide an increased amount of a functional dystrophin mRNA or protein and/or a decreased amount of an aberrant or truncated dystrophin mRNA or protein. Increased preferably means increased of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more. Decreased preferably means decreased of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more. An increase or a decrease of said protein is preferably assessed in a muscular tissue or in a muscular cell of an individual by comparison to the amount present in said individual before treatment with said compound exhibiting a readthrough activity. Alternatively, the comparison can be made with a muscular tissue or cell of said individual, which has not yet been treated with said compound in case the treatment is local. The assessment of an amount at the protein level is preferably carried out using western blot analysis.

[0137] Preferred compounds exhibiting a readthrough activity comprise or consist of aminoglycosides, including, but not limited to, geneticin (G418), paromomycin, gentamycin and/or 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid), and derivatives thereof (references 64, 65). A more preferred compound exhibiting a readthrough activity comprises or consists of PTC124.TM., and/or a functional equivalent thereof. PTC124.TM. is a registered trademark of PTC Therapeutics, Inc. South Plainfield, N.J., 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) also known as PTC124.TM. (references 16, 17) belongs to a new class of small molecules that mimics at lower concentrations the readthrough activity of gentamicin (reference 55). A functional equivalent of 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) or of gentamicin is a compound which is able to exhibit a readthrough activity as earlier defined herein. Most preferably, a compound exhibiting a readthrough activity comprises or consists of gentamycin and/or 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) also known as PTC124.TM.. A composition further comprising a compound exhibiting a readthrough activity, preferably comprising or consisting of gentamycin and/or 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. The usual doses of a compound exhibiting a readthrough activity, preferably 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) or of gentamicin are ranged between 3 mg/kg/day to 200 mg/kg/day, preferred dosages are between 10 mg/kg to 50 mg/kg per day or twice a day.

[0138] In a more preferred embodiment, a compound exhibiting a readthrough activity is combined with at least one of the previously identified adjunct compounds.

[0139] In another preferred embodiment, a composition further comprises a compound, which is capable of enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing. Small chemical compounds, such as for instance specific indole derivatives, have been shown to selectively inhibit spliceosome assembly and splicing.sup.38, for instance by interfering with the binding of serine- and arginine-rich (SR) proteins to their cognate splicing enhancers (ISEs or ESEs) and/or by interfering with the binding of splicing repressors to silencer sequences (ESSs or ISSs). These compounds are therefore suitable for applying as adjunct compounds that enhance exon skipping. A composition further comprising a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the compound, which is capable of enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing, the skilled person will know which quantities are preferably used. In a more preferred embodiment, a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing is combined with a ACE inhibitor and/or with any adjunct compounds as identified earlier herein.

[0140] The invention thus provides a composition further comprising an adjunct compound, wherein said adjunct compound comprises a steroid, an ACE inhibitor (preferably perindopril), angiotensin II type 1 receptor blocker Losartan, a tumour necrosis factor-alpha (TNF.alpha.) inhibitor, a source of mIGF-1, preferably mIGF-1, a compound for enhancing mIGF-1 expression, a compound for enhancing mIGF-1 activity, an antioxidant, an ion channel inhibitor, a protease inhibitor, L-arginine, a compound exhibiting a readthrough activity and/or inhibiting spliceosome assembly and/or splicing.

[0141] In one embodiment an individual is further provided with a functional dystrophin protein using a vector, preferably a viral vector, comprising a micro-mini-dystrophin gene. Most preferably, a recombinant adeno-associated viral (rAAV) vector is used. AAV is a single-stranded DNA parvovirus that is non-pathogenic and shows a helper-dependent life cycle. In contrast to other viruses (adenovirus, retrovirus, and herpes simplex virus), rAAV vectors have demonstrated to be very efficient in transducing mature skeletal muscle. Application of rAAV in classical DMD "gene addition" studies has been hindered by its restricted packaging limits (<5 kb). Therefore, rAAV is preferably applied for the efficient delivery of a much smaller micro- or mini-dystrophin gene. Administration of such micro- or mini-dystrophin gene results in the presence of an at least partially functional dystrophin protein. Reference is made to.sup.18-20.

[0142] Each constituent of a composition can be administered to an individual in any order. In one embodiment, each constituent is administered simultaneously (meaning that each constituent is administered within 10 hours, preferably within one hour). This is however not necessary. In one embodiment at least one adjunct compound is administered to an individual in need thereof before administration of an oligonucleotide. Alternatively, an oligonucleotide is administered to an individual in need thereof before administration of at least one adjunct compound.

[0143] Use

[0144] In a further aspect, there is provided the use of a oligonucleotide or of a composition as defined herein for the manufacture of a medicament for preventing or treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual. Each feature of said use has earlier been defined herein.

[0145] A treatment in a use or in a method according to the invention is 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 an oligonucleotide, composition, compound or adjunct compound of the invention may depend on several parameters such as the age of the patient, the mutation of the patient, the number of molecules (i.e. dose), the formulation of said molecule. The frequency may be ranged between at least once in two weeks, or three weeks or four weeks or five weeks or a longer time period.

[0146] Method

[0147] In a further aspect, there is provided a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual or alleviate one or more characteristic(s) of a myogenic or muscle cell of said individual, the method comprising administering to said individual an oligonucleotide or a composition as defined herein.

[0148] There is further provided a method for enhancing, inducing or promoting skipping of an exon from a dystrophin pre-mRNA in a cell expressing said pre-mRNA in an individual suffering from Duchenne Muscular Dystrophy or Becker Muscular Dystrophy, the method comprising administering to said individual an oligonucleotide or a composition as defined herein. Further provided is a method for increasing the production of a functional dystrophin protein and/or decreasing the production of an aberrant dystrophin protein in a cell, said cell comprising a pre-mRNA of a dystrophin gene encoding an aberrant dystrophin protein, the method comprising providing said cell with an oligonucleotide or composition of the invention and allowing translation of mRNA produced from splicing of said pre-mRNA. In one embodiment, said method is performed in vitro, for instance using a cell culture. Preferably, said method is in vivo.

[0149] In this context, increasing the production of a functional dystrophin protein has been earlier defined herein.

[0150] Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

[0151] 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 compound or adjunct compound 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.

[0152] 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".

[0153] The word "approximately" or "about" when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.

[0154] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Each embodiment as identified herein may be combined together unless otherwise indicated.

[0155] The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0156] FIG. 1. In human control myotubes, PS220 and PS305 both targeting an identical sequence within exon 45, were directly compared for relative skipping efficiencies. PS220 reproducibly induced highest levels of exon 45 skipping (up to 73%), whereas with PS305 maximum exon 45 skipping levels of up to 46% were obtained. No exon 45 skipping was observed in non-treated cells. (M: DNA size marker; NT: non-treated cells)

[0157] FIG. 2. Graph showing relative exon 45 skipping levels of inosine-containing AONs as assessed by RT-PCR analysis. In human control myotubes, a series of new AONs, all targeting exon 45 and containing one inosine for guanosine substitution were tested for relative exon 45 skipping efficiencies when compared with PS220 and PS305 (see FIG. 1). All new inosine-containing AONs were effective, albeit at variable levels (between 4% and 25%). PS220 induced highest levels of exon 45 skipping (up to 72%), whereas with PS305 maximum exon 45 skipping levels of up to 63% were obtained. No exon 45 skipping was observed in non-treated cells. (M: DNA size marker; NT: non-treated cells).

EXAMPLES

Example 1

Materials and Methods

[0158] AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program, on (partly) overlapping putative 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.

[0159] Tissue Culturing, Transfection and RT-PCR Analysis

[0160] 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 200 nM for each AON (PS220 and PS305). Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 .mu.l UNIFectylin per .mu.g AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking the targeted exon 45. PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the DNA 1000 LabChip Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA).

[0161] Results

[0162] DMD exon 45 skipping.

[0163] Two AONs, PS220 (SEQ ID NO: 76; 5'-UUUGCCGCUGCCCAAUGCCAUCCUG-3') and PS305 (SEQ ID NO: 557; 5'-UUUGCCICUGCCCAAUGCCAUCCUG-3') both targeting an identical sequence within exon 45, were directly compared for relative skipping efficiencies in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that both AONs were indeed capable of inducing exon 45 skipping. PS220, consisting a GCCGC stretch, reproducibly induced highest levels of exon 45 skipping (up to 73%), as shown in FIG. 1. However, PS305, which is identical to PS220 but containing an inosine for a G substitution at position 4 within that stretch is also effective and leading to exon 45 skipping levels of up to 46%. No exon 46 skipping was observed in non-treated cells (NT).

Example 2

Materials and Methods

[0164] AON design was based on (partly) overlapping open secondary structures of the target exon 45 RNA as predicted by the m-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, full-length phosphorothioate (PS) backbones and one inosine for guanosine substitution.

[0165] Tissue Culturing, Transfection and RT-PCR Analysis

[0166] Myotube cultures derived from a healthy individual ("human control") 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 200 nM for each AON. Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 .mu.l UNIFectylin per .mu.g AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking the targeted exon 45. PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the DNA 1000 LabChip Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA).

[0167] Results

[0168] DMD exon 45 skipping.

[0169] An additional series of AONs targeting exon 45 and containing one inosine-substitution were tested in healthy control myotube cultures for exon 45 skipping efficiencies, and directly compared to PS220 (without inosine; SEQ ID NO: 76)) and PS305 (identical sequence as PS220 but with inosine substitution; SEQ ID NO: 557). Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that all new AONs (PS309 to PS316) were capable of inducing exon 45 skipping between 4% (PS311) and 25% (PS310) as shown in FIG. 2. When compared to PS220 and PS305, PS220 induced highest levels of exon 45 skipping (up to 72%). Of the new inosine-containing AONs PS305 was most effective, showing exon 45 skipping levels of up to 63%. No exon 45 skipping was observed in non-treated cells (NT).

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TABLE-US-00001

[0234] Sequence listing DMD gene amino acid sequence SEQ ID NO 1: MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGL TGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIIL HWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIH SHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYIT SLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSP KPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEV LSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGT GKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELNDWLT KTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSG DHATAALEEQLKVLGDRWANICRWTEDRWVLLQDILLKWQRLTEEQCLFSAWLSEKE DAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVT QKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQPSLTQTTVMETVTTVTTREQILV KHAQEELPPPPPQKKRQITVDSEIRKRLDVDITELHSWITRSEAVLQSPEFAIFRKEGNF SDLKEKVNAIEREKAEKFRKLQDASRSAQALVEQMVNEGVNADSIKQASEQLNSRWIE FCQLLSERLNWLEYQNNIIAFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKIC KDEVNRLSCLQPQIERLKIQSIALKEKGQGPMFLDADFVAFTNHFKQVFSDVQAREKEL QTIFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQSSLQE QQSGLYYLSTTVKEMSKKAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEEQMNK LRKIQNHIQTLKKWMAEVDVFLKEEWPALGDSEILKKQLKQCRLLVSDIQTIQPSLNSV NEGGQKIKNEAEPEFASRLETELKELNTQWDHMCQQVYARKEALKGGLEKTVSLQKD LSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKRAKEEAQQKEAKVKLLTESV NSVIAQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLEEVWACWHELLSYLEKAN KWLNEVEFKLKTTENIPGGAEEISEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMD ELINEELETFNSRWRELHEEAVRRQKLLEQSIQSAQETEKSLHLIQESLTFIDKQLAAYI ADKVDAAQMPQEAQKIQSDLTSHEISLEEMKKHNQGKEAAQRVLSQIDVAQKKLQDVS MKFRLFQKPANFEQRLQESKMILDEVKMHLPALETKSVEQEVVQSQLNHCVNLYKSLS EVKSEVEMVIKTGRQIVQKKQTENPKELDERVTALKLHYNELGAKVTERKQQLEKCLK LSRKMRKEMNVLTEWLAATDMELTKRSAVECMPSNLDSEVAWCKATQKEIEKQKVH LKSITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLNLLLEYQKHMETFD QNVDHITKWIIQADTLLDESEKKKPQQKEDVLKRLKAELNDIRPKVDSTRDQAANLMA NRGDHCRKLVEPQISELNHRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAEIQQ GVNLKEEDFNKDMNEDNEGTVKELLQRGDNLQQRITDERKREEIKIKQQLLQTKHNA LKDLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERKIKEIDREL QKKKEELNAVRRQAECLSEDCAAMAVEPTQIQLSKRWREIESKFAQFRRLNFAQIHTV REETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQE ESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKD RQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGI GQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKN ILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQGILKQL NETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKK LEDLEEQLNHLLLWLSPIRNQLEIYNQPNQECPFDVQETEIAVQAKQPDVEEILSKCQH LYKEKPATQPVKRKLEDLSSEWKAVNRLLQELRAKQPDLAPGLTTIGASPTQTVTLVTQ PVVTKETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQRVMVGDLEDI NEMIIKQKATMQDLEQRRPQLEELITAAQNLKNKTSNQEARTIITDRIERIQNQWDEVQ EHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLGQARQKLESWKEGPYTVDAIQKKITE TKQLAKDLRQWQTNVDVANDLALKLLRDYSADDTRKVHMITENINASWRSIHKRVSER EAALEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKCVKELMKQ WQDLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSL NIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFK RELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEV NTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLID SLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQ VAVEDRVRQLHEAHRDFGPASQHFLSTSVQCPWERAISPNKVPYYINHETQTTCWDHP KMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQ PMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTG IISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSV RSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNK FRTKRYFAKHPRMCYLPVQTVLECDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRI EHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILIS LESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRD AELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSS PSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRG RNTPGKPMREDTM DMD Gene Exon 51 SEQ ID NO 2 GUACCUCCAACAUCAAGGAAGAUGG SEQ ID NO 39 GAGAUGGCAGUUUCCUUAGUAACCA SEQ ID NO 3 UACCUCCAACAUCAAGGAAGAUGGC SEQ ID NO 40 AGAUGGCAGUUUCCUUAGUAACCAC SEQ ID NO 4 ACCUCCAACAUCAAGGAAGAUGGCA SEQ ID NO 41 GAUGGCAGUUUCCUUAGUAACCACA SEQ ID NO 5 CCUCCAACAUCAACGAAGAUGGCAU SEQ ID NO 42 AUGGCAGUUUCCUUAGUAACCACAG SEQ ID NO 6 CUCCAACAUCAACGAAGAUGGCAUU SEQ ID NO 43 UGGCAGUUUCCUUAGUAACCACAGG SEQ ID NO 7 UCCAACAUCAAGGAAGAUGGCAUUU SEQ ID NO 44 GGCAGUUUCCUUAGUAACCACAGGU SEQ ID NO 8 CCAACAUCAAGGAAGAUGGCAUUUC SEQ ID NO 45 GCAGUUUCCUUAGUAACCACAGGUU SEQ ID NO 9 CAACAUCAAGGAAGAUGGCAUUUCU SEQ ID NO 46 CAGUUUCCUUAGUAACCACAGGUUG SEQ ID NO 10 AACAUCAAGGAAGAUGGCAUUUCUA SEQ ID NO 47 AGUUUCCUUAGUAACCACAGGUUGU SEQ ID NO 11 ACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO 48 GUUUCCUUAGUAACCACAGGUUGUG SEQ ID NO 12 CAUCAAGGAAGAUGGCAUUUCUAGU SEQ ID NO 49 UUUCCUUAGUAACCACAGGUUGUGU SEQ ID NO 13 AUCAAGGAAGAUGGCAUUUCUAGUU SEQ ID NO 50 UUCCUUAGUAACCACAGGUUGUGUC SEQ ID NO 14 UCAAGGAAGAUGGCAUUUCUAGUUU SEQ ID NO 51 UCCUUAGUAACCACAGGUUGUGUCA SEQ ID NO 15 CAAGGAAGAUGGCAUUUCUAGUUUG SEQ ID NO 52 CCUUAGUAACCACAGGUUGUGUCAC SEQ ID NO 16 AAGGAAGAUGGCAUUUCUAGUUUGG SEQ ID NO 53 CUUAGUAACCACAGGUUGUGUCACC SEQ ID NO 17 AGGAAGAUGGCAUUUCUAGUUUGGA SEQ ID NO 54 UUAGUAACCACAGGUUGUGUCACCA SEQ ID NO 18 GGAAGAUGGCAUUUCUAGUUUGGAG SEQ ID NO 55 UAGUAACCACAGGUUGUGUCACCAG SEQ ID NO 19 GAAGAUGGCAUUUCUAGUUUGGAGA SEQ ID NO 56 AGUAACCACAGGUUGUGUCACCAGA SEQ ID NO 20 AAGAUGGCAUUUCUAGUUUGGAGAU SEQ ID NO 57 GUAACCACAGGUUGUGUCACCAGAG SEQ ID NO 21 AGAUGGCAUUUCUAGUUUGGAGAUG SEQ ID NO 58 UAACCACAGGUUGUGUCACCAGAGU SEQ ID NO 22 GAUGGCAUUUCUAGUUUGGAGAUGG SEQ ID NO 59 AACCACAGGUUGUGUCACCAGAGUA SEQ ID NO 21 AUGGCAUUUCUAOUUUGOAGAUGGC SEQ ID NO 60 ACCACAGGUUGUGUCACCAGAGUAA SEQ ID NO 24 UGGCAUUUCUAGUUUGGAGAUGGCA SEQ ID NO 61 CCACAGGUUGUGUCACCAGAGUAAC SEQ ID NO 25 GGCAUUUCUAGUUUGGAGAUGGCAG SEQ ID NO 62 CACAGGUUGUGUCACCAGAGUAACA SEQ ID NO 26 GCAUUUCUAGUUUGGAGAUGGCAGU SEQ ID NO 63 ACAGGUUGUGUCACCAGAGUAACAG SEQ ID NO 27 CAUUUCUAGUUUGGAGAUGGCAGUU SEQ ID NO 64 CAGGUUGUGUCACCAGAGUAACAGU SEQ ID NO 28 AUUUCUAGUUUGGAGAUGGCAGUUU SEQ ID NO 65 AGGUUGUGUCACCAGAGUAACAGUC SEQ ID NO 29 UUUCUAGUUUGGAGAUGGCAGUUUC SEQ ID NO 66 GGUUGUGUOACCAGAGUAACAGUCU SEQ ID NO 30 UUCUAGUUUGGAGAUGGCAGUUUCC SEQ ID NO 67 GUUGUGUCACCAGAGUAACAGUCUG SEQ ID NO 31 UCUAGUUUGGAGAUGGCAGUUUCCU SEQ ID NO 68 UUGUGUCACCAGAGUAACAGUCUGA SEQ ID NO 32 CUAGUUUGGAGAUGGCAGUUUCCUU SEQ ID NO 69 UGUGUCACCAGAGUAACAGUCUGAG SEQ ID NO 33 UAGUUUGGAGAUGGCAGUUUCCUUA SEQ ID NO 70 GUGUCACCAGAGUAACAGUCUGAGU SEQ ID NO 34 AGUUUGGAGAUGGOAGUUUCCUUAG SEQ ID NO 71 UGUCACCAGAGUAACAGUCUGAGUA SEQ ID NO 35 GUUUGGAGAUGGCAGUUUCCUUAGU SEQ ID NO 72 CUCACCAGAGUAACAGUCUGAGUAG SEQ ID NO 36 UUUGGAGAUGGCAGUUUCCUUAGUA SEQ ID NO 73 UCACCAGAGUAACAGUCUGAGUAGG SEQ ID NO 37 UUGGAGAUGGCAGUUUCCUUAGUAA SEQ ID NO 74 CACCAGAGUAACAGUCUGAGUAGGA SEQ ID NO 38 UGGAGAUGGCAGUUUCCUUAGUAAC SEQ ID NO 75 ACCAGAGUAACAGUCUGAGUAGGAG SEQ ID NO 539 UCAAGGAAGAUGGCAUUUCU SEQ ID NO 548 UCAAGGAAGAUGGCAUIUCU SEQ ID NO 540 UCAA1GAAGAUGGCAUUUCU SEQ ID NO 549 UCAAGGAAGAUGGCAUUICU

