METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA

Abstract

The invention relates to a method for inducing or promoting skipping of exon 45 of DMD pre-mRNA in a Duchenne Muscular Dystrophy patient, preferably in an isolated (muscle) cell, the method comprising providing an isolate muscle cell with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon. The invention further relates to such molecule used in the method.

Description

PRIORITY

[0001] This application is a U.S. continuation patent application of U.S. patent application Ser. No. 13/094,548 filed Apr. 26, 2011, which is a U.S. continuation patent application of PCT/NL2009/050006, filed on Jan. 13, 2009, which claims priority to PCT/NL2008/050673, filed on Oct. 27, 2008, the entirety of which is incorporated herein by reference.

FIELD

[0002] The invention relates to the field of genetics, more specifically human genetics. The invention in particular relates to human Duchenne Muscular Dystrophy.

BACKGROUND OF THE INVENTION

[0003] Myopathies are disorders that result in functional impairment of muscles. Muscular dystrophy (MD) refers to genetic diseases that are characterized by progressive weakness and degeneration of skeletal muscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. They are recessive disorders and because the gene responsible for DMD and BMD resides on the X-chromosome, mutations mainly affect males with an incidence of about 1 in 3500 boys.

[0004] DMD and BMD are caused by genetic defects in the DMD gene encoding dystrophin, a muscle protein that is required for interactions between the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction. DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and DMD patients often die before the age of thirty due to respiratory- or heart failure. In contrast. BMD patients often remain ambulatory until later in life, and have near normal life expectancies. DMD mutations in the DMD gene are mainly characterized by frame shifting insertions or deletions or nonsense point mutations, resulting in the absence of functional dystrophin. BMD mutations in general keep the reading frame intact, allowing synthesis of a partly functional dystrophin.

[0005] During the last decade, specific modification of splicing in order to restore the disrupted reading frame of the DMD transcript has emerged as a promising therapy for Duchenne muscular dystrophy (DMD) (van Ommen, van Deutekom. Aartsma-Rus. Curr Opin Mol Ther. 2008; 10(2):140-9, Yokota, Duddy, Partidge. Acta Myol. 2007; 26(3): 179-84, van Deutekom et al., N Engl J Med. 2007; 357(26):2677-86. Using antisense oligonucleotides (AONs) interfering with splicing signals the skipping of specific exons can be induced in the DMD pre-mRNA, thus restoring the open reading frame and converting the severe DMD into a milder BMD phenotype (van Deutekom et al. Hum Mol Genet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet 2003; 12(8):907-14.). In vivo proof-of-concept was first obtained in the mdx mouse model, which is dystrophin-deficient due to a nonsense mutation in exon 23. Intramuscular and intravenous injections of AONs targeting the mutated exon 23 restored dystrophin expression for at least three months (Lu et al. Nat Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci USA. 2005; 102(1):198-203). This was accompanied by restoration of dystrophin-associated proteins at the fiber membrane as well as functional improvement of the treated muscle. In vivo skipping of human exons has also been achieved in the hDMD mouse model, which contains a complete copy of the human DMD gene integrated in chromosome 5 of the mouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; 't Hoen et al. J Biol Chem. 2008; 283: 5899-907).

[0006] As the majority of DMD patients have deletions that cluster in hotspot regions, the skipping of a small number of exons is applicable to relatively large numbers of patients. The actual applicability of exon skipping can be determined for deletions, duplications and point mutations reported in DMD mutation databases such as the Leiden DMD mutation database available at www.dmd.nl. Therapeutic skipping of exon 45 of the DMD pre-mRNA would restore the open reading frame of DMD patients having deletions including but not limited to exons 12-44, 18-44, 44, 46, 46-47, 46-48, 46-49, 46-51, 46-53, 46-55, 46-59, 46-60 of the DMD pre-mRNA, occurring in a total of 16% of all DMD patients with a deletion (Aartsma-Rus and van Deutekom, 2007. Antisense Elements (Genetics) Research Focus. 2007 Nova Science Publishers, Inc). Furthermore, for some DMD patients the simultaneous skipping of one of more exons in addition to exon 45, such as exons 51 or 53 is required to restore the correct reading frame. None-limiting examples include patients with a deletion of exons 46-50 requiring the co-skipping of exons 45 and 51, or with a deletion of exons 46-52 requiring the co-skipping of exons 45 and 53.