SEQ ID NO 541 UCAAGIAAGAUGGCAUUUCU SEQ ID NO 550 UCAAGGAAGAUGGCAUUUCI SEQ ID NO 542 UCAAGGAAIAUGGCAUUUCU SEQ ID NO 551 UCIAGGAAGAUGGCAUUUCU SEQ ID NO 543 UCAAGGAAGAUIGCAUUUCU SEQ ID NO 552 UCAIGGAAGAUGGCAUUUCU SEQ ID NO 544 UCAAGGAAGAUGICAUUUCU SEQ ID NO 553 UCAAGGIAGAUGGCAUUUCU SEQ ID NO 545 ICAAGGAAGAUGGCAUUUCU SEQ ID NO 554 UCAAGGAIGAUGGCAUUUCU SEQ ID NO 546 UCAAGGAAGAIGGCAUUUCU SEQ ID NO 555 UCAAGGAAGIUCGCAUUUCU SEQ ID NO 547 UCAAGGAAGAUGOCAIUUCU SEQ ID NO 556 UCAAGGAAGAUGGCIUULCU SEQ ID NO 76 UUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 109 GUUGCAUUCAAUGUUCUGACAACAG PS220 SEQ ID NO 77 AUUCAAUGUUCUGACAACAGUUUGC SEQ ID NO 110 UUGCAUUCAAUGUUCUGACAACAGU SEQ ID NO 78 CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID NO 111 UGCAUUCAAUGUUCUGACAACAGUU SEQ ID NO 79 CAGUUGCAUUCAAUGUUCUGAC SEQ ID NO 112 GCAUUCAAUGUUCUGACAACAGUUU SEQ ID NO 80 AGUUGCAUUCAAUGUUCUGA SEQ ID NO 113 CAUUCAAUGUUCUGACAACAGUUUG SEQ ID NO 81 GAUUGCUGAAUUAUUUCUUCC SEQ ID NO 114 AUUCAAUGUUCUGACAACAGUUUGC SEQ ID NO 82 CAUUCCUGAAUUAUUUCUUCCCCAC SEQ ID NO 115 UCAAUGUUCUGACAACAGUUUGCCG SEQ ID NO 83 AUUGCUGAAUUAUUUCUUCCCCAGU SEQ ID NO 116 CAAUGUUCUGACAACAGUUUGCCGC SEQ ID NO 84 UUGCUGAAUUAUUUCUUCCCCAGUU SEQ ID NO 117 AAUGUUCUGACAACAGUUUGCCGCU SEQ ID NO 85 UGCUGAAUUAUUUCUUCCCCAGUUG SEQ ID NO 118 AUGUUCUGACAACAGUUUGCCGCUG SEQ ID NO 86 GCUGAAUUAUUUCUUCCCCAGUUGC SEQ ID NO 119 UGUUCUGACAACAGUUUGCCGCUGC SEQ ID NO 87 CUGAAUUAUUUCUUCCCCAGUUGCA SEQ ID NO 120 GUUCUGACAACAGUUUGCCGCUGCC SEQ ID NO 88 UGAAUUAUUUCUUCCCCAGUUGCAU SEQ ID NO 121 UUCUGACAACAGUUUGCCGCUGCCC SEQ ID NO 89 GAAUUAUUUCUUCCCCAGUUGCAUU SKQ ID NO 122 UCUGACAACAGUUUGCCGCUGCCCA SEQ ID NO 90 AAUUAUUUCUUCCCCAGUUGCAUUC SEQ ID NO 123 CUGACAACAGUUUGCCGCUGCCCAA SEQ ID NO 91 AUUAUUUCUUCCCCAGUUGCAUUCA SEQ ID NO 124 UGACAACAGUUUGCCGCUGCCCAAU SEQ ID NO 92 UUAUUUCUUCCCCAGUUGCAUUCAA SEQ ID NO 125 GACAACAGUUUGCCGCUGCCCAAUG SEQ ID NO 93 UAUUUCUUCCCCAGUUGCAUUCAAU SEQ ID NO 126 ACAACAGUUUGCCGCUGCCCAAUGC SEQ ID NO 94 AUUUCUUCCCCAGUUGCAUUCAAUG SEQ ID NO 127 CAACAGUUUGCCGCUGCCCAAUGCC SEQ ID NO 95 UUUCUUCCCCAGUUGCAUUCAAUGU SEQ ID NO 128 AACAGUUUGCCGCUGCCCAAUGCCA SEQ ID NO 96 UUCUUCCCCAGUUGCAUUCAAUGUU SEQ ID NO 129 ACAGUUUGCCGCUGCCCAAUGCCAU SEQ ID NO 97 UCUUCCCCAGUUGCAUUCAAUGUUC SEQ ID NO 130 CAGUUUGCCGCUGCCGAAUGGCAUC SEQ ID NO 98 CUUCCCCAGUUGCAUUCAAUGUUCU SEQ ID NO 131 AGUUUGCCGCUGCCCAAUGCCAUCC SEQ ID NO 99 UUCCCCAGUUGCAUUCAAUGUUCUG SEQ ID NO 132 GUUUGCCGCUGCCCAAUGCCAUCCU SEQ ID NO 100 UCCCCAGUUGCAUUCAAUGUUCUGA SEQ ID NO 133 UUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 101 CCCCAGUUGCAUUCAAUGUUCUGAC SEQ ID NO 134 UUGCCGCUGCCCAAUGCCAUCCUGG SEQ ID NO 102 CCCAGUUGCAUUCAAUGUUCUGACA SEQ ID NO 135 UGCCGCUGCCCAAUGCCAUCCUGGA SEQ ID NO 103 CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID NO 136 GCCGCUGCCCAAUGCCAUCCUGGAG SEQ ID NO 104 CAGUUGCAUUCAAUGUUCUGACAAC SEQ ID NO 137 CCGCUGCCCAAUGCCAUCCUGGAGU SEQ ID NO 105 AGUUGCAUUCAAUGUUCUGACAACA SEQ ID NO 138 CGCUGCCCAAUGCCAUCCUGGAGUU SEQ ID NO 106 UCC UGU AGA AUA CUG GCA UC SEQ ID NO 139 UGUUUUUGAGGAUUGCUGAA SEQ ID NO 107 UGCAGACCUCCUGCCACCGCAGAUUCA SEQ ID NO 140 UGUUCUGACAACAGUUUGCCGCUGC CCAAUGCCAUCCUGG SEQ ID NO 108 UUGCAGACCUCCUGCCACCGCAGAUUC SEQ ID NO 557 UUUGCCICUGCCCAAUGCCAUCCUG AGGCUUC PS305 SEQ ID NO 558 UUUGCCGCUICCCAAUGCCAUCCUG SEQ ID NO 566 UUUGCCGCUGCCCAIUGCCAUCCUG SEQ ID NO 559 UUUGCCGCUGCCCAAUICCAUCCUG SEQ ID NO 567 UUUGCCGCUGCCCAAUGCCIUCCUG SEQ ID NO 500 UUUICCGCUGCCCAAUGCCAUCCUG SEQ ID NO 568 UUUICCICUGCCCAAUGCCAUCCUG SEQ ID NO 561 UUUGCCGCUGCCCAAUGCCAUCCUI SEQ ID NO 569 UUUGCCGCUGCCGAAIGCCAUCCUG SEQ ID NO 562 IUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 570 UUUGCCGCUGCCCAAUGCCAICCUG SEQ ID NO 563 UIUGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 571 UUUGCCGCUGCCCAAUGCCAUCCIG SEQ ID NO 564 UUIGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 572 UUUGCCGCUGCCCIAUGCCAUCCUG SEQ ID NO 565 UUUGCCGCIGCCCAAUGCCAUCCUG D.MD Gene Exon 53 SEQ ID NO 141 CUCUGGCCUGUCGUAAGACCUGCUC SEQ ID NO 165 CAGCUUCUUCCUUAGCUUCCAGCCA SEQ ID NO 142 UCUGGCCUGUCCUAAGACCUGCUCA SEQ ID NO 166 AGCUUCUUCCCUAGCUUCCAGCCAU SEQ ID NO 143 CUGGCCUGUCCUAAGACCUGCUCAG SEQ ID NO 167 GCUUCUUCCUUAGCUUCCAGCCAUU SEQ ID NO 144 UGGCCUGUCCUAAGACCUGCUCAGC SEQ ID NO 168 CUUCUUCCUUAGCUUCCAGCCAUUG SEQ ID NO 145 GGCCUGUCCUAAGACCUGCUCAGCU SEQ ID NO 160 UUCUUCCUUAGCUUCCAGCCAUUGU SEQ ID NO 146 GCCUGUCCUAAGACCUGCUCAGOUU SEQ ID NO 170 UCUUCCUUAGCUUCCAGCCAUUGUG SEQ ID NO 147 CCUGUCCUAAGACCUGCUCAGCUUC SEQ ID NO 171 CUUCCUUAGCUUCCAGCCAUUGUGU SEQ ID NO 148 CUGUCCUAAGACCUGCUCAGCUUCU SEQ ID NO 172 UUCCUUAGCUUCCAGCCAUUGUGUU SEQ ID NO 149 UGUCCUAAGACCUGCUCAGCUUCUU SEQ ID NO 173 UCCUUAGCUUCCAGCCAUUGUGUUG SEQ ID NO 150 GUCCUAAGACCUGCUCAGCUUCUUC SEQ ID NO 174 CCUUAGCUUCCAGCCAUUGUGUUGA SEQ ID NO 151 UCCUAAGACCUGCUCAGCUUCUUCC SEQ ID NO 175 CUUAGCUUCCAGCCAUUGUGUUGAA SEQ ID NO 152 CCUAAGACCUGCUCAGCUUCUUCCU SEQ ID NO 176 UUAGCUUCCAGCCAUUGUGUUGAAU SEQ ID NO 153 CUAAGACCUGCUCAGCUUCUUCCUU SEQ ID NO 177 UAGCUUCCAGCCAUUGUGUUGAAUC SEQ ID NO 154 UAAGACCUGCUCAGCUUCUUCCUUA SEQ ID NO 178 AGCUUCCAGCCAUUGUGUUGAAUCC SEQ ID NO 155 AAGACCUGCUCAGCUUCUUCCUUAG SEQ ID NO 179 GCUUCCAGCCAUUGUGUUGAAUCCU SEQ ID NO 150 AGACCUGCUCAGCUUCUUCCUUAGC SEQ ID NO 180 CUUCCAGCCAUUGUGUUGAAUCCUU SEQ ID NO 157 GACCUGCUCAGCUUCUUCCUUAGCU SEQ ID NO 181 UUCCAGCCAUUGUGUUGAAUCCUUU SEQ ID NO 158 ACCUGCUCACCUUCUUCCUUAGCUU SEQ ID NO 182 UCCAGCCAUUCUCUUCAAUCCUUUA SEQ ID NO 159 CCUGCUCAGCUUCUUCCUUAGCUUC SEQ ID NO 183 CCAGCCAUUGUGUUGAAUCCUUUAA SEQ ID NO 160 CUGCUCAGCUUCUUCCUUAGCUUCC SEQ ID NO 184 CAGCCAUUGUGUUGAAUCCUUUAAC SEQ ID NO 161 UGCUCAGCUUCUUCCUUAGCUUCCA SEQ ID NO 185 AGCCAUUOUGUUGAAUCCUUUAACA SEQ ID NO 162 GCUCAGGUUCUUCCUUAGCUUCCAG SEQ ID NO 186 GCGAUUGUGUUGAAUCCUUUAACAU SEQ ID NO 163 CUCAGCUUCUUCCUUAGCUUCCAGG SEQ ID NO 187 CCAUUGUGUUGAAUCCUUUAACAUU SEQ ID NO 164 UCAGCUUCUUCCUUAGCUUCCAGCC SEQ ID NO 188 CAUUGUGUUGAAUCCUUUAACAUUU DMD Gone Exon 44 SEQ ID NO 189 UCAGCUUCUGUUAGCCACUG SEQ ID NO 214 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 190 UUCAGCUUCUGUUAGCCACU SEQ ID NO 215 CAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 191 UUCAGCUUCUGUUAGCCACUG SEQ ID NO 216 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 192 UCAGCUUCUGUUAGCCACUGA SEQ ID NO 217 AGCUUCUGUUAGCCACUGAU SEQ ID NO 193 UUCAGCUUCUGUUAGCCACUGA SEQ ID NO 218 GCUUCUGUUAGCCACUGAUU SEQ ID NO 194 UCAGCUUCUGUUAGCCACUGA SEQ ID NO 219 AGCUUCUGUUAGCCACUGAUU SEQ ID NO 195 UUCAGCUUCUGUUAGCCACUGA SEQ ID NO 220 GCUUCUGUUAGCCACUGAUUA SEQ ID NO 196 UCAGCUUCUGUUAGCCACUGAU SEQ ID NO 221 AGCUUCUGUUAGCCACUGAUUA SEQ ID NO 197 UUCAGCUUCUGUUAGCCACUGAU SEQ ID NO 222 GCUUCUGUUAGCCACUGAUUAA SEQ ID NO 198 UCAGCUUCUGUUAGCCACUGAUU SEQ ID NO 223 AGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 199 UUCAGCUUCUGUUAGCCACUGAUU SEQ ID NO 224 GCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 200 UCAGCUUCUGUUAGCCACUGAUUA SEQ ID NO 225 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 201 UUCAGCUUCUGUUAGCCACUGAUA SEQ ID NO 226 GCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 202 UCAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 227 CCAUUUGUAUUUAGCAUGUUCCC SEQ ID NO 203 UUCAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 228 AGAUACCAUUUGUAUUUAGC