[0007] Recently, a first-in-man study was successfully completed where an AON inducing the skipping of exon 51 was injected into a small area of the tibialis anterior muscle of four DMD patients. Novel dystrophin expression was observed in the majority of muscle fibers in all four patients treated, and the AON was safe and well tolerated (van Deutekom et al. N Engl J Med. 2007: 357: 2677-86).

[0008] Most AONs studied contain up to 20 nucleotides, and it has been argued that this relatively short size improves the tissue distribution and/or cell penetration of an AON. However, such short AONs will result in a limited specificity due to an increased risk for the presence of identical sequences elsewhere in the genome, and a limited target binding or target affinity due to a low free energy of the AON-target complex. Therefore the inventors decided to design new and optionally improved oligonucleotides that would not exhibit all of these drawbacks.

DESCRIPTION OF THE INVENTION

Method

[0009] In a first aspect, the invention provides a method for inducing and/or promoting skipping of exon 45 of DMD pre-mRNA in a patient, preferably in an isolated cell of said patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon. Accordingly, a method is herewith provided for inducing and/or promoting skipping of exon 45 of DMD pre-mRNA, preferably in an isolated cell of a patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon.

[0010] It is to be understood that said method encompasses an in vitro, in vivo or ex vivo method. As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient. The DMD gene or protein corresponds to the dystrophin gene or protein.

[0011] A patient is preferably intended to mean a patient having DMD or BMD as later defined herein or a patient susceptible to develop DMD or BMD due to his or her genetic background.

[0012] Exon skipping refers to the induction in a cell of a mature mRNA that does not contain a particular exon that is normally present therein. Exon skipping is achieved by providing a cell expressing the pre-mRNA of said mRNA with a molecule capable of interfering with sequences such as, for example, the splice donor or splice acceptor sequence that are both required for allowing the enzymatic process of splicing, or a molecule that is capable of interfering with an exon inclusion signal required for recognition of a stretch of nucleotides as an exon to be included in the mRNA. The term pre-mRNA refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template in the cell nucleus by transcription.

[0013] Within the context of the invention inducing and/or promoting skipping of an exon as indicated herein means that at least 1%. 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more (muscle) cells of a treated patient will not contain said exon. This is preferably assessed by PCR as described in the examples.

[0014] Preferably, a method of the invention by inducing or promoting skipping of exon 45 of the DMD pre-mRNA in one or more cells of a patient provides said patient with a functional dystrophin protein and/or decreases the production of an aberrant dystrophin protein in said patient. Therefore a preferred method is a method, wherein a patient or a cell of said patient is provided with a functional dystrophin protein and/or wherein the production of an aberrant dystrophin protein in said patient or in a cell of said patient is decreased Decreasing the production of an aberrant dystrophin may be assessed at the mRNA level and preferably means that 99%. 90%, 80%, 70%, 60%, 50%, 40%, 30%. 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional dystrophin mRNA or protein. A non functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein.

[0015] Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional dystrophin mRNA is detectable by RT PCR. In another embodiment, 1%. 5%, 10%, 20%, 30%. 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional dystrophin mRNA.

[0016] 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%, 100%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein.

[0017] 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 other words, a functional dystrophin is a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. "At least to some extent" preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin. In this context, an activity of a functional dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC) (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immunofluorescence analysis of cross-sections, from a muscle biopsy, as known to the skilled person.

[0018] Individuals or patients suffering from Duchenne muscular dystrophy typically have a mutation in the DMD gene that prevent synthesis of the complete dystrophin protein, i.e of a premature stop prevents the synthesis of the C-terminus. In Becker muscular dystrophy the DMD gene also comprises a mutation compared to the wild type gene but the mutation does typically not induce a premature stop and the C-terminus is typically synthesized. As a result a functional dystrophin protein is synthesized that has at least the same activity in kind as the wild type protein, not although not necessarily the same amount of activity. The genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a 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 (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Exon-skipping for the treatment of DMD is typically directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-shaped domain to correct the reading frame and allow synthesis of the remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain. In a preferred embodiment, an individual having DMD and being treated by a method as defined herein will be provided a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. More preferably, if said individual is a Duchenne patient or is suspected to be a Duchenne patient, a functional dystrophin is a dystrophin of an individual having BMD: typically said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006). Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). The central rod-shaped domain of wild type dystrophin comprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). For example, a central rod-shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC.

[0019] A method of the invention may alleviate one or more characteristics of a muscle cell from a DMD patient comprising deletions including but not limited to exons 12-44, 18-44, 44, 46, 46-47, 46-48, 46-49, 46-51, 46-53, 46-55, 46-59, 46-60 of the DMD pre-mRNA of said patient (Aartsma-Rus and van Deutekom, 2007, Antisense Elements (Genetics) Research Focus, 2007 Nova Science Publishers, Inc) as well as from DMD patients requiring the simultaneous skipping of one of more exons in addition to exon 45 including but not limited to patients with a deletion of exons 46-50 requiring the co-skipping of exons 45 and 51, or with a deletion of exons 46-52 requiring the co-skipping of exons 45 and 53.

[0020] In a preferred method, one or more symptom(s) or characteristic(s) of a myogenic cell or muscle cell from a DMD patient is/are alleviated. Such symptoms or characteristics may be assessed at the cellular, tissue level or on the patient self.

[0021] An alleviation of one or more symptoms or 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.

[0022] The improvement of muscle fiber function, integrity and/or survival may also 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.

[0023] Creatine kinase may be detected in blood as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). A detectable decrease in creatine kinase may mean a decrease of 5%, 10/%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same DMD patient before treatment.

[0024] A detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006) using biopsy cross-sections. A detectable decrease of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%. 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same DMD patient before treatment.

[0025] A detectable increase of the homogeneity of the diameter of muscle fibers is preferably assessed in a muscle biopsy cross-section, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). The increase is measured by comparison to the homogeneity of the diameter of muscle fibers in a muscle biopsy cross-section of a same DMD patient before treatment.

[0026] An alleviation of one or more symptoms or characteristics may be assessed by any of the following assays on the patient self: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur at al (Manzur A Y et al. (2008), Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review), Wiley publishers, The Cochrane collaboration.) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy has been alleviated in an individual using a method of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). Alternatively, the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.

[0027] A treatment in a method according to the invention may have a duration of at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more. The frequency of administration of an oligonucleotide, composition, compound of the invention may depend on several parameters such as the age of the patient, the type of mutation, the number of molecules (dose), the formulation of said molecule. The frequency may be ranged between at least once in a two weeks, or three weeks or four weeks or five weeks or a longer time period.

[0028] 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 and may be administered directly in vivo, ex vivo or in vitro. An oligonucleotide as used herein 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, 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, and may be administered directly or indirectly in vivo, ex vivo or in vitro. As Duchenne muscular dystrophy has 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.

[0029] A molecule or oligonucleotide or equivalent thereof can be delivered as is to a cell. When administering said molecule, oligonucleotide or equivalent thereof to an individual, it is preferred that it is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred for a method of the invention is the use of an excipient that will further enhance delivery of said molecule, oligonucleotide or functional equivalent thereof as defined herein, to a cell and into a cell, preferably a muscle cell. Preferred excipient are defined in the section entitled "pharmaceutical composition". In vitro, we obtained very good results using polyethylenimine (PEI, ExGen500, MBI Fermentas) as shown in the example.