SEQ ID NO 204 UCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 229 GCCAUUUCUCAACAGAUCU SEQ ID NO 205 UUCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 230 GCCAUUUCUCAACAGAUCUGUCA SEQ ID NO 206 CAGCUUCUGUUAGCCACUG SEQ ID NO 231 AUUCUCAGGAAUUUGUGUCUUUC SEQ ID NO 207 CAGCUUCUGUUAGCCACUGAU SEQ ID NO 232 UCUCAGGAAUUUGUGUCUUUC SEQ ID NO 208 AGCUUCUGUUAGCCACUGAUU SEQ ID NO 233 GUUCAGCUUCUGUUAGCC SEQ ID NO 209 CAGCUUCUGUUAGCCACUGAUU SEQ ID NO 234 CUGAUUAAAUAUCUUUAUAUC SEQ ID NO 210 AGCUUCUGUUACCCACUCAUUA SEQ ID NO 235 CCCGCCAUUUCUCAACAG SEQ ID NO 211 CAGCUUCUGUUAGCCACUGAUUA SEQ ID NO 236 GUAUUUAGCAUGUUCCCA SEQ ID NO 212 AGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 237 CAGGAAUUUGUGUCUUUC SEQ ID NO 218 CAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 575 UCAICUUCUGUUAGCCACUG SEQ ID NO 573 UCAGCUUCUIUUAGCCACUG SEQ ID NO 576 UCAGCUUCUGUUAGCCACUI SEQ ID NO 574 UCAGCUUCUGUUAICCACUG DMD Gene Exon 46 SEQ ID NO 238 GCUUUUCUUUUAGUUGCUGCUCUUU SEQ ID NO 265 CCAGGUUCAAGUGGGAUACUAGCAA SEQ ID NO 239 CUUUUCUUUUAGUUGCUGCUCUUUU SEQ ID NO 266 CAGGUUCAAGUGGGAUACUAGCAAU SEQ ID NO 240 UUUUGUUUUAGUUGCUGCUCUUUUC SEQ ID NO 267 AGGUUCAAGUGGGAUACUAGCAAUG SEQ ID NO 241 UUUCUUUUAGUUGCUGCUCUUUUCC SEQ ID NO 268 GGUUCAAGUGGGAUACUAGCAAUGU SEQ ID NO 242 UUCUUUUAGUUGCUGCUCUUUUCCA SEQ ID NO 269 GUUCAAGUGGGAUACUAGCAAUGUU SEQ ID NO 243 UCUUUUAGUUGCUGCUCUUUUCCAG SEQ ID NO 270 UUCAAGUGGGAUACUAGCAAUGUUA SEQ ID NO 244 CUUUUAGUUGCUGCUCUUUUCCAGG SEQ ID NO 271 UCAAGUGGGAUACUAGCAAUGUUAU SEQ ID NO 245 UUUUAGUUGCUGCUCUUUUCCAGGU SEQ ID NO 272 CAAGUGGGAUACUAGCAAUGUUAUC SEQ ID NO 246 UUUAGUUGCUGCUCUUUUCCAGGUU SEQ ID NO 273 AAGUGGGAUACUAGCAAUGUUAUCU SEQ ID NO 247 UUAGUUGCUGCUCUUUUCCAGGUUC SEQ ID NO 274 AGUGGGAUACUAGCAAUGUUAUCUG SEQ ID NO 248 UAGUUGCUGCUCUUUUCCAGGUUCA SEQ ID NO 275 GUGGGAUACUAGCAAUGUUAUCUGC SEQ ID NO 249 AGUUGCUGCUCUUUUCCAGGUUCAA SEQ ID NO 276 UGGGAUACUAGCAAUGUUAUCUGCU SEQ ID NO 250 GUUGCUGCUCUUUUCCAGGUUCAAG SEQ ID NO 277 GGGAUACUAGCAAUGUUAUCUGCUU SEQ ID NO 251 UUGCUGCUCUUUUCCAGGUUCAAGU SEQ ID NO 278 GGAUACUAGCAAUGUUAUCUGCUUC SEQ ID NO 252 UGCUGCUCUUUUCCAGGUUCAAGUG SEQ ID NO 279 GAUACUAGCAAUGUUAUCUGCUUCC SEQ ID NO 253 GCUGCUCUUUUCCAGGUUCAAGUGG SEQ ID NO 280 AUACUAGCAAUGUUAUCUGCUUCCU SEQ ID NO 251 CUGCUCUUUUCCAGGUUCAAGUGGG SEQ ID NO 281 UACUAGCAAUGUUAUCUGCUUCCUC SEQ ID NO 255 UGCUCUUUUCCAGGUUCAAGUGGGA SEQ ID NO 282 ACUAGCAAUGUUAUCUGCUUCCUCC SEQ ID NO 256 GCUCUUUUCCAGGUUCAAGUGGGAC SEQ ID NO 283 CUAGCAAUGUUAUCUGCUUCCUCCA SEQ ID NO 257 CUCUUUUCCAOOUUCAAOUGGGAUA SEQ ID NO 284 UAGCAAUGUUAUCUGCUUCCUCCAA SEQ ID NO 258 UCUUUUCCAGGUUCAAGUGGGAUAC SEQ ID NO 285 AGCAAUGUUAUCUGCUUCCUCCAAC SEQ ID NO 259 UCUUUUCCAGGUUCAAGUGG SEQ ID NO 286 GCAAUGUUAUCUGCUUCCUCCAACC SEQ ID NO 260 CUUUUCCAGGUUCAAGUGGGAUACU SEQ ID NO 287 CAAUGUUAUCUGCUUOCUCCAACCA SEQ ID NO 261 UUUUCCAGGUUCAAGUGGGAUACUA SEQ ID NO 288 AAUGUUAUCUGCUUCCUCCAACCAU SEQ ID NO 262 UUUCCAGGUUCAAGUGGGAUACUAG SEQ ID NO 289 AUGUUAUCUGCUUCCUCCAACCAUA SEQ ID NO 263 UUCCAGGUUCAAGUGGGAUACUAGC SEQ ID NO 290 UGUUAUGUGGDUCCUCCAACGAUAA SEQ ID NO 264 UCCAGGUUCAAGUGGGAUACUAGCA DMD Gene Exon 52 SEQ ID NO 291 AGCCUCUUGAUUGCUGGUCUUGUUU SEQ ID NO 326 UUGGGCAGCGGUAAUGAGUUCUUCC SEQ ID NO 292 GCCUCUUGAUUGCUGGUCUUGUUUU SEQ ID NO 327 UGGGCAGCGGUAAUGAGUUCUUCCA SEQ ID NO 293 CCUCUUGAUUGCUGGUCUUGUUUUU SEQ ID NO 328 GGGCAGCGGUAAUGAGUUCUUCCAA SEQ ID NO 294 CCUCUUGAUUGCUGGUCUUG SEQ ID NO 329 UGCAGCGGUAAUGAGUUCUUCCAAC SEQ ID NO 295 CUCUUGAUUGCUGGUCUUGUUUUUC SEQ ID NO 330 GCAGCGGUAAUGAGUUCUUCCAACU SEQ ID NO 296 UCUUGAUUGCUGGUCUUGUUUUUCA SEQ ID NO 331 CAGCGGUAAUGAGUUCUUCCAACUG SEQ ID NO 297 CUUGAUUGCUGGUCUUGUUUUUCAA SEQ ID NO 332 AGCGGUAAUGAGUUCUUCCAACUGG SEQ ID NO 298 UUGAUUGCUGGUCUUGUUUUUCAAA SEQ ID NO 333 GCGGUAAUGAGUUCUUCCAACUGGG SEQ ID NO 299 UGAUUGCUGGUCUUGUUUUUCAAAU SEQ ID NO 334 CGGUAAUGAGUUCUUCCAACUGGGG SEQ ID NO 300 GAUUGCUGGUCUUGUUUUUCAAAUU SEQ ID NO 335 GGUAAUGAGUUCUUCCAACUGGGGA SEQ ID NO 301 GAUUGCUGGUCUUGUUUUUC SEQ ID NO 336 GGUAAUGAGUUCUUCCAACUGG SEQ ID NO 302 AUUGCUGGUCUUGUUUUUCAAAUUU SEQ ID NO 337 GUAAUCAGUUCUUCCAACUGGGGAC SEQ ID NO 303 UUCCUCCUCUUGUUUUUCAAAUUUU SEQ ID NO 338 UAAUCAGUUCUUCCAACUGGGGACC SEQ ID NO 304 UGCUGGUCUUGUUUUUCAAAUUUUG SEQ ID NO 339 AAUGAGUUCUUCCAACUGGGGACGC SEQ ID NO 305 GCUGGUCUUGUUUUUGAAAUUUUGG SEQ ID NO 340 AUGAGUUCUUCCAACUGGGGACGCC SEQ ID NO 306 CUGGUCUUGUUUUUCAAAUUUUGGG SEQ ID NO 341 UGAGUUCUUCCAACUGGGGACGCCU SEQ ID NO 307 UGGUCUUGUUUUUCAAAUUUUGGGC SEQ ID NO 342 GAGUUCUUCCAACUGGGGACGCCUC SEQ ID NO 308 GGUCUUGUUUUUCAAAUUUUGGGCA SEQ ID NO 343 AGUUCUUCCAACUGGGGACGCCUCU SEQ ID NO 309 GUCUUGUUUUUCAAAUUUUGGGCAG SEQ ID NO 344 GUUCUUCCAACUGGGGACGCCUCUG SEQ ID NO 310 UCUUGUUUUUCAAAUUUUGGGCAGC SEQ ID NO 345 UUCUUCCAACUGGGGACGCCUCUGU SEQ ID NO 311 CUUGUUUUUCAAAUUUUGGGCAGCG SEQ ID NO 346 UCUUCCAACUGGGGACGCCUCUGUU SEQ ID NO 312 UUGUUUUUCAAAUUUUGGGCAGCGG SEQ ID NO 347 CUUCCAACUGGGGACGCCUCUGUUC SEQ ID NO 313 UGUUUUUCAAAUUUUGGGCAGCGGU SEQ ID NO 348 UUCCAACUGGGGACGCCUCUGUUCC SEQ ID NO 314 GUUUUUCAAAUUUUGGGCAGCGGUA SEQ ID NO 349 UCCAACUGGGGACGCCUCUGUUCCA SEQ ID NO 315 UUUUUCAAAUUUUGGGCAGCGGUAA SEQ ID NO 350 CCAACUGGGGACGCCUCUGUUCCAA SEQ ID NO 316 UUUUCAAAUUUUGGGCAGCGGUAAU SEQ ID NO 351 CAACUGGGGACGCCUCUGUUCCAAA SEQ ID NO 317 UUUCAAAUUUUGGGCAGCGGUAAUG SEQ ID NO 352 AACUGGGGACGCCUCUGUUCCAAAU SEQ ID NO 318 UUCAAAUUUUGGGCAGCGGUAAUGA SEQ ID NO 353 ACUCCGGACCCCUCUGUUCCAAAUC SEQ ID NO 319 UCAAAUUUUGGGCAGCGGUAAUGAG SEQ ID NO 354 CUGGGGACGCCUCUGUUCCAAAUCC SEQ ID NO 320 CAAAUUUUGGGCAGCGGUAAUGAGU SEQ ID NO 355 UGGGGACGCCUCUGUUCCAAAUCCU SEQ ID NO 321 AAAUUUUGGGCAGCGGUAAUGAGUU SEQ ID NO 356 GGGGACGCCUCUGUUCCAAAUCCUG SEQ ID NO 322 AAUUUUGGGCAGCGGUAAUGAGUUC SEQ ID NO 357 GGGACGCCUCUGUUCCAAAUCCUGC SEQ ID NO 323 AUUUUGGGCAGCGGUAAUGAGUUCU SEQ ID NO 358 GGACGCCUCUGUUCCAAAUCCUGCA SEQ ID NO 324 UUUUGGGCAGCGGUAAUGAGUUCUU SEQ ID NO 359 GACGCCUCUGUUCCAAAUCCUGCAU SEQ ID NO 325 UUUGGGCAGCGGUAAUGAGUUCUUC DMD Gene Exon 50 SEQ ID NO 360 CCAAUAGUGGUCAGUCCAGGAGCUA SEQ ID NO 386 CUAGGUCAGGCUGCUUUGCCCUCAG SEQ ID NO 361 CAAUAGUGGUCAGUCCAGGAGCUAG SEQ ID NO 387 UAGGUCAGGCUGCUUUGCCCUCAGC SEQ ID NO 362 AAUAGUGGUCAGUCCAGGAGCUAGG SEQ ID NO 388 AGGUCAGGCUGCUUUGCCCUCAGCU SEQ ID NO 363 AUAGUGGUCAGUCCAGGAGCUAGGU SEQ ID NO 389 GGUCAGGCUGCUUUGCCCUCAGCUC SEQ ID NO 364 AUAGUGGUCAGUCCAGGAGCU SEQ ID NO 390 GUCAGGCUGCUUUGCCCUCAGCUCU SEQ ID NO 365 UAGUGGUCAGUCCAGGAGCUAGGUC SEQ ID NO 391 UCAGGCUGCUUUGCCCUCAGCUCUU SEQ ID NO 366 AGUGGUCAGUCCAGGAGCUAGGUCA SEQ ID NO 392 CAGGCUGCUUUGCCCUCAGCUCUUG SEQ ID NO 367 GUGGUCAGUCCAGGAGCUAGGUCAG SEQ ID NO 393 AGGCUGCUUUGCCCUCAGCUCUUGA SEQ ID NO 368 UGGUCAGUCCAGGAGCUAGGUCAGG SEQ ID NO 394 GGCUGCUUUGCCCUCAGCUCUUGAA SEQ ID NO 369 GGUCAGUCCAGGAGCUAGGUCAGGC SEQ ID NO 395 GCUGCUUUGCCCUCAGCUCUUGAAG SEQ ID NO 370 GUCAGUCCAGGAGCUAGGUCAGGCU SEQ ID NO 396 CUGCUUUGCCCUCAGCUCUUGAAGU SEQ ID NO 371 UCAGUCCAGGAGCUAGGUCAGGCUG SEQ ID NO 397 UGCUUUGCCCUCAGCUCUUGAAGUA SEQ ID NO 372 CAGUCCAGGAGCUAGGUCAGGCUGC SEQ ID NO 398 GCUUUGCCCUCAGCUCUUGAAGUAA

SEQ ID NO 373 AGUCCAGGAGCUAGGUCAGGCUGCU SEQ ID NO 399 CUUUGCCCUCAGCUCUUGAAGUAAA SEQ ID NO 374 GUCCAGGAGCUAGGUCAGGCUGCUU SEQ ID NO 400 UUUGUCCUCAGCUCUUGAAGUAAAC SEQ ID NO 375 UCCAGGAGCUAGGUCAGGCUGCUUU SEQ ID NO 401 UUGCCCUCAGCUCUUGAAGUAAACG SEQ ID NO 376 CCAGGAGCUAGGUCAGGCUGCUUUG SEQ ID NO 402 UGCCCUCAGCUCUUGAAGUAAACGG SEQ ID NO 377 CAGGAGCUAGGUCAGGCUGCUUUGC SEQ ID NO 403 GCCCUCAGCUCUUGAAGUAAACGGU SEQ ID NO 378 AGGAGCUAGGUCAGGCUGCUUUGCC SEQ ID NO 404 CCCUCAGCUCUUGAAGUAAACGGUU SEQ ID NO 379 GGAGCUAGCUCAGGCUGCUUUGCCC SEQ ID NO 405 CCUCAGCUCUUGAACUAAAC SEQ ID NO 380 GAGCUAGGUCAGGCUGCUUUGCCCU SEQ ID NO 406 CCUCAGCUCUUGAAGUAAACG SEQ ID NO 381 AGCUAGGUCAGGCUGCUUUGCCCUC SEQ ID NO 407 CUCAGCUCUUGAAGUAAACG SEQ ID NO 382 GCUAGGUCAGGCUGCUUUGCCCUCA SEQ ID NO 408 CCUCAGCUCUUGAAGUAAACGGUUU SEQ ID NO 383 CUCAGCUCUUGAAGUAAACGGUUUA SEQ ID NO 409 UCAGCUCUUGAAGUAAACGGUUUAC SEQ ID NO 384 CAGCUCUUGAAGUAAACGGUUUACC SEQ ID NO 410 AGCUCUUGAAGUAAACGGUUUACCG SEQ ID NO 385 GCUCUUGAAGUAAACGGUUUACCGC SEQ ID NO 411 CUCUUGAAGUAAACGGUUUACCGCC DMD Gene Exon 43 SEQ ID NO 412 CCACAGGCGUUGCACUUUGCAAUGC SEQ ID NO 443 UCUUCUUGCUAUGAAUAAUGUCAAU SEQ ID NO 413 CACAGGCGUUGCACUUUGCAAUGCU SEQ ID NO 444 CUUCUUGCUAUGAAUAAUGUCAAUC SEQ ID NO 414 ACAGGCGUUGCACUUUGCAAUGCUG SEQ ID NO 445 UUCUUGCUAUGAAUAAUGUCAAUCC SEQ ID NO 415 CAGGCGUUGCACUUUGCAAUGCUGC SEQ ID NO 446 UCUUGCUAUGAAUAAUGUCAAUCCG SEQ ID NO 116 AGGCGUUGCACUUUGCAAUGCUGCU SEQ ID NO 447 CUUGCUAUGAAUAAUGUCAAUCCGA SEQ ID NO 417 GGCGUUGCACUUUGCAAUGCUGCUG SEQ ID NO 448 UUGCUAUGAAUAAUGUCAAUCCGAC SEQ ID NO 4IS GCGUUGCACUUUGCAAUGCUGCUGU SEQ ID NO 449 UGCUAUGAAUAAUGUCAAUCCGACC SEQ ID NO 419 CGUUGCACUUUGCAAUGCUGCUGUC SEQ ID NO 450 GCUAUGAAUAAUGUCAAUCCGACCU SEQ ID NO 420 CGUUGCACUUUGCAAUGCUGCUG SEQ ID NO 451 CUAUGAAUAAUGUCAAUCCGACCUG SEQ ID NO 421 GUUGCACUUUGCAAUGCUGCUGUCU SEQ ID NO 452 UAUGAAUAAUGUCAAUCCGACCUGA SEQ ID NO 422 UUGCACUUUGCAAUGCUGCUGUCUU SEQ ID NO 453 AUGAAUAAUGUCAAUCCGACCUGAG SEQ ID NO 423 UGCACUUUGCAAUGCUCCUGUCUUC SEQ ID NO 454 UGAAUAAUGUCAAUCCGACCUGAGC SEQ ID NO 424 GCACUUUGCAAUGCUGCUGUCUUCU SEQ ID NO 455 GAAUAAUGUCAAUCCGACCUGAGCU SEQ ID NO 425 CACUUUGCAAUGCUGCUGUCUUCUU SEQ ID NO 456 AAUAAUGUCAAUCCGACCUGAGCUU SEQ ID NO 426 ACUUUGCAAUGCUGCUGUCUUCUUG SEQ ID NO 457 AUAAUGUCAAUCCCACCUGACCUUU SEQ ID NO 427 CUUUGCAAUGCUGCUCUCUUCUUGC SEQ ID NO 458 UAAUGUCAAUCCGACCUGAGCUUUG SEQ ID NO 428 UUUGCAAUGCUGCUGUCUUCUUGCU SEQ ID NO 459 AAUGUCAAUCCGACCUGAGCUUUGU SEQ ID NO 429 UUGCAAUGCUGCUGUCUUCUUGCUA SEQ ID NO 460 AUGUCAAUCCGACCUGAGCUUUGUU SEQ ID NO 430 UGCAAUGCUGCUGUCUUCUUGCUAU SEQ ID NO 461 UGUCAAUCCGACCUGAGCUUUGUUG SEQ ID NO 431 GCAAUGCUGCUGUCUUCUUGCUAUG SEQ ID NO 462 GUCAAUCCGACCUGAGCUUUGUUGU SEQ ID NO 432 CAAUGCUGCUGUCUUCUUGCUAUGA SEQ ID NO 463 UCAAUCCGACCUGAGCUUUGUUGUA SEQ ID NO 433 AAUGCUGCUGUCUUCUUGCUAUGAA SEQ ID NO 464 CAAUCCGACCUGAGCUUUGUUGUAG SEQ ID NO 434 AUGCUGCUGUCUUCUUGCUAUGAAU SEQ ID NO 465 AAUCCGACCUGAGCUUUGUUGUAGA SEQ ID NO 435 UGCUGCUGUCUUCUUGCUAUGAAUA SEQ ID NO 466 AUCCGACCUGAGCUUUGUUGUAGAC SEQ ID NO 436 GCUGCUGUCUUCUUGCUAUGAAUAA SEQ ID NO 467 UCCGACCUGAGCUUUGUUGUAGACU SEQ ID NO 437 CUGCUGUCUUCUUGCUAUGAAUAAU SEQ ID NO 468 CCGACCUGAGCUUUGUUCUAGACUA SEQ ID NO 438 UGCUGUCUUCUUGCUAUGAAUAAUG SEQ ID NO 469 CGACCUGAGCUUUGUUGUAG SEQ ID NO 439 GCUGUCUUCUUGCUAUGAAUAAUGU SEQ ID NO 470 CGACCUGAGCUUUGUUGUAGACUAU SEQ ID NO 440 CUGUCUUCUUGCUAUGAAUAAUGUC SEQ ID NO 471 GACCUGAGCUUUGUUGUAGACUAUC SEQ ID NO 441 UGUCUUCUUGCUAUGAAUAAUGUCA SEQ ID NO 472 ACCUGAGCUUUGUUGUAGACUAUCA SEQ ID NO 442 GUCUUCUUGCUAUGAAUAAUGUCAA SEQ ID NO 473 CCUGA GCUUU GUUGU AGACU AUC DMD Gene Exon 6 SEQ ID NO 474 CAUUUUUGACCUACAUGUGG SEQ ID NO 479 AUUUUUGACCUACAUGGGAAAG SEQ ID NO 475 UUUGACCUACAUGUGGAAAG SEQ ID NO 480 UACGAGUUGAUUGUCGGACCCAG SEQ ID NO 476 UACAUUUUUGACCUACAUGUGGAAAG SEQ ID NO 481 GUGGUCUCCUUACCUAUGACUGUGG SEQ ID NO 477 GGUCUCCUUACCUAUGA SEQ ID NO 482 UGUCUCAGUAAUCUUCUUACCUAU SEQ ID NO 478 UCUUACCUAUGACUAUGGAUGAGA DMD Gene Exon 7 SEQ ID NO 483 UGCAUGUUCCAGUCGUUGUGUGG SEQ ID NO 485 AUUUACCAACCUUCAGGAUCGAGUA SEQ ID NO 484 CACUAUUCCAGUCAAAUAGGUCUGG SEQ ID NO 486 GGCCUAAAACACAUACACAUA DMD Gene Exon 8 SEQ ID NO 487 GAUAGGUGGUAUCAACAUCUGUAA SEQ ID NO 490 UGUUGUUGUUUAUGCUCAUU SEQ ID NO 488 GAUAGGUGGUAUCAACAUCUG SEQ ID NO 491 GUACAUUAAGAUGGACUUC SEQ ID NO 489 CUUCCUGGAUGGCUUGAAU DMD Gene Exon 55 SEQ ID NO 492 CUGUUGCAGUAAUCUAUGAC SEQ ID NO 495 UCCCAUUCUUUCAUCACCUCUUU SEQ ID NO 493 UGCAGUAAUCUAUGAGUUUC SEQ ID NO 496 UCCUGUACGACAUUGGCAGU SEQ ID NO 494 GAGUCUUCUAGGAGCCUU SEQ ID NO 497 CUUGGAGUCUUCUAGGAGCC DMD Gene Exon 2 SEQ ID NO 498 CCAUUUUGUGAAUGUUUUCUUUUGAACAUC SEQ ID NO 500 GAAAAUUGUGCAUUUACCCAUUUU SEQ ID NO 499 CCCAUUUUGUGAAUGUUUUCUUUU SEQ ID NO 501 UUGUGCAUUUACCCAUUUUGUG DMD Gene Exon 11 SEQID NO 502 CCCUGAGGCAUUCCCAUCUUGAAU SEQ ID NO 504 CUUGAAUUUAGGAGAUUCAUCUG SEQID NO 503 AGGACUUACUUGCUUUGUUU SEQ ID NO 505 CAUCUUCUGAUAAUUUUCCUGUU DMD Gene Exon 17 SEQ ID NO 506 CCAUUACAGUUGUCUGUGUU SEQ ID NO 508 UAAUCUGCCUCUUCUUUUGG SEQ ID NO 507 UGACAGCCUGUGAAAUCUGUGAG DMD Gene Exon 19 SEQ ID NO 509 CAGCAGUAGUUGUCAUCUGC SEQ ID NO 511 GCCUGAGCUGAUCUGCUGGCAUCUUGCAGUU SEQ ID NO 510 GCCUGAGCUGAUCUGCUGGCAUCUUGC SEQ ID NO 512 UCUGCUGGCAUCUUGC DMD Gene Exon 21 SEQ ID NO 513 GCCGGUUGACUUCAUCCUGUGC SEQ ID NO 516 CUGCAUCCAGGAACAUGGGUCC SEQ ID NO 514 GUCUGCAUCCAGGAACAUGGGUC SEQ ID NO 517 GUUGAAGAUCUGAUAGCCGGUUGA SEQ ID NO 515 UACUUACUGUCUGUAGCUCUUUCU DMD Gene Exon 57 SEQ ID NO 518 UAGGUGCCUGCCGGCUU SEQ ID NO 520 CUGAACUGCUGGAAAGUCGCC SEQ ID NO 519 UUCAGCUGUAGCCACACC SEQ ID NO 521 CUGGCUUCCAAAUGGGACCUGAAAAAGAAC DMD Gene Exon 59 SEQ ID NO 522 CAAUUUUUCCCACUCAGUAUU SEQ ID NO 521 UCCUCAGGAGGCAGCUCUAAAU SEQ ID NO 523 UUGAAGUUCCUGGAGUCUU DMD Gene Exon 62 SEQ ID NO 525 UGGCUCUCUCCCAGGG SEQ ID NO 527 GCGCACUUUGUUUGGCG SEQ ID NO 526 GAGAUGGCUCUCUCCCAGGGACCCUGG DMD Gene Exon 63 SEQ ID NO 528 GGUCCCAGCAAGUUGUUUG SEQ ID NO 530 GUAGAGCUCUGUCAUUUUGGG SEQ ID NO 529 UGGGAUGGUCCCAGCAAGUUGUUUG DMD Gene Exon 65 SEQ ID NO 531 GCUCAAGAGAUCCACUGCAAAAAAC SEQ ID NO 533 UCUGCAGGAUAUCCAUGGGCUGGUC SEQ ID NO 532 GCCAUACGUACGUAUCAUAAACAUUC DMD Gene Exon 66 SEQ ID NO 534 GAUCCUCCCUGUUCGUCCCCUAUUAUG DMD Gene Exon 69 SEQ ID NO 535 UGCUUUAGACUCCUGUACCUGAUA DMD Gene Exon 75 SEQ ID NO 536 GGCGGCCUUUGUGUUGAC SEQ ID NO 538 CCUUUAUGUUCGUGCUGCU SEQ ID NO 587 GGACAGGCCUUUAUGUUCGUGCUGC Human IGF-1 Isoform 4 amino acid sequence SEQ ID NO 577: MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELV DALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA RSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM

Sequence CWU 1

1

57713685PRTHomo sapiens 1Met Leu Trp Trp Glu Glu Val Glu Asp Cys Tyr Glu Arg Glu Asp Val 1 5 10 15 Gln Lys Lys Thr Phe Thr Lys Trp Val Asn Ala Gln Phe Ser Lys Phe 20 25 30 Gly Lys Gln His Ile Glu Asn Leu Phe Ser Asp Leu Gln Asp Gly Arg 35 40 45 Arg Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln Lys Leu Pro Lys 50 55 60 Glu Lys Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Lys Ala 65 70 75 80 Leu Arg Val Leu Gln Asn Asn Asn Val Asp Leu Val Asn Ile Gly Ser 85 90 95 Thr Asp Ile Val Asp Gly Asn His Lys Leu Thr Leu Gly Leu Ile Trp 100 105 110 Asn Ile Ile Leu His Trp Gln Val Lys Asn Val Met Lys Asn Ile Met 115 120 125 Ala Gly Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu Ser Trp Val 130 135 140 Arg Gln Ser Thr Arg Asn Tyr Pro Gln Val Asn Val Ile Asn Phe Thr 145 150 155 160 Thr Ser Trp Ser Asp Gly Leu Ala Leu Asn Ala Leu Ile His Ser His 165 170 175 Arg Pro Asp Leu Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala 180 185 190 Thr Gln Arg Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly 195 200 205 Ile Glu Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp 210 215 220 Lys Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu Pro 225 230 235 240 Gln Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu Met Leu Pro Arg 245 250 255 Pro Pro Lys Val Thr Lys Glu Glu His Phe Gln Leu His His Gln Met 260 265 270 His Tyr Ser Gln Gln Ile Thr Val Ser Leu Ala Gln Gly Tyr Glu Arg 275 280 285 Thr Ser Ser Pro Lys Pro Arg Phe Lys Ser Tyr Ala Tyr Thr Gln Ala 290 295 300 Ala Tyr Val Thr Thr Ser Asp Pro Thr Arg Ser Pro Phe Pro Ser Gln 305 310 315 320 His Leu Glu Ala Pro Glu Asp Lys Ser Phe Gly Ser Ser Leu Met Glu 325 330 335 Ser Glu Val Asn Leu Asp Arg Tyr Gln Thr Ala Leu Glu Glu Val Leu 340 345 350 Ser Trp Leu Leu Ser Ala Glu Asp Thr Leu Gln Ala Gln Gly Glu Ile 355 360 365 Ser Asn Asp Val Glu Val Val Lys Asp Gln Phe His Thr His Glu Gly 370 375 380 Tyr Met Met Asp Leu Thr Ala His Gln Gly Arg Val Gly Asn Ile Leu 385 390 395 400 Gln Leu Gly Ser Lys Leu Ile Gly Thr Gly Lys Leu Ser Glu Asp Glu 405 410 415 Glu Thr Glu Val Gln Glu Gln Met Asn Leu Leu Asn Ser Arg Trp Glu 420 425 430 Cys Leu Arg Val Ala Ser Met Glu Lys Gln Ser Asn Leu His Arg Val 435 440 445 Leu Met Asp Leu Gln Asn Gln Lys Leu Lys Glu Leu Asn Asp Trp Leu 450 455 460 Thr Lys Thr Glu Glu Arg Thr Arg Lys Met Glu Glu Glu Pro Leu Gly 465 470 475 480 Pro Asp Leu Glu Asp Leu Lys Arg Gln Val Gln Gln His Lys Val Leu 485 490 495 Gln Glu Asp Leu Glu Gln Glu Gln Val Arg Val Asn Ser Leu Thr His 500 505 510 Met Val Val Val Val Asp Glu Ser Ser Gly Asp His Ala Thr Ala Ala 515 520 525 Leu Glu Glu Gln Leu Lys Val Leu Gly Asp Arg Trp Ala Asn Ile Cys 530 535 540 Arg Trp Thr Glu Asp Arg Trp Val Leu Leu Gln Asp Ile Leu Leu Lys 545 550 555 560 Trp Gln Arg Leu Thr Glu Glu Gln Cys Leu Phe Ser Ala Trp Leu Ser 565 570 575 Glu Lys Glu Asp Ala Val Asn Lys Ile His Thr Thr Gly Phe Lys Asp 580 585 590 Gln Asn Glu Met Leu Ser Ser Leu Gln Lys Leu Ala Val Leu Lys Ala 595 600 605 Asp Leu Glu Lys Lys Lys Gln Ser Met Gly Lys Leu Tyr Ser Leu Lys 610 615 620 Gln Asp Leu Leu Ser Thr Leu Lys Asn Lys Ser Val Thr Gln Lys Thr 625 630 635 640 Glu Ala Trp Leu Asp Asn Phe Ala Arg Cys Trp Asp Asn Leu Val Gln 645 650 655 Lys Leu Glu Lys Ser Thr Ala Gln Ile Ser Gln Ala Val Thr Thr Thr 660 665 670 Gln Pro Ser Leu Thr Gln Thr Thr Val Met Glu Thr Val Thr Thr Val 675 680 685 Thr Thr Arg Glu Gln Ile Leu Val Lys His Ala Gln Glu Glu Leu Pro 690 695 700 Pro Pro Pro Pro Gln Lys Lys Arg Gln Ile Thr Val Asp Ser Glu Ile 705 710 715 720 Arg Lys Arg Leu Asp Val Asp Ile Thr Glu Leu His Ser Trp Ile Thr 725 730 735 Arg Ser Glu Ala Val Leu Gln Ser Pro Glu Phe Ala Ile Phe Arg Lys 740 745 750 Glu Gly Asn Phe Ser Asp Leu Lys Glu Lys Val Asn Ala Ile Glu Arg 755 760 765 Glu Lys Ala Glu Lys Phe Arg Lys Leu Gln Asp Ala Ser Arg Ser Ala 770 775 780 Gln Ala Leu Val Glu Gln Met Val Asn Glu Gly Val Asn Ala Asp Ser 785 790 795 800 Ile Lys Gln Ala Ser Glu Gln Leu Asn Ser Arg Trp Ile Glu Phe Cys 805 810 815 Gln Leu Leu Ser Glu Arg Leu Asn Trp Leu Glu Tyr Gln Asn Asn Ile 820 825 830 Ile Ala Phe Tyr Asn Gln Leu Gln Gln Leu Glu Gln Met Thr Thr Thr 835 840 845 Ala Glu Asn Trp Leu Lys Ile Gln Pro Thr Thr Pro Ser Glu Pro Thr 850 855 860 Ala Ile Lys Ser Gln Leu Lys Ile Cys Lys Asp Glu Val Asn Arg Leu 865 870 875 880 Ser Gly Leu Gln Pro Gln Ile Glu Arg Leu Lys Ile Gln Ser Ile Ala 885 890 895 Leu Lys Glu Lys Gly Gln Gly Pro Met Phe Leu Asp Ala Asp Phe Val 900 905 910 Ala Phe Thr Asn His Phe Lys Gln Val Phe Ser Asp Val Gln Ala Arg 915 920 925 Glu Lys Glu Leu Gln Thr Ile Phe Asp Thr Leu Pro Pro Met Arg Tyr 930 935 940 Gln Glu Thr Met Ser Ala Ile Arg Thr Trp Val Gln Gln Ser Glu Thr 945 950 955 960 Lys Leu Ser Ile Pro Gln Leu Ser Val Thr Asp Tyr Glu Ile Met Glu 965 970 975 Gln Arg Leu Gly Glu Leu Gln Ala Leu Gln Ser Ser Leu Gln Glu Gln 980 985 990 Gln Ser Gly Leu Tyr Tyr Leu Ser Thr Thr Val Lys Glu Met Ser Lys 995 1000 1005 Lys Ala Pro Ser Glu Ile Ser Arg Lys Tyr Gln Ser Glu Phe Glu 1010 1015 1020 Glu Ile Glu Gly Arg Trp Lys Lys Leu Ser Ser Gln Leu Val Glu 1025 1030 1035 His Cys Gln Lys Leu Glu Glu Gln Met Asn Lys Leu Arg Lys Ile 1040 1045 1050 Gln Asn His Ile Gln Thr Leu Lys Lys Trp Met Ala Glu Val Asp 1055 1060 1065 Val Phe Leu Lys Glu Glu Trp Pro Ala Leu Gly Asp Ser Glu Ile 1070 1075 1080 Leu Lys Lys Gln Leu Lys Gln Cys Arg Leu Leu Val Ser Asp Ile 1085 1090 1095 Gln Thr Ile Gln Pro Ser Leu Asn Ser Val Asn Glu Gly Gly Gln 1100 1105 1110 Lys Ile Lys Asn Glu Ala Glu Pro Glu Phe Ala Ser Arg Leu Glu 1115 1120 1125 Thr Glu Leu Lys Glu Leu Asn Thr Gln Trp Asp His Met Cys Gln 1130 1135 1140 Gln Val Tyr Ala Arg Lys Glu Ala Leu Lys Gly Gly Leu Glu Lys 1145 1150 1155 Thr Val Ser Leu Gln Lys Asp Leu Ser Glu Met His Glu Trp Met 1160 1165 1170 Thr Gln Ala Glu Glu Glu Tyr Leu Glu Arg Asp Phe Glu Tyr Lys 1175 1180 1185 Thr Pro Asp Glu Leu Gln Lys Ala Val Glu Glu Met Lys Arg Ala 1190 1195 1200 Lys Glu Glu Ala Gln Gln Lys Glu Ala Lys Val Lys Leu Leu Thr 1205 1210 1215 Glu Ser Val Asn Ser Val Ile Ala Gln Ala Pro Pro Val Ala Gln 1220 1225 1230 Glu Ala Leu Lys Lys Glu Leu Glu Thr Leu Thr Thr Asn Tyr Gln 1235 1240 1245 Trp Leu Cys Thr Arg Leu Asn Gly Lys Cys Lys Thr Leu Glu Glu 1250 1255 1260 Val Trp Ala Cys Trp His Glu Leu Leu Ser Tyr Leu Glu Lys Ala 1265 1270 1275 Asn Lys Trp Leu Asn Glu Val Glu Phe Lys Leu Lys Thr Thr Glu 1280 1285 1290 Asn Ile Pro Gly Gly Ala Glu Glu Ile Ser Glu Val Leu Asp Ser 1295 1300 1305 Leu Glu Asn Leu Met Arg His Ser Glu Asp Asn Pro Asn Gln Ile 1310 1315 1320 Arg Ile Leu Ala Gln Thr Leu Thr Asp Gly Gly Val Met Asp Glu 1325 1330 1335 Leu Ile Asn Glu Glu Leu Glu Thr Phe Asn Ser Arg Trp Arg Glu 1340 1345 1350 Leu His Glu Glu Ala Val Arg Arg Gln Lys Leu Leu Glu Gln Ser 1355 1360 1365 Ile Gln Ser Ala Gln Glu Thr Glu Lys Ser Leu His Leu Ile Gln 1370 1375 1380 Glu Ser Leu Thr Phe Ile Asp Lys Gln Leu Ala Ala Tyr Ile Ala 1385 1390 1395 Asp Lys Val Asp Ala Ala Gln Met Pro Gln Glu Ala Gln Lys Ile 1400 1405 1410 Gln Ser Asp Leu Thr Ser His Glu Ile Ser Leu Glu Glu Met Lys 1415 1420 1425 Lys His Asn Gln Gly Lys Glu Ala Ala Gln Arg Val Leu Ser Gln 1430 1435 1440 Ile Asp Val Ala Gln Lys Lys Leu Gln Asp Val Ser Met Lys Phe 1445 1450 1455 Arg Leu Phe Gln Lys Pro Ala Asn Phe Glu Gln Arg Leu Gln Glu 1460 1465 1470 Ser Lys Met Ile Leu Asp Glu Val Lys Met His Leu Pro Ala Leu 1475 1480 1485 Glu Thr Lys Ser Val Glu Gln Glu Val Val Gln Ser Gln Leu Asn 1490 1495 1500 His Cys Val Asn Leu Tyr Lys Ser Leu Ser Glu Val Lys Ser Glu 1505 1510 1515 Val Glu Met Val Ile Lys Thr Gly Arg Gln Ile Val Gln Lys Lys 1520 1525 1530 Gln Thr Glu Asn Pro Lys Glu Leu Asp Glu Arg Val Thr Ala Leu 1535 1540 1545 Lys Leu His Tyr Asn Glu Leu Gly Ala Lys Val Thr Glu Arg Lys 1550 1555 1560 Gln Gln Leu Glu Lys Cys Leu Lys Leu Ser Arg Lys Met Arg Lys 1565 1570 1575 Glu Met Asn Val Leu Thr Glu Trp Leu Ala Ala Thr Asp Met Glu 1580 1585 1590 Leu Thr Lys Arg Ser Ala Val Glu Gly Met Pro Ser Asn Leu Asp 1595 1600 1605 Ser Glu Val Ala Trp Gly Lys Ala Thr Gln Lys Glu Ile Glu Lys 1610 1615 1620 Gln Lys Val His Leu Lys Ser Ile Thr Glu Val Gly Glu Ala Leu 1625 1630 1635 Lys Thr Val Leu Gly Lys Lys Glu Thr Leu Val Glu Asp Lys Leu 1640 1645 1650 Ser Leu Leu Asn Ser Asn Trp Ile Ala Val Thr Ser Arg Ala Glu 1655 1660 1665 Glu Trp Leu Asn Leu Leu Leu Glu Tyr Gln Lys His Met Glu Thr 1670 1675 1680 Phe Asp Gln Asn Val Asp His Ile Thr Lys Trp Ile Ile Gln Ala 1685 1690 1695 Asp Thr Leu Leu Asp Glu Ser Glu Lys Lys Lys Pro Gln Gln Lys 1700 1705 1710 Glu Asp Val Leu Lys Arg Leu Lys Ala Glu Leu Asn Asp Ile Arg 1715 1720 1725 Pro Lys Val Asp Ser Thr Arg Asp Gln Ala Ala Asn Leu Met Ala 1730 1735 1740 Asn Arg Gly Asp His Cys Arg Lys Leu Val Glu Pro Gln Ile Ser 1745 1750 1755 Glu Leu Asn His Arg Phe Ala Ala Ile Ser His Arg Ile Lys Thr 1760 1765 1770 Gly Lys Ala Ser Ile Pro Leu Lys Glu Leu Glu Gln Phe Asn Ser 1775 1780 1785 Asp Ile Gln Lys Leu Leu Glu Pro Leu Glu Ala Glu Ile Gln Gln 1790 1795 1800 Gly Val Asn Leu Lys Glu Glu Asp Phe Asn Lys Asp Met Asn Glu 1805 1810 1815 Asp Asn Glu Gly Thr Val Lys Glu Leu Leu Gln Arg Gly Asp Asn 1820 1825 1830 Leu Gln Gln Arg Ile Thr Asp Glu Arg Lys Arg Glu Glu Ile Lys 1835 1840 1845 Ile Lys Gln Gln Leu Leu Gln Thr Lys His Asn Ala Leu Lys Asp 1850 1855 1860 Leu Arg Ser Gln Arg Arg Lys Lys Ala Leu Glu Ile Ser His Gln 1865 1870 1875 Trp Tyr Gln Tyr Lys Arg Gln Ala Asp Asp Leu Leu Lys Cys Leu 1880 1885 1890 Asp Asp Ile Glu Lys Lys Leu Ala Ser Leu Pro Glu Pro Arg Asp 1895 1900 1905 Glu Arg Lys Ile Lys Glu Ile Asp Arg Glu Leu Gln Lys Lys Lys 1910 1915 1920 Glu Glu Leu Asn Ala Val Arg Arg Gln Ala Glu Gly Leu Ser Glu 1925 1930 1935 Asp Gly Ala Ala Met Ala Val Glu Pro Thr Gln Ile Gln Leu Ser 1940 1945 1950 Lys Arg Trp Arg Glu Ile Glu Ser Lys Phe Ala Gln Phe Arg Arg 1955 1960 1965 Leu Asn Phe Ala Gln Ile His Thr Val Arg Glu Glu Thr Met Met 1970 1975 1980 Val Met Thr Glu Asp Met Pro Leu Glu Ile Ser Tyr Val Pro Ser 1985 1990 1995 Thr Tyr Leu Thr Glu Ile Thr His Val Ser Gln Ala Leu Leu Glu 2000 2005 2010 Val Glu Gln Leu Leu Asn Ala Pro Asp Leu Cys Ala Lys Asp Phe 2015 2020 2025 Glu Asp Leu Phe Lys Gln Glu Glu Ser Leu Lys Asn Ile Lys Asp 2030 2035 2040 Ser Leu Gln Gln Ser Ser Gly Arg Ile Asp Ile Ile His Ser Lys 2045 2050 2055 Lys Thr Ala Ala Leu Gln Ser Ala Thr Pro Val Glu Arg Val Lys 2060 2065 2070 Leu Gln Glu Ala Leu Ser Gln Leu Asp Phe Gln Trp Glu Lys Val 2075 2080 2085 Asn Lys Met Tyr Lys Asp Arg Gln Gly Arg Phe Asp Arg Ser Val 2090 2095 2100 Glu Lys Trp Arg Arg Phe His Tyr Asp Ile Lys Ile Phe Asn Gln 2105 2110 2115 Trp Leu Thr Glu Ala Glu Gln Phe Leu Arg Lys Thr Gln Ile Pro 2120 2125 2130 Glu Asn Trp Glu His Ala Lys Tyr Lys Trp Tyr Leu Lys Glu Leu 2135 2140 2145 Gln Asp Gly Ile Gly Gln Arg Gln Thr Val Val Arg Thr Leu Asn 2150 2155 2160 Ala Thr Gly Glu Glu Ile Ile Gln Gln Ser Ser Lys Thr Asp Ala 2165 2170 2175 Ser Ile Leu Gln Glu Lys Leu Gly Ser Leu Asn Leu Arg Trp Gln 2180 2185 2190 Glu Val Cys Lys Gln Leu Ser Asp Arg Lys Lys Arg Leu Glu Glu 2195 2200 2205 Gln Lys Asn Ile Leu Ser Glu Phe Gln Arg Asp Leu Asn Glu Phe 2210 2215 2220 Val Leu Trp Leu Glu Glu Ala Asp Asn Ile Ala Ser Ile Pro Leu 2225 2230 2235 Glu Pro Gly Lys Glu Gln Gln