[0030] In a preferred method of the invention, an additional molecule is used which is able to induce and/or promote skipping of a distinct exon of the DMD pre-mRNA of a patient. Preferably, the second exon is selected from: exon 7, 44, 46, 51, 53, 59, 67 of the dystrophin pre-mRNA of a patient. Molecules which can be used are depicted in table 2. Preferred molecules comprise or consist of any of the oligonucleotides as disclosed in table 2. Several oligonucleotides may also be used in combination. This way, inclusion of two or more exons of a DMD pre-mRNA in mRNA produced from this pre-mRNA is prevented. This embodiment is further referred to as double- or multi-exon skipping (Aartsma-Rus A, Janson A A, Kaman W E, et al. Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet 2004; 74(1):83-92, Aartsma-Rus A, Kaman W E, Weij R, den Dunnen J T, van Ommen G J, van Deutekom J C. Exploring the frontiers of therapeutic exon skipping for Duchenne muscular dystrophy by double targeting within one or multiple exons. Mol Ther 2006: 14(3):401-7). In most cases double-exon skipping results in the exclusion of only the two targeted exons from the 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-skipping was notably so for the combination of oligonucleotides derived from the DMD gene, wherein one oligonucleotide for exon 45 and one oligonucleotide for exon 51 was added to a cell transcribing the DMD gene. Such a set-up resulted in mRNA being produced that did not contain exons 45 to 51. Apparently, the structure of the pre-mRNA in the presence of the mentioned oligonucleotides was such that the splicing machinery was stimulated to connect exons 44 and 52 to each other. It is possible to specifically promote the skipping of also the intervening exons by providing a linkage between the two complementary oligonucleotides. Hence, in one embodiment stretches of nucleotides complementary to at least two dystrophin exons are separated by a linking moiety. The at least two stretches of nucleotides are thus linked in this embodiment so as to form a single molecule.

[0031] In case, more than one compounds are used in a method of the invention, said compounds can be administered to an individual in any order. In one embodiment, said compounds are administered simultaneously (meaning that said compounds are administered within 10 hours, preferably within one hour). This is however not necessary. In another embodiment, said compounds are administered sequentially.

Molecule

[0032] In a second aspect, there is provided a molecule for use in a method as described in the previous section entitled "Method". This molecule preferably comprises or consists of an oligonucleotide, Said oligonucleotide is preferably an antisense oligonucleotide (AON) or antisense oligoribonucleotide.

[0033] It was found by the present investigators that especially exon 45 is specifically skipped at a high frequency using a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon. Although this effect can be associated with a higher binding affinity of said molecule, compared to a molecule that binds to a continuous stretch of less than 21 nucleotides, there could be other intracellular parameters involved that favor thermodynamic, kinetic, or structural characteristics of the hybrid duplex. In a preferred embodiment, a molecule that binds to a continuous stretch of at least 21, 25, 30, 35, 40, 45, 50 nucleotides within said exon is used.

[0034] In a preferred embodiment, a molecule or an oligonucleotide of the invention which comprises a sequence that is complementary to a part of exon 45 of DMD pre-mRNA is such that the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% and most preferably up to 100%. "A part of exon 45" preferably means a stretch of at least 21 nucleotides. In a most preferred embodiment, an oligonucleotide of the invention consists of a sequence that is complementary to part of exon 45 dystrophin pre-mRNA as defined herein. Alternatively, an oligonucleotide may comprise a sequence that is complementary to part of exon 45 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 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. Several types of flanking sequences may be used. Preferably, additional flanking sequences are used to modify the binding of a protein to said molecule or oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity. In another preferred embodiment, additional flanking sequences are complementary to sequences of the DMD pre-mRNA which are not present in exon 45. Such flanking sequences are preferably complementary to sequences comprising or consisting of the splice site acceptor or donor consensus sequences of exon 45. In a preferred embodiment, such flanking sequences are complementary to sequences comprising or consisting of sequences of an intron of the DMD pre-mRNA which is adjacent to exon 45; i.e. intron 44 or 45.

[0035] A continuous stretch of at least 21, 25, 30, 35, 40, 45, 50 nucleotides within exon 45 is preferably selected from the sequence:

TABLE-US-00001 (SEQ ID NO 2) 5'-CCAGGAUGGCAUUGGGCAGCGGCAAACUGUUGUCAGA ACAUUGAAUGCAACUGGGGAAGAAAUAAUUCAGCAAUC-3'.

[0036] It was found that a molecule that binds to a nucleotide sequence comprising or consisting of a continuous stretch of at least 21, 25, 30, 35, 40, 45, 50 nucleotides of SEQ ID NO. 2 results in highly efficient skipping of exon 45 in a cell provided with this molecule. Molecules that bind to a nucleotide sequence comprising a continuous stretch of less than 21 nucleotides of SEQ ID NO:2 were found to induce exon skipping in a less efficient way than the molecules of the invention. Therefore, in a preferred embodiment, a method is provided wherein a molecule binds to a continuous stretch of at least 21, 25, 30, 35 nucleotides within SEQ ID NO:2. Contrary to what was generally thought, the inventors surprisingly found that a higher specificity and efficiency of exon skipping may be reached using an oligonucleotides having a length of at least 21 nucleotides. None of the indicated sequences is derived from conserved parts of splice-junction sites. Therefore, said molecule is not likely to mediate differential splicing of other exons from the DMD pre-mRNA or exons from other genes.

[0037] In one embodiment, a molecule of the invention capable of interfering with the inclusion of exon 45 of the DMD pre-mRNA is a compound molecule that binds to the specified sequence, or a protein such as an RNA-binding protein or a non-natural zinc-finger protein that has been modified to be able to bind to the indicated nucleotide sequence on a RNA molecule. Methods for screening compound molecules that bind specific nucleotide sequences are for example disclosed in PCT/NL01/00697 and U.S. Pat. No. 6,875,736, which are herein enclosed by reference. Methods for designing RNA-binding Zinc-finger proteins that bind specific nucleotide sequences are disclosed by Friesen and Darby, Nature Structural Biology 5: 543-546 (1998) which is herein enclosed by reference.

[0038] In a further embodiment, a molecule of the invention capable of interfering with the inclusion of exon 45 of the DMD pre-mRNA comprises an antisense oligonucleotide that is complementary to and can base-pair with the coding strand of the pre-mRNA of the DMD gene. Said antisense oligonucleotide preferably contains a RNA residue, a DNA residue, and/or a nucleotide analogue or equivalent, as will be further detailed herein below.

[0039] A preferred molecule of the invention comprises a nucleotide-based or nucleotide or an antisense oligonucleotide sequence of between 21 and 50 nucleotides or bases, more preferred between 21 and 40 nucleotides, more preferred between 21 and 30 nucleotides, such as 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides. 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides. 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides. 45 nucleotides. 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides or 50 nucleotides.

[0040] A most preferred molecule of the invention comprises a nucleotide-based sequence of 25 nucleotides.

[0041] In a preferred embodiment, a molecule of the invention binds to a continuous stretch of or is complementary to or is antisense to at least a continuous stretch of at least 21 nucleotides within the nucleotide sequence SEQ ID NO:2.

[0042] In a certain embodiment, the invention provides a molecule comprising or consisting of an antisense nucleotide sequence selected from the antisense nucleotide sequences as depicted in Table 1, except SEQ ID NO:68. A molecule of the invention that is antisense to the sequence of SEQ ID NO 2, which is present in exon 45 of the DMD gene preferably comprises or consists of the antisense nucleotide sequence of SEQ ID NO 3; SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42. SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53. SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66 and/or SEQ ID NO:67.

[0043] In a more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 3; SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and/or SEQ ID NO 8.

[0044] In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 3. It was found that this molecule is very efficient in modulating splicing of exon 45 of the DMD pre-mRNA in a muscle cell.

[0045] A nucleotide sequence of a molecule of the invention may contain a RNA residue, a DNA residue, a nucleotide analogue or equivalent as will be further detailed herein below. In addition, a molecule of the invention may encompass a functional equivalent of a molecule of the invention as defined herein.