Leu Lys Glu Lys Leu Glu Gln Val 2240 2245 2250 Lys Leu Leu Val Glu Glu Leu Pro Leu Arg Gln Gly Ile Leu Lys 2255 2260 2265 Gln Leu Asn Glu Thr Gly Gly Pro Val Leu Val Ser Ala Pro Ile 2270 2275 2280 Ser Pro Glu Glu Gln Asp Lys Leu Glu Asn Lys Leu Lys Gln Thr 2285 2290 2295 Asn Leu Gln Trp Ile Lys Val Ser Arg Ala Leu Pro Glu Lys Gln 2300 2305 2310 Gly Glu Ile Glu Ala Gln Ile Lys Asp Leu Gly Gln Leu Glu Lys 2315 2320 2325 Lys Leu Glu Asp Leu Glu Glu Gln Leu Asn His Leu Leu Leu Trp 2330 2335 2340 Leu Ser Pro Ile Arg Asn Gln Leu Glu Ile Tyr Asn Gln Pro Asn 2345 2350 2355 Gln Glu Gly Pro Phe Asp Val Gln Glu Thr Glu Ile Ala Val Gln 2360 2365 2370 Ala Lys Gln Pro Asp Val Glu Glu Ile Leu Ser Lys Gly Gln His 2375 2380 2385 Leu Tyr Lys Glu Lys Pro Ala Thr Gln Pro Val Lys Arg Lys Leu 2390 2395 2400 Glu Asp Leu Ser Ser Glu Trp Lys Ala Val Asn Arg Leu Leu Gln 2405 2410 2415 Glu Leu Arg Ala Lys Gln Pro Asp Leu Ala Pro Gly Leu Thr Thr 2420 2425 2430 Ile Gly Ala Ser Pro Thr Gln Thr Val Thr Leu Val Thr Gln Pro 2435 2440 2445 Val Val Thr Lys Glu Thr Ala Ile Ser Lys Leu Glu Met Pro Ser 2450 2455 2460 Ser Leu Met Leu Glu Val Pro Ala Leu Ala Asp Phe Asn Arg Ala 2465 2470 2475 Trp Thr Glu Leu Thr Asp Trp Leu Ser Leu Leu Asp Gln Val Ile 2480 2485 2490 Lys Ser Gln Arg Val Met Val Gly Asp Leu Glu Asp Ile Asn Glu 2495 2500 2505 Met Ile Ile Lys Gln Lys Ala Thr Met Gln Asp Leu Glu Gln Arg 2510 2515 2520 Arg Pro Gln Leu Glu Glu Leu Ile Thr Ala Ala Gln Asn Leu Lys 2525 2530 2535 Asn Lys Thr Ser Asn Gln Glu Ala Arg Thr Ile Ile Thr Asp Arg 2540 2545 2550 Ile Glu Arg Ile Gln Asn Gln Trp Asp Glu Val Gln Glu His Leu 2555 2560 2565 Gln Asn Arg Arg Gln Gln Leu Asn Glu Met Leu Lys Asp Ser Thr 2570 2575 2580 Gln Trp Leu Glu Ala Lys Glu Glu Ala Glu Gln Val Leu Gly Gln 2585 2590 2595 Ala Arg Ala Lys Leu Glu Ser Trp Lys Glu Gly Pro Tyr Thr Val 2600 2605 2610 Asp Ala Ile Gln Lys Lys Ile Thr Glu Thr Lys Gln Leu Ala Lys 2615 2620 2625 Asp Leu Arg Gln Trp Gln Thr Asn Val Asp Val Ala Asn Asp Leu 2630 2635 2640 Ala Leu Lys Leu Leu Arg Asp Tyr Ser Ala Asp Asp Thr Arg Lys 2645 2650 2655 Val His Met Ile Thr Glu Asn Ile Asn Ala Ser Trp Arg Ser Ile 2660 2665 2670 His Lys Arg Val Ser Glu Arg Glu Ala Ala Leu Glu Glu Thr His 2675 2680 2685 Arg Leu Leu Gln Gln Phe Pro Leu Asp Leu Glu Lys Phe Leu Ala 2690 2695 2700 Trp Leu Thr Glu Ala Glu Thr Thr Ala Asn Val Leu Gln Asp Ala 2705 2710 2715 Thr Arg Lys Glu Arg Leu Leu Glu Asp Ser Lys Gly Val Lys Glu 2720 2725 2730 Leu Met Lys Gln Trp Gln Asp Leu Gln Gly Glu Ile Glu Ala His 2735 2740 2745 Thr Asp Val Tyr His Asn Leu Asp Glu Asn Ser Gln Lys Ile Leu 2750 2755 2760 Arg Ser Leu Glu Gly Ser Asp Asp Ala Val Leu Leu Gln Arg Arg 2765 2770 2775 Leu Asp Asn Met Asn Phe Lys Trp Ser Glu Leu Arg Lys Lys Ser 2780 2785 2790 Leu Asn Ile Arg Ser His Leu Glu Ala Ser Ser Asp Gln Trp Lys 2795 2800 2805 Arg Leu His Leu Ser Leu Gln Glu Leu Leu Val Trp Leu Gln Leu 2810 2815 2820 Lys Asp Asp Glu Leu Ser Arg Gln Ala Pro Ile Gly Gly Asp Phe 2825 2830 2835 Pro Ala Val Gln Lys Gln Asn Asp Val His Arg Ala Phe Lys Arg 2840 2845 2850 Glu Leu Lys Thr Lys Glu Pro Val Ile Met Ser Thr Leu Glu Thr 2855 2860 2865 Val Arg Ile Phe Leu Thr Glu Gln Pro Leu Glu Gly Leu Glu Lys 2870 2875 2880 Leu Tyr Gln Glu Pro Arg Glu Leu Pro Pro Glu Glu Arg Ala Gln 2885 2890 2895 Asn Val Thr Arg Leu Leu Arg Lys Gln Ala Glu Glu Val Asn Thr 2900 2905 2910 Glu Trp Glu Lys Leu Asn Leu His Ser Ala Asp Trp Gln Arg Lys 2915 2920 2925 Ile Asp Glu Thr Leu Glu Arg Leu Gln Glu Leu Gln Glu Ala Thr 2930 2935 2940 Asp Glu Leu Asp Leu Lys Leu Arg Gln Ala Glu Val Ile Lys Gly 2945 2950 2955 Ser Trp Gln Pro Val Gly Asp Leu Leu Ile Asp Ser Leu Gln Asp 2960 2965 2970 His Leu Glu Lys Val Lys Ala Leu Arg Gly Glu Ile Ala Pro Leu 2975 2980 2985 Lys Glu Asn Val Ser His Val Asn Asp Leu Ala Arg Gln Leu Thr 2990 2995 3000 Thr Leu Gly Ile Gln Leu Ser Pro Tyr Asn Leu Ser Thr Leu Glu 3005 3010 3015 Asp Leu Asn Thr Arg Trp Lys Leu Leu Gln Val Ala Val Glu Asp 3020 3025 3030 Arg Val Arg Gln Leu His Glu Ala His Arg Asp Phe Gly Pro Ala 3035 3040 3045 Ser Gln His Phe Leu Ser Thr Ser Val Gln Gly Pro Trp Glu Arg 3050 3055 3060 Ala Ile Ser Pro Asn Lys Val Pro Tyr Tyr Ile Asn His Glu Thr 3065 3070 3075 Gln Thr Thr Cys Trp Asp His Pro Lys Met Thr Glu Leu Tyr Gln 3080 3085 3090 Ser Leu Ala Asp Leu Asn Asn Val Arg Phe Ser Ala Tyr Arg Thr 3095 3100 3105 Ala Met Lys Leu Arg Arg Leu Gln Lys Ala Leu Cys Leu Asp Leu 3110 3115 3120 Leu Ser Leu Ser Ala Ala Cys Asp Ala Leu Asp Gln His Asn Leu 3125 3130 3135 Lys Gln Asn Asp Gln Pro Met Asp Ile Leu Gln Ile Ile Asn Cys 3140 3145 3150 Leu Thr Thr Ile Tyr Asp Arg Leu Glu Gln Glu His Asn Asn Leu 3155 3160 3165 Val Asn Val Pro Leu Cys Val Asp Met Cys Leu Asn Trp Leu Leu 3170 3175 3180 Asn Val Tyr Asp Thr Gly Arg Thr Gly Arg Ile Arg Val Leu Ser 3185 3190 3195 Phe Lys Thr Gly Ile Ile Ser Leu Cys Lys Ala His Leu Glu Asp 3200 3205 3210 Lys Tyr Arg Tyr Leu Phe Lys Gln Val Ala Ser Ser Thr Gly Phe 3215 3220 3225 Cys Asp Gln Arg Arg Leu Gly Leu Leu Leu His Asp Ser Ile Gln 3230 3235 3240 Ile Pro Arg Gln Leu Gly Glu Val Ala Ser Phe Gly Gly Ser Asn 3245 3250 3255 Ile Glu Pro Ser Val Arg Ser Cys Phe Gln Phe Ala Asn Asn Lys 3260 3265 3270 Pro Glu Ile Glu Ala Ala Leu Phe Leu Asp Trp Met Arg Leu Glu 3275 3280 3285 Pro Gln Ser Met Val Trp Leu Pro Val Leu His Arg Val Ala Ala 3290 3295 3300 Ala Glu Thr Ala Lys His Gln Ala Lys Cys Asn Ile Cys Lys Glu 3305 3310 3315 Cys Pro Ile Ile Gly Phe Arg Tyr Arg Ser Leu Lys His Phe Asn 3320 3325 3330 Tyr Asp Ile Cys Gln Ser Cys Phe Phe Ser Gly Arg Val Ala Lys 3335 3340 3345 Gly His Lys Met His Tyr Pro Met Val Glu Tyr Cys Thr Pro Thr 3350 3355 3360 Thr Ser Gly Glu Asp Val Arg Asp Phe Ala Lys Val Leu Lys Asn 3365 3370 3375 Lys Phe Arg Thr Lys Arg Tyr Phe Ala Lys His Pro Arg Met Gly 3380 3385 3390 Tyr Leu Pro Val Gln Thr Val Leu Glu Gly Asp Asn Met Glu Thr 3395 3400 3405 Pro Val Thr Leu Ile Asn Phe Trp Pro Val Asp Ser Ala Pro Ala 3410 3415 3420 Ser Ser Pro Gln Leu Ser His Asp Asp Thr His Ser Arg Ile Glu 3425 3430 3435 His Tyr Ala Ser Arg Leu Ala Glu Met Glu Asn Ser Asn Gly Ser 3440 3445 3450 Tyr Leu Asn Asp Ser Ile Ser Pro Asn Glu Ser Ile Asp Asp Glu 3455 3460 3465 His Leu Leu Ile Gln His Tyr Cys Gln Ser Leu Asn Gln Asp Ser 3470 3475 3480 Pro Leu Ser Gln Pro Arg Ser Pro Ala Gln Ile Leu Ile Ser Leu 3485 3490 3495 Glu Ser Glu Glu Arg Gly Glu Leu Glu Arg Ile Leu Ala Asp Leu 3500 3505 3510 Glu Glu Glu Asn Arg Asn Leu Gln Ala Glu Tyr Asp Arg Leu Lys 3515 3520 3525 Gln Gln His Glu His Lys Gly Leu Ser Pro Leu Pro Ser Pro Pro 3530 3535 3540 Glu Met Met Pro Thr Ser Pro Gln Ser Pro Arg Asp Ala Glu Leu 3545 3550 3555 Ile Ala Glu Ala Lys Leu Leu Arg Gln His Lys Gly Arg Leu Glu 3560 3565 3570 Ala Arg Met Gln Ile Leu Glu Asp His Asn Lys Gln Leu Glu Ser 3575 3580 3585 Gln Leu His Arg Leu Arg Gln Leu Leu Glu Gln Pro Gln Ala Glu 3590 3595 3600 Ala Lys Val Asn Gly Thr Thr Val Ser Ser Pro Ser Thr Ser Leu 3605 3610 3615 Gln Arg Ser Asp Ser Ser Gln Pro Met Leu Leu Arg Val Val Gly 3620 3625 3630 Ser Gln Thr Ser Asp Ser Met Gly Glu Glu Asp Leu Leu Ser Pro 3635 3640 3645 Pro Gln Asp Thr Ser Thr Gly Leu Glu Glu Val Met Glu Gln Leu 3650 3655 3660 Asn Asn Ser Phe Pro Ser Ser Arg Gly Arg Asn Thr Pro Gly Lys 3665 3670 3675 Pro Met Arg Glu Asp Thr Met 3680 3685 225RNAArtificialoligonucleotide 2guaccuccaa caucaaggaa gaugg 25325RNAArtificialoligonucleotide 3uaccuccaac aucaaggaag auggc 25425RNAArtificialoligonucleotide 4accuccaaca ucaaggaaga uggca 25525RNAArtificialoligonucleotide 5ccuccaacau caaggaagau ggcau 25625RNAArtificialoligonucleotide 6cuccaacauc aaggaagaug gcauu 25725RNAArtificialoligonucleotide 7uccaacauca aggaagaugg cauuu 25825RNAArtificialoligonucleotide 8ccaacaucaa ggaagauggc auuuc 25925RNAArtificialoligonucleotide 9caacaucaag gaagauggca uuucu 251025RNAArtificialoligonucleotide 10aacaucaagg aagauggcau uucua 251125RNAArtificialoligonucleotide 11acaucaagga agauggcauu ucuag 251225RNAArtificialoligonucleotide 12caucaaggaa gauggcauuu cuagu 251325RNAArtificialoligonucleotide 13aucaaggaag auggcauuuc uaguu 251425RNAArtificialoligonucleotide 14ucaaggaaga uggcauuucu aguuu 251525RNAArtificialoligonucleotide 15caaggaagau ggcauuucua guuug 251625RNAArtificialoligonucleotide 16aaggaagaug gcauuucuag uuugg 251725RNAArtificialoligonucleotide 17aggaagaugg cauuucuagu uugga 251825RNAArtificialoligonucleotide 18ggaagauggc auuucuaguu uggag 251925RNAArtificialoligonucleotide 19gaagauggca uuucuaguuu ggaga 252025RNAArtificialoligonucleotide 20aagauggcau uucuaguuug gagau 252125RNAArtificialoligonucleotide 21agauggcauu ucuaguuugg agaug 252225RNAArtificialoligonucleotide 22gauggcauuu cuaguuugga gaugg 252325RNAArtificialoligonucleotide 23auggcauuuc uaguuuggag auggc 252425RNAArtificialoligonucleotide 24uggcauuucu aguuuggaga uggca 252525RNAArtificialoligonucleotide 25ggcauuucua guuuggagau ggcag 252625RNAArtificialoligonucleotide 26gcauuucuag uuuggagaug gcagu 252725RNAArtificialoligonucleotide 27cauuucuagu uuggagaugg caguu 252825RNAArtificialoligonucleotide 28auuucuaguu uggagauggc aguuu 252925RNAArtificialoligonucleotide 29uuucuaguuu ggagauggca guuuc 253025RNAArtificialoligonucleotide 30uucuaguuug gagauggcag uuucc 253125RNAArtificialoligonucleotide 31ucuaguuugg agauggcagu uuccu 253225RNAArtificialoligonucleotide 32cuaguuugga gauggcaguu uccuu 253325RNAArtificialoligonucleotide 33uaguuuggag auggcaguuu ccuua 253425RNAArtificialoligonucleotide 34aguuuggaga uggcaguuuc cuuag 253525RNAArtificialoligonucleotide 35guuuggagau ggcaguuucc uuagu 253625RNAArtificialoligonucleotide 36uuuggagaug gcaguuuccu uagua 253725RNAArtificialoligonucleotide 37uuggagaugg caguuuccuu aguaa 253825RNAArtificialoligonucleotide 38uggagauggc aguuuccuua guaac 253925RNAArtificialoligonucleotide 39gagauggcag uuuccuuagu aacca 254025RNAArtificialoligonucleotide 40agauggcagu uuccuuagua accac 254125RNAArtificialoligonucleotide 41gauggcaguu uccuuaguaa ccaca 254225RNAArtificialoligonucleotide 42auggcaguuu ccuuaguaac cacag 254325RNAArtificialoligonucleotide 43uggcaguuuc cuuaguaacc acagg 254425RNAArtificialoligonucleotide 44ggcaguuucc uuaguaacca caggu 254525RNAArtificialoligonucleotide 45gcaguuuccu uaguaaccac agguu 254625RNAArtificialoligonucleotide 46caguuuccuu aguaaccaca gguug 254725RNAArtificialoligonucleotide 47aguuuccuua guaaccacag guugu 254825RNAArtificialoligonucleotide 48guuuccuuag uaaccacagg uugug 254925RNAArtificialoligonucleotide 49uuuccuuagu aaccacaggu ugugu 255025RNAArtificialoligonucleotide 50uuccuuagua accacagguu guguc 255125RNAArtificialoligonucleotide 51uccuuaguaa ccacagguug uguca 255225RNAArtificialoligonucleotide 52ccuuaguaac cacagguugu gucac 255325RNAArtificialoligonucleotide 53cuuaguaacc acagguugug ucacc 255425RNAArtificialoligonucleotide 54uuaguaacca cagguugugu cacca 255525RNAArtificialoligonucleotide 55uaguaaccac agguuguguc accag 255625RNAArtificialoligonucleotide 56aguaaccaca gguuguguca ccaga 255725RNAArtificialoligonucleotide 57guaaccacag guugugucac cagag 255825RNAArtificialoligonucleotide 58uaaccacagg uugugucacc agagu 255925RNAArtificialoligonucleotide

59aaccacaggu ugugucacca gagua 256025RNAArtificialoligonucleotide 60accacagguu gugucaccag aguaa 256125RNAArtificialoligonucleotide 61ccacagguug ugucaccaga guaac 256225RNAArtificialoligonucleotide 62cacagguugu gucaccagag uaaca 256325RNAArtificialoligonucleotide 63acagguugug ucaccagagu aacag 256425RNAArtificialoligonucleotide 64cagguugugu caccagagua acagu 256525RNAArtificialoligonucleotide 65agguuguguc accagaguaa caguc 256625RNAArtificialoligonucleotide 66gguuguguca ccagaguaac agucu 256725RNAArtificialoligonucleotide 67guugugucac cagaguaaca gucug 256825RNAArtificialoligonucleotide 68uugugucacc agaguaacag ucuga 256925RNAArtificialoligonucleotide 69ugugucacca gaguaacagu cugag 257025RNAArtificialoligonucleotide 70gugucaccag aguaacaguc ugagu 257125RNAArtificialoligonucleotide 71ugucaccaga guaacagucu gagua 257225RNAArtificialoligonucleotide 72gucaccagag uaacagucug aguag 257325RNAArtificialoligonucleotide 73ucaccagagu aacagucuga guagg 257425RNAArtificialoligonucleotide 74caccagagua acagucugag uagga 257525RNAArtificialoligonucleotide 75accagaguaa cagucugagu aggag 257625RNAArtificialoligonucleotide 76uuugccgcug cccaaugcca uccug 257725RNAArtificialoligonucleotide 77auucaauguu cugacaacag uuugc 257825RNAArtificialoligonucleotide 78ccaguugcau ucaauguucu gacaa 257922RNAArtificialoligonucleotide 79caguugcauu caauguucug ac 228020RNAArtificialoligonucleotide 80aguugcauuc aauguucuga 208121RNAArtificialoligonucleotide 81gauugcugaa uuauuucuuc c 218225RNAArtificialoligonucleotide 82gauugcugaa uuauuucuuc cccag 258325RNAArtificialoligonucleotide 83auugcugaau uauuucuucc ccagu 258425RNAArtificialoligonucleotide 84uugcugaauu auuucuuccc caguu 258525RNAArtificialoligonucleotide 85ugcugaauua uuucuucccc aguug 258625RNAArtificialoligonucleotide 86gcugaauuau uucuucccca guugc 258725RNAArtificialoligonucleotide 87cugaauuauu ucuuccccag uugca 258825RNAArtificialoligonucleotide 88ugaauuauuu cuuccccagu ugcau 258925RNAArtificialoligonucleotide 89gaauuauuuc uuccccaguu gcauu 259025RNAArtificialoligonucleotide 90aauuauuucu uccccaguug cauuc 259125RNAArtificialoligonucleotide 91auuauuucuu ccccaguugc auuca 259225RNAArtificialoligonucleotide 92uuauuucuuc cccaguugca uucaa 259325RNAArtificialoligonucleotide 93uauuucuucc ccaguugcau ucaau 259425RNAArtificialoligonucleotide 94auuucuuccc caguugcauu caaug 259525RNAArtificialoligonucleotide 95uuucuucccc aguugcauuc aaugu 259625RNAArtificialoligonucleotide 96uucuucccca guugcauuca auguu 259725RNAArtificialoligonucleotide 97ucuuccccag uugcauucaa uguuc 259825RNAArtificialoligonucleotide 98cuuccccagu ugcauucaau guucu 259925RNAArtificialoligonucleotide 99uuccccaguu gcauucaaug uucug 2510025RNAArtificialoligonucleotide 100uccccaguug cauucaaugu ucuga 2510125RNAArtificialoligonucleotide 101ccccaguugc auucaauguu cugac 2510225RNAArtificialoligonucleotide 102cccaguugca uucaauguuc ugaca 2510325RNAArtificialoligonucleotide 103ccaguugcau ucaauguucu gacaa 2510425RNAArtificialoligonucleotide 104caguugcauu caauguucug acaac 2510525RNAArtificialoligonucleotide 105aguugcauuc aauguucuga caaca 2510620RNAArtificialoligonucleotide 106uccuguagaa uacuggcauc 2010727RNAArtificialoligonucleotide 107ugcagaccuc cugccaccgc agauuca 2710834RNAArtificialoligonucleotide 108uugcagaccu ccugccaccg cagauucagg cuuc 3410925RNAArtificialoligonucleotide 109guugcauuca auguucugac aacag 2511025RNAArtificialoligonucleotide 110uugcauucaa uguucugaca acagu 2511125RNAArtificialoligonucleotide 111ugcauucaau guucugacaa caguu 2511225RNAArtificialoligonucleotide 112gcauucaaug uucugacaac aguuu 2511325RNAArtificialoligonucleotide 113cauucaaugu ucugacaaca guuug 2511425RNAArtificialoligonucleotide 114auucaauguu cugacaacag uuugc 2511525RNAArtificialoligonucleotide 115ucaauguucu gacaacaguu ugccg 2511625RNAArtificialoligonucleotide 116caauguucug acaacaguuu gccgc 2511725RNAArtificialoligonucleotide 117aauguucuga caacaguuug ccgcu 2511825RNAArtificialoligonucleotide 118auguucugac aacaguuugc cgcug 2511925RNAArtificialoligonucleotide 119uguucugaca acaguuugcc gcugc 2512025RNAArtificialoligonucleotide 120guucugacaa caguuugccg cugcc 2512125RNAArtificialoligonucleotide 121uucugacaac aguuugccgc ugccc 2512225RNAArtificialoligonucleotide 122ucugacaaca guuugccgcu gccca 2512325RNAArtificialoligonucleotide 123cugacaacag uuugccgcug cccaa 2512425RNAArtificialoligonucleotide 124ugacaacagu uugccgcugc ccaau 2512525RNAArtificialoligonucleotide 125gacaacaguu ugccgcugcc caaug 2512625RNAArtificialoligonucleotide 126acaacaguuu gccgcugccc aaugc 2512725RNAArtificialoligonucleotide 127caacaguuug ccgcugccca augcc 2512825RNAArtificialoligonucleotide 128aacaguuugc cgcugcccaa ugcca 2512925RNAArtificialoligonucleotide 129acaguuugcc gcugcccaau gccau 2513025RNAArtificialoligonucleotide 130caguuugccg cugcccaaug ccauc 2513125RNAArtificialoligonucleotide 131aguuugccgc ugcccaaugc caucc 2513225RNAArtificialoligonucleotide 132guuugccgcu gcccaaugcc auccu 2513325RNAArtificialoligonucleotide 133uuugccgcug cccaaugcca uccug 2513425RNAArtificialoligonucleotide 134uugccgcugc ccaaugccau ccugg 2513525RNAArtificialoligonucleotide 135ugccgcugcc caaugccauc cugga 2513625RNAArtificialoligonucleotide 136gccgcugccc aaugccaucc uggag 2513725RNAArtificialoligonucleotide 137ccgcugccca augccauccu ggagu 2513825RNAArtificialoligonucleotide 138cgcugcccaa ugccauccug gaguu 2513920RNAArtificialoligonucleotide 139uguuuuugag gauugcugaa 2014040RNAArtificialoligonucleotide 140uguucugaca acaguuugcc gcugcccaau gccauccugg 4014125RNAArtificialoligonucleotide 141cucuggccug uccuaagacc ugcuc 2514225RNAArtificialoligonucleotide 142ucuggccugu ccuaagaccu gcuca 2514325RNAArtificialoligonucleotide 143cuggccuguc cuaagaccug cucag 2514425RNAArtificialoligonucleotide 144uggccugucc uaagaccugc ucagc 2514525RNAArtificialoligonucleotide 145ggccuguccu aagaccugcu cagcu 2514625RNAArtificialoligonucleotide 146gccuguccua agaccugcuc agcuu 2514725RNAArtificialoligonucleotide 147ccuguccuaa gaccugcuca gcuuc 2514825RNAArtificialoligonucleotide 148cuguccuaag accugcucag cuucu 2514925RNAArtificialoligonucleotide 149uguccuaaga ccugcucagc uucuu 2515025RNAArtificialoligonucleotide 150guccuaagac cugcucagcu ucuuc 2515125RNAArtificialoligonucleotide 151uccuaagacc ugcucagcuu cuucc 2515225RNAArtificialoligonucleotide 152ccuaagaccu gcucagcuuc uuccu 2515325RNAArtificialoligonucleotide 153cuaagaccug cucagcuucu uccuu 2515425RNAArtificialoligonucleotide 154uaagaccugc ucagcuucuu ccuua 2515525RNAArtificialoligonucleotide 155aagaccugcu cagcuucuuc cuuag 2515625RNAArtificialoligonucleotide 156agaccugcuc agcuucuucc uuagc 2515725RNAArtificialoligonucleotide 157gaccugcuca gcuucuuccu uagcu 2515825RNAArtificialoligonucleotide 158accugcucag cuucuuccuu agcuu 2515925RNAArtificialoligonucleotide 159ccugcucagc uucuuccuua gcuuc 2516025RNAArtificialoligonucleotide 160cugcucagcu ucuuccuuag cuucc 2516125RNAArtificialoligonucleotide 161ugcucagcuu cuuccuuagc uucca 2516225RNAArtificialoligonucleotide 162gcucagcuuc uuccuuagcu uccag 2516325RNAArtificialoligonucleotide 163cucagcuucu uccuuagcuu ccagc 2516425RNAArtificialoligonucleotide 164ucagcuucuu ccuuagcuuc cagcc 2516525RNAArtificialoligonucleotide 165cagcuucuuc cuuagcuucc agcca 2516625RNAArtificialoligonucleotide 166agcuucuucc uuagcuucca gccau 2516725RNAArtificialoligonucleotide 167gcuucuuccu uagcuuccag ccauu 2516825RNAArtificialoligonucleotide 168cuucuuccuu agcuuccagc cauug 2516925RNAArtificialoligonucleotide 169uucuuccuua gcuuccagcc auugu 2517025RNAArtificialoligonucleotide 170ucuuccuuag cuuccagcca uugug 2517125RNAArtificialoligonucleotide 171cuuccuuagc uuccagccau ugugu 2517225RNAArtificialoligonucleotide 172uuccuuagcu uccagccauu guguu 2517325RNAArtificialoligonucleotide 173uccuuagcuu ccagccauug uguug 2517425RNAArtificialoligonucleotide 174ccuuagcuuc cagccauugu guuga 2517525RNAArtificialoligonucleotide 175cuuagcuucc agccauugug uugaa 2517625RNAArtificialoligonucleotide 176uuagcuucca gccauugugu ugaau 2517725RNAArtificialoligonucleotide 177uagcuuccag ccauuguguu gaauc 2517825RNAArtificialoligonucleotide 178agcuuccagc cauuguguug aaucc 2517925RNAArtificialoligonucleotide 179gcuuccagcc auuguguuga auccu 2518025RNAArtificialoligonucleotide 180cuuccagcca uuguguugaa uccuu 2518125RNAArtificialoligonucleotide 181uuccagccau uguguugaau ccuuu 2518225RNAArtificialoligonucleotide 182uccagccauu guguugaauc cuuua 2518325RNAArtificialoligonucleotide 183ccagccauug uguugaaucc uuuaa 2518425RNAArtificialoligonucleotide 184cagccauugu guugaauccu uuaac 2518525RNAArtificialoligonucleotide 185agccauugug uugaauccuu uaaca 2518625RNAArtificialoligonucleotide 186gccauugugu ugaauccuuu aacau 2518725RNAArtificialoligonucleotide 187ccauuguguu gaauccuuua acauu 2518825RNAArtificialoligonucleotide 188cauuguguug aauccuuuaa cauuu 2518920RNAArtificialoligonucleotide 189ucagcuucug uuagccacug 2019020RNAArtificialoligonucleotide 190uucagcuucu guuagccacu 2019121RNAArtificialoligonucleotide 191uucagcuucu guuagccacu g 2119221RNAArtificialoligonucleotide 192ucagcuucug uuagccacug a 2119322RNAArtificialoligonucleotide 193uucagcuucu guuagccacu ga 2219421RNAArtificialoligonucleotide 194ucagcuucug uuagccacug a 2119522RNAArtificialoligonucleotide 195uucagcuucu guuagccacu ga 2219622RNAArtificialoligonucleotide 196ucagcuucug uuagccacug au 2219723RNAArtificialoligonucleotide 197uucagcuucu guuagccacu gau 2319823RNAArtificialoligonucleotide 198ucagcuucug uuagccacug auu 2319924RNAArtificialoligonucleotide 199uucagcuucu guuagccacu gauu 2420024RNAArtificialoligonucleotide 200ucagcuucug uuagccacug auua 2420124RNAArtificialoligonucleotide 201uucagcuucu guuagccacu gaua 2420225RNAArtificialoligonucleotide 202ucagcuucug uuagccacug auuaa 2520326RNAArtificialoligonucleotide 203uucagcuucu guuagccacu gauuaa 2620426RNAArtificialoligonucleotide 204ucagcuucug uuagccacug auuaaa 2620527RNAArtificialoligonucleotide 205uucagcuucu guuagccacu gauuaaa 2720619RNAArtificialoligonucleotide 206cagcuucugu uagccacug 1920721RNAArtificialoligonucleotide 207cagcuucugu uagccacuga u 2120821RNAArtificialoligonucleotide 208agcuucuguu agccacugau u 2120922RNAArtificialoligonucleotide 209cagcuucugu uagccacuga uu 2221022RNAArtificialoligonucleotide 210agcuucuguu agccacugau ua 2221123RNAArtificialoligonucleotide 211cagcuucugu uagccacuga uua 2321223RNAArtificialoligonucleotide 212agcuucuguu agccacugau uaa 2321324RNAArtificialoligonucleotide 213cagcuucugu uagccacuga uuaa 2421424RNAArtificialoligonucleotide 214agcuucuguu agccacugau uaaa 2421525RNAArtificialoligonucleotide 215cagcuucugu uagccacuga uuaaa 2521624RNAArtificialoligonucleotide 216agcuucuguu agccacugau uaaa 2421720RNAArtificialoligonucleotide 217agcuucuguu agccacugau 2021820RNAArtificialoligonucleotide 218gcuucuguua gccacugauu 2021921RNAArtificialoligonucleotide 219agcuucuguu agccacugau u 2122021RNAArtificialoligonucleotide 220gcuucuguua gccacugauu a 2122122RNAArtificialoligonucleotide 221agcuucuguu agccacugau ua 2222222RNAArtificialoligonucleotide 222gcuucuguua gccacugauu aa 2222323RNAArtificialoligonucleotide 223agcuucuguu agccacugau uaa 2322423RNAArtificialoligonucleotide 224gcuucuguua gccacugauu aaa 2322524RNAArtificialoligonucleotide 225agcuucuguu agccacugau uaaa 2422623RNAArtificialoligonucleotide 226gcuucuguua gccacugauu aaa