[0046] It is preferred that a molecule of the invention comprises a or at least one residue that is modified to increase nuclease resistance, and/or to increase the affinity of the antisense nucleotide for the target sequence. Therefore, in a preferred embodiment, an antisense nucleotide sequence comprises a or at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.

[0047] In a preferred embodiment, a nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.

[0048] It is further preferred that the linkage between a residue in a backbone does not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.

[0049] A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495497). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566-568).

[0050] A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.

[0051] In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a substitution of at least one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester. H-phosphonate, methyl and other alkyl phosphonate including 3'-alkylene phosphonate, 5'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.

[0052] A further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2', 3' and/or 5' position such as a --OH; --F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; aminoxy, methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably a ribose or a derivative thereof, or deoxyribose or derivative thereof. Such preferred derivatized sugar moieties comprise Locked Nucleic Acid (LNA), in which the 2'-carbon atom is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2'-O,4'-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA.

[0053] It is understood by a skilled person that it is not necessary for all positions in an antisense oligonucleotide to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single antisense oligonucleotide or even at a single position within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents.

[0054] A preferred antisense oligonucleotide according to the invention comprises a 2'-O-alkyl phosphorothioate antisense oligonucleotide, such as 2'-O-methyl modified ribose (RNA), 2'-O-ethyl modified ribose, 2'-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.

[0055] A most preferred antisense oligonucleotide according to the invention comprises a 2'-O-methyl phosphorothioate ribose.

[0056] A functional equivalent of a molecule of the invention may be defined as an oligonucleotide as defined herein wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is inducing exon 45 skipping and providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by detection of exon 45 skipping and quantifying the amount of a functional dystrophin protein. A functional dystrophin is herein preferably defined as being a dystrophin able to bind actin and members of the DGC protein complex. The assessment of said activity of an oligonucleotide is preferably done by RT-PCR or by immunofluorescence or Western blot analysis. Said activity is preferably retained to at least some extent when it represents at least 50%, or at least 60%, or at least 70% or at least 80% or at least 90% or at least 95% or more of corresponding activity of said oligonucleotide the functional equivalent derives from. Throughout this application, when the word oligonucleotide is used it may be replaced by a functional equivalent thereof as defined herein.

[0057] It will also be understood by a skilled person that distinct antisense oligonucleotides can be combined for efficiently skipping of exon 45 of the human DMD pre-mRNA. In a preferred embodiment, a combination of at least two antisense oligonucleotides are used in a method of the invention, such as two distinct antisense oligonucleotides, three distinct antisense oligonucleotides, four distinct antisense oligonucleotides, or five distinct antisense oligonucleotides or even more. It is also encompassed by the present invention to combine several oligonucleotides or molecules as depicted in table 1 except SEQ ID NO:68.

[0058] An antisense oligonucleotide can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells, preferably myogenic cells or muscle cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.

[0059] A preferred antisense oligonucleotide comprises a peptide-linked PMO.

[0060] A preferred antisense oligonucleotide comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an antisense oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an antisense oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells. Therefore, in one embodiment it is preferred to use a combination of antisense oligonucleotides comprising different nucleotide analogs or equivalents for modulating skipping of exon 45 of the human DMD pre-mRNA.

[0061] A cell can be provided with a molecule capable of interfering with essential sequences that result in highly efficient skipping of exon 45 of the human DMD pre-mRNA by plasmid-derived antisense oligonucleotide expression or viral expression provided by viral-based vector. Such a viral-based vector comprises an expression cassette that drives expression of an antisense molecule as defined herein. Preferred virus-based vectors include adenovirus- or adeno-associated virus-based vectors. Expression is preferably driven by a polymerase III promoter, such as a U1, a U6, or a U7 RNA promoter. A muscle or myogenic cell can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution. Alternatively, a plasmid can be provided by transfection using known transfection agentia such as, for example, LipofectAMINE.TM. 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), or derivatives thereof.