2322723RNAArtificialoligonucleotide 227ccauuuguau uuagcauguu ccc 2322820RNAArtificialoligonucleotide 228agauaccauu uguauuuagc 2022919RNAArtificialoligonucleotide 229gccauuucuc aacagaucu 1923023RNAArtificialoligonucleotide 230gccauuucuc aacagaucug uca 2323123RNAArtificialoligonucleotide 231auucucagga auuugugucu uuc 2323221RNAArtificialoligonucleotide 232ucucaggaau uugugucuuu c 2123318RNAArtificialoligonucleotide 233guucagcuuc uguuagcc 1823421RNAArtificialoligonucleotide 234cugauuaaau aucuuuauau c 2123518RNAArtificialoligonucleotide 235gccgccauuu cucaacag 1823618RNAArtificialoligonucleotide 236guauuuagca uguuccca 1823718RNAArtificialoligonucleotide 237caggaauuug ugucuuuc 1823825RNAArtificialoligonucleotide 238gcuuuucuuu uaguugcugc ucuuu 2523925RNAArtificialoligonucleotide 239cuuuucuuuu aguugcugcu cuuuu 2524025RNAArtificialoligonucleotide 240uuuucuuuua guugcugcuc uuuuc 2524125RNAArtificialoligonucleotide 241uuucuuuuag uugcugcucu uuucc 2524225RNAArtificialoligonucleotide 242uucuuuuagu ugcugcucuu uucca 2524325RNAArtificialoligonucleotide 243ucuuuuaguu gcugcucuuu uccag 2524425RNAArtificialoligonucleotide 244cuuuuaguug cugcucuuuu ccagg 2524525RNAArtificialoligonucleotide 245uuuuaguugc ugcucuuuuc caggu 2524625RNAArtificialoligonucleotide 246uuuaguugcu gcucuuuucc agguu 2524725RNAArtificialoligonucleotide 247uuaguugcug cucuuuucca gguuc 2524825RNAArtificialoligonucleotide 248uaguugcugc ucuuuuccag guuca 2524925RNAArtificialoligonucleotide 249aguugcugcu cuuuuccagg uucaa 2525025RNAArtificialoligonucleotide 250guugcugcuc uuuuccaggu ucaag 2525125RNAArtificialoligonucleotide 251uugcugcucu uuuccagguu caagu 2525225RNAArtificialoligonucleotide 252ugcugcucuu uuccagguuc aagug 2525325RNAArtificialoligonucleotide 253gcugcucuuu uccagguuca agugg 2525425RNAArtificialoligonucleotide 254cugcucuuuu ccagguucaa guggg 2525525RNAArtificialoligonucleotide 255ugcucuuuuc cagguucaag uggga 2525625RNAArtificialoligonucleotide 256gcucuuuucc agguucaagu gggac 2525725RNAArtificialoligonucleotide 257cucuuuucca gguucaagug ggaua 2525825RNAArtificialoligonucleotide 258ucuuuuccag guucaagugg gauac 2525920RNAArtificialoligonucleotide 259ucuuuuccag guucaagugg 2026025RNAArtificialoligonucleotide 260cuuuuccagg uucaaguggg auacu 2526125RNAArtificialoligonucleotide 261uuuuccaggu ucaaguggga uacua 2526225RNAArtificialoligonucleotide 262uuuccagguu caagugggau acuag 2526325RNAArtificialoligonucleotide 263uuccagguuc aagugggaua cuagc 2526425RNAArtificialoligonucleotide 264uccagguuca agugggauac uagca 2526525RNAArtificialoligonucleotide 265ccagguucaa gugggauacu agcaa 2526625RNAArtificialoligonucleotide 266cagguucaag ugggauacua gcaau 2526725RNAArtificialoligonucleotide 267agguucaagu gggauacuag caaug 2526825RNAArtificialoligonucleotide 268gguucaagug ggauacuagc aaugu 2526925RNAArtificialoligonucleotide 269guucaagugg gauacuagca auguu 2527025RNAArtificialoligonucleotide 270uucaaguggg auacuagcaa uguua 2527125RNAArtificialoligonucleotide 271ucaaguggga uacuagcaau guuau 2527225RNAArtificialoligonucleotide 272caagugggau acuagcaaug uuauc 2527325RNAArtificialoligonucleotide 273aagugggaua cuagcaaugu uaucu 2527425RNAArtificialoligonucleotide 274agugggauac uagcaauguu aucug 2527525RNAArtificialoligonucleotide 275gugggauacu agcaauguua ucugc 2527625RNAArtificialoligonucleotide 276ugggauacua gcaauguuau cugcu 2527725RNAArtificialoligonucleotide 277gggauacuag caauguuauc ugcuu 2527825RNAArtificialoligonucleotide 278ggauacuagc aauguuaucu gcuuc 2527925RNAArtificialoligonucleotide 279gauacuagca auguuaucug cuucc 2528025RNAArtificialoligonucleotide 280auacuagcaa uguuaucugc uuccu 2528125RNAArtificialoligonucleotide 281uacuagcaau guuaucugcu uccuc 2528225RNAArtificialoligonucleotide 282acuagcaaug uuaucugcuu ccucc 2528325RNAArtificialoligonucleotide 283cuagcaaugu uaucugcuuc cucca 2528425RNAArtificialoligonucleotide 284uagcaauguu aucugcuucc uccaa 2528525RNAArtificialoligonucleotide 285agcaauguua ucugcuuccu ccaac 2528625RNAArtificialoligonucleotide 286gcaauguuau cugcuuccuc caacc 2528725RNAArtificialoligonucleotide 287caauguuauc ugcuuccucc aacca 2528825RNAArtificialoligonucleotide 288aauguuaucu gcuuccucca accau 2528925RNAArtificialoligonucleotide 289auguuaucug cuuccuccaa ccaua 2529025RNAArtificialoligonucleotide 290uguuaucugc uuccuccaac cauaa 2529125RNAArtificialoligonucleotide 291agccucuuga uugcuggucu uguuu 2529225RNAArtificialoligonucleotide 292gccucuugau ugcuggucuu guuuu 2529325RNAArtificialoligonucleotide 293ccucuugauu gcuggucuug uuuuu 2529420RNAArtificialoligonucleotide 294ccucuugauu gcuggucuug 2029525RNAArtificialoligonucleotide 295cucuugauug cuggucuugu uuuuc 2529625RNAArtificialoligonucleotide 296ucuugauugc uggucuuguu uuuca 2529725RNAArtificialoligonucleotide 297cuugauugcu ggucuuguuu uucaa 2529825RNAArtificialoligonucleotide 298uugauugcug gucuuguuuu ucaaa 2529925RNAArtificialoligonucleotide 299ugauugcugg ucuuguuuuu caaau 2530025RNAArtificialoligonucleotide 300gauugcuggu cuuguuuuuc aaauu 2530120RNAArtificialoligonucleotide 301gauugcuggu cuuguuuuuc 2030225RNAArtificialoligonucleotide 302auugcugguc uuguuuuuca aauuu 2530325RNAArtificialoligonucleotide 303uugcuggucu uguuuuucaa auuuu 2530425RNAArtificialoligonucleotide 304ugcuggucuu guuuuucaaa uuuug 2530525RNAArtificialoligonucleotide 305gcuggucuug uuuuucaaau uuugg 2530625RNAArtificialoligonucleotide 306cuggucuugu uuuucaaauu uuggg 2530725RNAArtificialoligonucleotide 307uggucuuguu uuucaaauuu ugggc 2530825RNAArtificialoligonucleotide 308ggucuuguuu uucaaauuuu gggca 2530925RNAArtificialoligonucleotide 309gucuuguuuu ucaaauuuug ggcag 2531025RNAArtificialoligonucleotide 310ucuuguuuuu caaauuuugg gcagc 2531125RNAArtificialoligonucleotide 311cuuguuuuuc aaauuuuggg cagcg 2531225RNAArtificialoligonucleotide 312uuguuuuuca aauuuugggc agcgg 2531325RNAArtificialoligonucleotide 313uguuuuucaa auuuugggca gcggu 2531425RNAArtificialoligonucleotide 314guuuuucaaa uuuugggcag cggua 2531525RNAArtificialoligonucleotide 315uuuuucaaau uuugggcagc gguaa 2531625RNAArtificialoligonucleotide 316uuuucaaauu uugggcagcg guaau 2531725RNAArtificialoligonucleotide 317uuucaaauuu ugggcagcgg uaaug 2531825RNAArtificialoligonucleotide 318uucaaauuuu gggcagcggu aauga 2531925RNAArtificialoligonucleotide 319ucaaauuuug ggcagcggua augag 2532025RNAArtificialoligonucleotide 320caaauuuugg gcagcgguaa ugagu 2532125RNAArtificialoligonucleotide 321aaauuuuggg cagcgguaau gaguu 2532225RNAArtificialoligonucleotide 322aauuuugggc agcgguaaug aguuc 2532325RNAArtificialoligonucleotide 323auuuugggca gcgguaauga guucu 2532425RNAArtificialoligonucleotide 324uuuugggcag cgguaaugag uucuu 2532525RNAArtificialoligonucleotide 325uuugggcagc gguaaugagu ucuuc 2532625RNAArtificialoligonucleotide 326uugggcagcg guaaugaguu cuucc 2532725RNAArtificialoligonucleotide 327ugggcagcgg uaaugaguuc uucca 2532825RNAArtificialoligonucleotide 328gggcagcggu aaugaguucu uccaa 2532925RNAArtificialoligonucleotide 329ggcagcggua augaguucuu ccaac 2533025RNAArtificialoligonucleotide 330gcagcgguaa ugaguucuuc caacu 2533125RNAArtificialoligonucleotide 331cagcgguaau gaguucuucc aacug 2533225RNAArtificialoligonucleotide 332agcgguaaug aguucuucca acugg 2533325RNAArtificialoligonucleotide 333gcgguaauga guucuuccaa cuggg 2533425RNAArtificialoligonucleotide 334cgguaaugag uucuuccaac ugggg 2533525RNAArtificialoligonucleotide 335gguaaugagu ucuuccaacu gggga 2533622RNAArtificialoligonucleotide 336gguaaugagu ucuuccaacu gg 2233725RNAArtificialoligonucleotide 337guaaugaguu cuuccaacug gggac 2533825RNAArtificialoligonucleotide 338uaaugaguuc uuccaacugg ggacg 2533925RNAArtificialoligonucleotide 339aaugaguucu uccaacuggg gacgc 2534025RNAArtificialoligonucleotide 340augaguucuu ccaacugggg acgcc 2534125RNAArtificialoligonucleotide 341ugaguucuuc caacugggga cgccu 2534225RNAArtificialoligonucleotide 342gaguucuucc aacuggggac gccuc 2534325RNAArtificialoligonucleotide 343aguucuucca acuggggacg ccucu 2534425RNAArtificialoligonucleotide 344guucuuccaa cuggggacgc cucug 2534525RNAArtificialoligonucleotide 345uucuuccaac uggggacgcc ucugu 2534625RNAArtificialoligonucleotide 346ucuuccaacu ggggacgccu cuguu 2534725RNAArtificialoligonucleotide 347cuuccaacug gggacgccuc uguuc 2534825RNAArtificialoligonucleotide 348uuccaacugg ggacgccucu guucc 2534925RNAArtificialoligonucleotide 349uccaacuggg gacgccucug uucca 2535025RNAArtificialoligonucleotide 350ccaacugggg acgccucugu uccaa 2535125RNAArtificialoligonucleotide 351caacugggga cgccucuguu ccaaa 2535225RNAArtificialoligonucleotide 352aacuggggac gccucuguuc caaau 2535325RNAArtificialoligonucleotide 353acuggggacg ccucuguucc aaauc 2535425RNAArtificialoligonucleotide 354cuggggacgc cucuguucca aaucc 2535525RNAArtificialoligonucleotide 355uggggacgcc ucuguuccaa auccu 2535625RNAArtificialoligonucleotide 356ggggacgccu cuguuccaaa uccug 2535725RNAArtificialoligonucleotide 357gggacgccuc uguuccaaau ccugc 2535825RNAArtificialoligonucleotide 358ggacgccucu guuccaaauc cugca 2535925RNAArtificialoligonucleotide 359gacgccucug uuccaaaucc ugcau 2536025RNAArtificialoligonucleotide 360ccaauagugg ucaguccagg agcua 2536125RNAArtificialoligonucleotide 361caauaguggu caguccagga gcuag 2536225RNAArtificialoligonucleotide 362aauagugguc aguccaggag cuagg 2536325RNAArtificialoligonucleotide 363auagugguca guccaggagc uaggu 2536421RNAArtificialoligonucleotide 364auagugguca guccaggagc u 2136525RNAArtificialoligonucleotide 365uaguggucag uccaggagcu agguc 2536625RNAArtificialoligonucleotide 366aguggucagu ccaggagcua gguca 2536725RNAArtificialoligonucleotide 367guggucaguc caggagcuag gucag 2536825RNAArtificialoligonucleotide 368uggucagucc aggagcuagg ucagg 2536925RNAArtificialoligonucleotide 369ggucagucca ggagcuaggu caggc 2537025RNAArtificialoligonucleotide 370gucaguccag gagcuagguc aggcu 2537125RNAArtificialoligonucleotide 371ucaguccagg agcuagguca ggcug 2537225RNAArtificialoligonucleotide 372caguccagga gcuaggucag gcugc 2537325RNAArtificialoligonucleotide 373aguccaggag cuaggucagg cugcu 2537425RNAArtificialoligonucleotide 374guccaggagc uaggucaggc ugcuu 2537525RNAArtificialoligonucleotide 375uccaggagcu aggucaggcu gcuuu 2537625RNAArtificialoligonucleotide 376ccaggagcua ggucaggcug cuuug 2537725RNAArtificialoligonucleotide 377caggagcuag gucaggcugc uuugc 2537825RNAArtificialoligonucleotide 378aggagcuagg ucaggcugcu uugcc 2537925RNAArtificialoligonucleotide 379ggagcuaggu caggcugcuu ugccc 2538025RNAArtificialoligonucleotide 380gagcuagguc aggcugcuuu gcccu 2538125RNAArtificialoligonucleotide 381agcuagguca ggcugcuuug cccuc 2538225RNAArtificialoligonucleotide 382gcuaggucag gcugcuuugc ccuca 2538325RNAArtificialoligonucleotide 383cucagcucuu gaaguaaacg guuua 2538425RNAArtificialoligonucleotide 384cagcucuuga aguaaacggu uuacc 2538525RNAArtificialoligonucleotide 385gcucuugaag uaaacgguuu accgc 2538625RNAArtificialoligonucleotide 386cuaggucagg cugcuuugcc cucag 2538725RNAArtificialoligonucleotide 387uaggucaggc ugcuuugccc ucagc 2538825RNAArtificialoligonucleotide 388aggucaggcu gcuuugcccu cagcu 2538925RNAArtificialoligonucleotide 389ggucaggcug cuuugcccuc agcuc 2539025RNAArtificialoligonucleotide 390gucaggcugc uuugcccuca gcucu 2539125RNAArtificialoligonucleotide 391ucaggcugcu uugcccucag cucuu 2539225RNAArtificialoligonucleotide 392caggcugcuu ugcccucagc ucuug 2539325RNAArtificialoligonucleotide 393aggcugcuuu gcccucagcu cuuga

2539425RNAArtificialoligonucleotide 394ggcugcuuug cccucagcuc uugaa 2539525RNAArtificialoligonucleotide 395gcugcuuugc ccucagcucu ugaag 2539625RNAArtificialoligonucleotide 396cugcuuugcc cucagcucuu gaagu 2539725RNAArtificialoligonucleotide 397ugcuuugccc ucagcucuug aagua 2539825RNAArtificialoligonucleotide 398gcuuugcccu cagcucuuga aguaa 2539925RNAArtificialoligonucleotide 399cuuugcccuc agcucuugaa guaaa 2540025RNAArtificialoligonucleotide 400uuugcccuca gcucuugaag uaaac 2540125RNAArtificialoligonucleotide 401uugcccucag cucuugaagu aaacg 2540225RNAArtificialoligonucleotide 402ugcccucagc ucuugaagua aacgg 2540325RNAArtificialoligonucleotide 403gcccucagcu cuugaaguaa acggu 2540425RNAArtificialoligonucleotide 404cccucagcuc uugaaguaaa cgguu 2540520RNAArtificialoligonucleotide 405ccucagcucu ugaaguaaac 2040621RNAArtificialoligonucleotide 406ccucagcucu ugaaguaaac g 2140720RNAArtificialoligonucleotide 407cucagcucuu gaaguaaacg 2040825RNAArtificialoligonucleotide 408ccucagcucu ugaaguaaac gguuu 2540925RNAArtificialoligonucleotide 409ucagcucuug aaguaaacgg uuuac 2541025RNAArtificialoligonucleotide 410agcucuugaa guaaacgguu uaccg 2541125RNAArtificialoligonucleotide 411cucuugaagu aaacgguuua ccgcc 2541225RNAArtificialoligonucleotide 412ccacaggcgu ugcacuuugc aaugc 2541325RNAArtificialoligonucleotide 413cacaggcguu gcacuuugca augcu 2541425RNAArtificialoligonucleotide 414acaggcguug cacuuugcaa ugcug 2541525RNAArtificialoligonucleotide 415caggcguugc acuuugcaau gcugc 2541625RNAArtificialoligonucleotide 416aggcguugca cuuugcaaug cugcu 2541725RNAArtificialoligonucleotide 417ggcguugcac uuugcaaugc ugcug 2541825RNAArtificialoligonucleotide 418gcguugcacu uugcaaugcu gcugu 2541925RNAArtificialoligonucleotide 419cguugcacuu ugcaaugcug cuguc 2542023RNAArtificialoligonucleotide 420cguugcacuu ugcaaugcug cug 2342125RNAArtificialoligonucleotide 421guugcacuuu gcaaugcugc ugucu 2542225RNAArtificialoligonucleotide 422uugcacuuug caaugcugcu gucuu 2542325RNAArtificialoligonucleotide 423ugcacuuugc aaugcugcug ucuuc 2542425RNAArtificialoligonucleotide 424gcacuuugca augcugcugu cuucu 2542525RNAArtificialoligonucleotide 425cacuuugcaa ugcugcuguc uucuu 2542625RNAArtificialoligonucleotide 426acuuugcaau gcugcugucu ucuug 2542725RNAArtificialoligonucleotide 427cuuugcaaug cugcugucuu cuugc 2542825RNAArtificialoligonucleotide 428uuugcaaugc ugcugucuuc uugcu 2542925RNAArtificialoligonucleotide 429uugcaaugcu gcugucuucu ugcua 2543025RNAArtificialoligonucleotide 430ugcaaugcug cugucuucuu gcuau 2543125RNAArtificialoligonucleotide 431gcaaugcugc ugucuucuug cuaug 2543225RNAArtificialoligonucleotide 432caaugcugcu gucuucuugc uauga 2543325RNAArtificialoligonucleotide 433aaugcugcug ucuucuugcu augaa 2543425RNAArtificialoligonucleotide 434augcugcugu cuucuugcua ugaau 2543525RNAArtificialoligonucleotide 435ugcugcuguc uucuugcuau gaaua 2543625RNAArtificialoligonucleotide 436gcugcugucu ucuugcuaug aauaa 2543725RNAArtificialoligonucleotide 437cugcugucuu cuugcuauga auaau 2543825RNAArtificialoligonucleotide 438ugcugucuuc uugcuaugaa uaaug 2543925RNAArtificialoligonucleotide 439gcugucuucu ugcuaugaau aaugu 2544025RNAArtificialoligonucleotide 440cugucuucuu gcuaugaaua auguc 2544125RNAArtificialoligonucleotide 441ugucuucuug cuaugaauaa uguca 2544225RNAArtificialoligonucleotide 442gucuucuugc uaugaauaau gucaa 2544325RNAArtificialoligonucleotide 443ucuucuugcu augaauaaug ucaau 2544425RNAArtificialoligonucleotide 444cuucuugcua ugaauaaugu caauc 2544525RNAArtificialoligonucleotide 445uucuugcuau gaauaauguc aaucc 2544625RNAArtificialoligonucleotide 446ucuugcuaug aauaauguca auccg 2544725RNAArtificialoligonucleotide 447cuugcuauga auaaugucaa uccga 2544825RNAArtificialoligonucleotide 448uugcuaugaa uaaugucaau ccgac 2544925RNAArtificialoligonucleotide 449ugcuaugaau aaugucaauc cgacc 2545025RNAArtificialoligonucleotide 450gcuaugaaua augucaaucc gaccu 2545125RNAArtificialoligonucleotide 451cuaugaauaa ugucaauccg accug 2545225RNAArtificialoligonucleotide 452uaugaauaau gucaauccga ccuga 2545325RNAArtificialoligonucleotide 453augaauaaug ucaauccgac cugag 2545425RNAArtificialoligonucleotide 454ugaauaaugu caauccgacc ugagc 2545525RNAArtificialoligonucleotide 455gaauaauguc aauccgaccu gagcu 2545625RNAArtificialoligonucleotide 456aauaauguca auccgaccug agcuu 2545725RNAArtificialoligonucleotide 457auaaugucaa uccgaccuga gcuuu 2545825RNAArtificialoligonucleotide 458uaaugucaau ccgaccugag cuuug 2545925RNAArtificialoligonucleotide 459aaugucaauc cgaccugagc uuugu 2546025RNAArtificialoligonucleotide 460augucaaucc gaccugagcu uuguu 2546125RNAArtificialoligonucleotide 461ugucaauccg accugagcuu uguug 2546225RNAArtificialoligonucleotide 462gucaauccga ccugagcuuu guugu 2546325RNAArtificialoligonucleotide 463ucaauccgac cugagcuuug uugua 2546425RNAArtificialoligonucleotide 464caauccgacc ugagcuuugu uguag 2546525RNAArtificialoligonucleotide 465aauccgaccu gagcuuuguu guaga 2546625RNAArtificialoligonucleotide 466auccgaccug agcuuuguug uagac 2546725RNAArtificialoligonucleotide 467uccgaccuga gcuuuguugu agacu 2546825RNAArtificialoligonucleotide 468ccgaccugag cuuuguugua gacua 2546920RNAArtificialoligonucleotide 469cgaccugagc uuuguuguag 2047025RNAArtificialoligonucleotide 470cgaccugagc uuuguuguag acuau 2547125RNAArtificialoligonucleotide 471gaccugagcu uuguuguaga cuauc 2547225RNAArtificialoligonucleotide 472accugagcuu uguuguagac uauca 2547323RNAArtificialoligonucleotide 473ccugagcuuu guuguagacu auc 2347420RNAArtificialoligonucleotide 474cauuuuugac cuacaugugg 2047520RNAArtificialoligonucleotide 475uuugaccuac auguggaaag 2047626RNAArtificialoligonucleotide 476uacauuuuug accuacaugu ggaaag 2647717RNAArtificialoligonucleotide 477ggucuccuua ccuauga 1747824RNAArtificialoligonucleotide 478ucuuaccuau gacuauggau gaga 2447922RNAArtificialoligonucleotide 479auuuuugacc uacaugggaa ag 2248023RNAArtificialoligonucleotide 480uacgaguuga uugucggacc cag 2348125RNAArtificialoligonucleotide 481guggucuccu uaccuaugac ugugg 2548224RNAArtificialoligonucleotide 482ugucucagua aucuucuuac cuau 2448323RNAArtificialoligonucleotide 483ugcauguucc agucguugug ugg 2348425RNAArtificialoligonucleotide 484cacuauucca gucaaauagg ucugg 2548525RNAArtificialoligonucleotide 485auuuaccaac cuucaggauc gagua 2548621RNAArtificialoligonucleotide 486ggccuaaaac acauacacau a 2148724RNAArtificialoligonucleotide 487gauagguggu aucaacaucu guaa 2448821RNAArtificialoligonucleotide 488gauagguggu aucaacaucu g 2148919RNAArtificialoligonucleotide 489cuuccuggau ggcuugaau 1949020RNAArtificialoligonucleotide 490uguuguuguu uaugcucauu 2049119RNAArtificialoligonucleotide 491guacauuaag auggacuuc 1949220RNAArtificialoligonucleotide 492cuguugcagu aaucuaugag 2049320RNAArtificialoligonucleotide 493ugcaguaauc uaugaguuuc 2049418RNAArtificialoligonucleotide 494gagucuucua ggagccuu 1849523RNAArtificialoligonucleotide 495ugccauuguu ucaucagcuc uuu 2349620RNAArtificialoligonucleotide 496uccuguagga cauuggcagu 2049720RNAArtificialoligonucleotide 497cuuggagucu ucuaggagcc 2049830RNAArtificialoligonucleotide 498ccauuuugug aauguuuucu uuugaacauc 3049924RNAArtificialoligonucleotide 499cccauuuugu gaauguuuuc uuuu 2450024RNAArtificialoligonucleotide 500gaaaauugug cauuuaccca uuuu 2450122RNAArtificialoligonucleotide 501uugugcauuu acccauuuug ug 2250224RNAArtificialoligonucleotide 502cccugaggca uucccaucuu gaau 2450320RNAArtificialoligonucleotide 503aggacuuacu ugcuuuguuu 2050423RNAArtificialoligonucleotide 504cuugaauuua ggagauucau cug 2350523RNAArtificialoligonucleotide 505caucuucuga uaauuuuccu guu 2350620RNAArtificialoligonucleotide 506ccauuacagu ugucuguguu 2050723RNAArtificialoligonucleotide 507ugacagccug ugaaaucugu gag 2350820RNAArtificialoligonucleotide 508uaaucugccu cuucuuuugg 2050920RNAArtificialoligonucleotide 509cagcaguagu ugucaucugc 2051027RNAArtificialoligonucleotide 510gccugagcug aucugcuggc aucuugc 2751131RNAArtificialoligonucleotide 511gccugagcug aucugcuggc aucuugcagu u 3151216RNAArtificialoligonucleotide 512ucugcuggca ucuugc 1651322RNAArtificialoligonucleotide 513gccgguugac uucauccugu gc 2251423RNAArtificialoligonucleotide 514gucugcaucc aggaacaugg guc 2351524RNAArtificialoligonucleotide 515uacuuacugu cuguagcucu uucu 2451622RNAArtificialoligonucleotide 516cugcauccag gaacaugggu cc 2251724RNAArtificialoligonucleotide 517guugaagauc ugauagccgg uuga 2451817RNAArtificialoligonucleotide 518uaggugccug ccggcuu 1751918RNAArtificialoligonucleotide 519uucagcugua gccacacc 1852021RNAArtificialoligonucleotide 520cugaacugcu ggaaagucgc c 2152130RNAArtificialoligonucleotide 521cuggcuucca aaugggaccu gaaaaagaac 3052221RNAArtificialoligonucleotide 522caauuuuucc cacucaguau u 2152319RNAArtificialoligonucleotide 523uugaaguucc uggagucuu 1952422RNAArtificialoligonucleotide 524uccucaggag gcagcucuaa au 2252516RNAArtificialoligonucleotide 525uggcucucuc ccaggg 1652627RNAArtificialoligonucleotide 526gagauggcuc ucucccaggg acccugg 2752717RNAArtificialoligonucleotide 527gggcacuuug uuuggcg 1752819RNAArtificialoligonucleotide 528ggucccagca aguuguuug 1952925RNAArtificialoligonucleotide 529ugggaugguc ccagcaaguu guuug 2553021RNAArtificialoligonucleotide 530guagagcucu gucauuuugg g 2153125RNAArtificialoligonucleotide 531gcucaagaga uccacugcaa aaaac 2553226RNAArtificialoligonucleotide 532gccauacgua cguaucauaa acauuc 2653325RNAArtificialoligonucleotide 533ucugcaggau auccaugggc ugguc 2553427RNAArtificialoligonucleotide 534gauccucccu guucgucccc uauuaug 2753524RNAArtificialoligonucleotide 535ugcuuuagac uccuguaccu gaua 2453618RNAArtificialoligonucleotide 536ggcggccuuu guguugac 1853725RNAArtificialoligonucleotide 537ggacaggccu uuauguucgu gcugc 2553819RNAArtificialoligonucleotide 538ccuuuauguu cgugcugcu 1953920RNAArtificialoligonucleotide 539ucaaggaaga uggcauuucu 2054020RNAArtificialoligonucleotide 540ucaangaaga uggcauuucu 2054120RNAArtificialoligonucleotide 541ucaagnaaga uggcauuucu 2054220RNAArtificialoligonucleotide 542ucaaggaana uggcauuucu 2054320RNAArtificialoligonucleotide 543ucaaggaaga ungcauuucu 2054420RNAArtificialoligonucleotide 544ucaaggaaga ugncauuucu 2054520RNAArtificialoligonucleotide 545ncaaggaaga uggcauuucu 2054620RNAArtificialoligonucleotide 546ucaaggaaga nggcauuucu 2054720RNAArtificialoligonucleotide 547ucaaggaaga uggcanuucu 2054820RNAArtificialoligonucleotide 548ucaaggaaga uggcaunucu 2054920RNAArtificialoligonucleotide 549ucaaggaaga uggcauuncu 2055020RNAArtificialoligonucleotide 550ucaaggaaga uggcauuucn 2055120RNAArtificialoligonucleotide 551ucnaggaaga uggcauuucu 2055220RNAArtificialoligonucleotide 552ucanggaaga uggcauuucu 2055320RNAArtificialoligonucleotide 553ucaaggnaga uggcauuucu 2055420RNAArtificialoligonucleotide 554ucaagganga uggcauuucu 2055520RNAArtificialoligonucleotide 555ucaaggaagn uggcauuucu 2055620RNAArtificialoligonucleotide 556ucaaggaaga uggcnuuucu 2055725RNAArtificialoligonucleotide 557uuugccncug cccaaugcca uccug 2555825RNAArtificialoligonucleotide 558uuugccgcun cccaaugcca uccug 2555925RNAArtificialoligonucleotide 559uuugccgcug cccaauncca uccug 2556025RNAArtificialoligonucleotide 560uuunccgcug cccaaugcca uccug 2556125RNAArtificialoligonucleotide

561uuugccgcug cccaaugcca uccun 2556225RNAArtificialoligonucleotide 562nuugccgcug cccaaugcca uccug 2556325RNAArtificialoligonucleotide 563unugccgcug cccaaugcca uccug 2556425RNAArtificialoligonucleotide 564uungccgcug cccaaugcca uccug 2556525RNAArtificialoligonucleotide 565uuugccgcng cccaaugcca uccug 2556625RNAArtificialoligonucleotide 566uuugccgcug cccanugcca uccug 2556725RNAArtificialoligonucleotide 567uuugccgcug cccaaugccn uccug 2556825RNAArtificialoligonucleotide 568uuunccncug cccaaugcca uccug 2556925RNAArtificialoligonucleotide 569uuugccgcug cccaangcca uccug 2557025RNAArtificialoligonucleotide 570uuugccgcug cccaaugcca nccug 2557125RNAArtificialoligonucleotide 571uuugccgcug cccaaugcca uccng 2557225RNAArtificialoligonucleotide 572uuugccgcug cccnaugcca uccug 2557320RNAArtificialoligonucleotide 573ucagcuucun uuagccacug 2057420RNAArtificialoligonucleotide 574ucagcuucug uuanccacug 2057520RNAArtificialoligonucleotide 575ucancuucug uuagccacug 2057620RNAArtificialoligonucleotide 576ucagcuucug uuagccacun 20577153PRTHomo sapiens 577Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe 1 5 10 15 Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu 20 25 30 Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 35 40 45 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 50 55 60 Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 65 70 75 80 Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 85 90 95 Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 100 105 110 Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp 115 120 125 Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly 130 135 140 Ser Ala Gly Asn Lys Asn Tyr Arg Met 145 150

Claims

1. An isolated antisense oligonucleotide 13 to 50 nucleotides in length comprising an inosine base, wherein said oligonucleotide is capable of binding to an exon of the human dystrophin pre-mRNA so as to induce skipping of said exon.

2. The oligonucleotide of claim 1, said oligonucleotide being 20-50 nucleotides in length.

3. The oligonucleotide of claim 1, said oligonucleotide being 14-25 nucleotides in length.

4. The oligonucleotide of claim 1, said oligonucleotide being 20-25 nucleotides in length.

5. The oligonucleotide of claim 1, said oligonucleotide comprising from one to four inosine bases.

6. The oligonucleotide of claim 1, further comprising a base and/or sugar modification.

7. The oligonucleotide of claim 1, wherein said oligonucleotide is a 2'-O-methyl phosphorothioate oligonucleotide.

8. The oligonucleotide of claim 1, wherein said exon is selected from the group consisting of 51, 45, 53, 44, 46, 52, 50, 43 and 55.

9. The oligonucleotide of claim 1, said oligonucleotide being RNA.

10. An isolated antisense oligonucleotide consisting of a base or a nucleotide sequence selected from the group consisting of: SEQ ID NO: 2-473, 539-576, wherein said oligonucleotide comprises an inosine base.

11. The oligonucleotide of claim 8, further comprising a base and/or sugar modification.

12. The oligonucleotide of claim 8, wherein said oligonucleotide is a 2'-O-methyl phosphorothioate oligonucleotide.

13. The oligonucleotide of claim 8, said oligonucleotide being RNA.

14. A method for inducing skipping of an exon of human dystrophin pre-mRNA in a muscle cell, the method comprising contacting said cell with an oligonucleotide of claim 1 for a time and under conditions which permit exon skipping.

15. The oligonucleotide of claim 1, which is a locked nucleic acid oligonucleotide (LNA).

16. A method for inducing skipping of an exon of human dystrophin pre-mRNA in a human subject, the method comprising administering an oligonucleotide of claim 1 to said subject in an amount and for a time which is effective to induce exon skipping.

17. A method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual, the method comprising administering to said individual an oligonucleotide of claim 1, wherein said oligonucleotide induces skipping of an exon of a dystrophin pre-mRNA.

18. A method for inducing and/or promoting skipping of exon 43, 44, 45, 46, 50, 51, 52 or 53 of the dystrophin pre-mRNA in a patient, the method comprising administering an oligonucleotide of claim 4 to said patient.

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