[0062] One preferred antisense oligonucleotide expression system is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-based vectors have been developed that can be used for prolonged expression of small antisense nucleotide sequences for highly efficient skipping of exon 45 of the DMD pre-mRNA.

[0063] A preferred AAV-based vector comprises an expression cassette that is driven by a polymerase Ill-promoter (Pol III). A preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA promoter.

[0064] The invention therefore also provides a viral-based vector, comprising a Pol III-promoter driven expression cassette for expression of one or more antisense sequences of the invention for inducing skipping of exon 45 of the human DMD pre-mRNA.

Pharmaceutical Composition

[0065] If required, a molecule or a vector expressing an antisense oligonucleotide of the invention can be incorporated into a pharmaceutically active mixture or composition by adding a pharmaceutically acceptable carrier.

[0066] Therefore, in a further aspect, the invention provides a composition, preferably a pharmaceutical composition comprising a molecule comprising an antisense oligonucleotide according to the invention, and/or a viral-based vector expressing the antisense sequence(s) according to the invention and a pharmaceutically acceptable carrier.

[0067] A preferred pharmaceutical composition comprises a molecule as defined herein and/or a vector as defined herein, and a pharmaceutical acceptable carrier or excipient, optionally combined with a molecule and/or a vector which is able to modulate skipping of exon 7, 44, 46, 51, 53, 59, 67 of the DMD pre-mRNA.

[0068] Preferred excipients include excipients capable of forming complexes, vesicles and/or liposomes that deliver such a molecule as defined herein, preferably an oligonucleotide complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine and derivatives, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils, Lipofectin.TM., DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such molecule, preferably an oligonucleotide as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver (oligonucleotide such as antisense) nucleic acids to a wide variety of cultured cells, including muscle cells. We obtained very good results using polyethylenimine (PEI, ExGen500, MBI Fermentas) as shown in the example. 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.

[0069] 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.

[0070] Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver a molecule or a compound as defined herein, preferably an oligonucleotide across cell membranes into cells.

[0071] In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate a compound as defined herein, preferably an oligonucleotide as colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of a compound as defined herein, preferably an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver a compound as defined herein, preferably an oligonucleotide for use in the current invention to deliver said compound for the treatment of Duchenne Muscular Dystrophy in humans.

[0072] In addition, a compound as defined herein, preferably an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake 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 a compound as defined herein, preferably an oligonucleotide from vesicles, e.g. endosomes or lysosomes.

[0073] Therefore, in a preferred embodiment, a compound as defined herein, preferably an oligonucleotide are formulated in a medicament which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising a compound as defined herein, preferably an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery.

[0074] It is to be understood that a molecule or compound or oligonucleotide may not be formulated in one single composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each compound.

[0075] In a preferred embodiment, an in vitro concentration of a molecule or an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 .quadrature.M is used. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM, molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If several molecules or oligonucleotides are used, these concentrations may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.

[0076] More preferably, a compound preferably an oligonucleotide and an adjunct compound to be used in the invention to prevent, treat DMD are synthetically produced and administered directly to a cell, a tissue, an organ and/or patients in formulated form in a pharmaceutically acceptable composition or preparation. The delivery of a pharmaceutical composition to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body.

Use

[0077] In yet a further aspect, the invention provides the use of an antisense oligonucleotide or molecule according to the invention, and/or a viral-based vector that expresses one or more antisense sequences according to the invention and/or a pharmaceutical composition, for inducing and/or promoting splicing of the DMD pre-mRNA. The splicing is preferably modulated in a human myogenic cell or a muscle cell in vitro. More preferred is that splicing is modulated in human a myogenic cell or muscle cell in vivo.

[0078] Accordingly, the invention further relates to the use of the molecule as defined herein and/or the vector as defined herein and/or or the pharmaceutical composition as defined herein for inducing and/or promoting splicing of the DMD pre-mRNA or for the preparation of a medicament for the treatment of a DMD patient.

[0079] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of" meaning that a molecule or a viral-based vector or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0080] Each embodiment as identified herein may be combined together unless otherwise indicated.

[0081] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0082] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1. In human control myotubes, a series of AONs (PS220 to PS225: SEQ ID NO: 3 to 8), all binding to a continuous stretch of at least 21 nucleotides within a specific sequence of exon 45 (i.e. SEQ ID NO:2), were tested at two different concentrations (200 and 500 nM). All six AONs were effective in inducing specific exon 45 skipping, as confirmed by sequence analysis (not shown). PS220 (SEQ ID NO:3) however, reproducibly induced highest levels of exon 45 skipping (see FIG. 2). (NT: non-treated cells, M: size marker).

[0084] FIG. 2. In human control myotubes, 25-mer PS220 (SEQ ID NO: 3) was tested at increasing concentration. Levels of exon 45 skipping of up to 75% (at 400 nM) were observed reproducibly, as assessed by Agilent LabChip Analysis.

[0085] FIG. 3. In human control myotubes, the efficiencies of a "short" 17-mer AON45-5 (SEQ ID NO:68) and its overlapping "long" 25-mer counterpart PS220 were directly compared at 200 nM and 500 nM. PS220 was markedly more efficient at both concentrations: 63% when compared to 3% obtained with 45-5. (NT: non-treated cells, M: size marker).

EXAMPLES

Examples 1 and 2

Materials and Methods

[0086] AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program (Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res., 31, 3406-3415), and on (partly) overlapping putative SR-protein binding sites as predicted by numerous software programs such as ESEfinder (Cartegni, L. et al. (2003) ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res, 31, 3568-71; Smith, P. J. et al. (2006) An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. Hum. Mol. Genet., 15, 2490-2508) that predicts binding sites for the four most abundant SR proteins (SF2/ASF, SC35, SRp40 and SRp55). AONs were synthesized by Prosensa Therapeutics B. V. (Leiden, Netherlands), and contain 2'-O-methyl RNA and full-length phosphorothioate (PS) backbones.

Tissue Culturing, Transfection and RT-PCR Analysis

[0087] Myotube cultures derived from a healthy individual ("human control") were obtained as described previously (Aartsma-Rus et al. Hum Mol Genet 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 0 to 500 nM of each AON. The transfection reagent polyethylenimine (PEI, ExGen500 MBI Fermentas) was used according to manufacturer's instructions, with 2 .mu.l PEI per .mu.g AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking exon 45. PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the Agilent DNA 1000 LabChip Kit and the Agilent 2100 bioanalyzer (Agilent Technologies, USA).

Results

[0088] A series of AONs targeting sequences within SEQ ID NO:2 within exon 45 were designed and tested in normal myotube cultures, by transfection and subsequent RT-PCR and sequence analysis of isolated RNA. PS220 (SEQ ID NO: 3) reproducibly induced highest levels of exon 45 skipping, when compared to PS221-PS225 (FIG. 1). High levels of exon 45 skipping of up to 75% were already obtained at 400 nM PS220 (FIG. 2). In a direct comparison, PS220 (a 25-mer) was reproducibly more efficient in inducing exon 45 skipping than its shorter 17-mer counterpart AON 45-5 (SEQ ID NO: 68; previously published as h45AON5 (Aartsma-Rus et al. Am J Hum Genet 2004; 74: 83-92)), at both AON concentrations of 200 nM and 500 nM and with 63% versus 3% respectively at 500 nM (FIG. 3). This result is probably due to the fact that the extended length of PS220, in fact completely overlapping AON 45-5, increases the free energy of the AON-target complex such that the efficiency of inducing exon 45 skipping is also increased.

TABLE-US-00002 TABLE 1 AONs in exon 45 SEQ ID NO 3 UUUGCCGCUGCCCAAUGCCAUCCUG (PS220) SEQ ID NO 4 AUUCAAUGUUCUGACAACAGUUUGC (PS221) SEQ ID NO 5 CCAGUUGCAUUCAAUGUUCUGACAA (PS222) SEQ ID NO 6 CAGUUGCAUUCAAUGUUCUGAC (PS223) SEQ ID NO 7 AGUUGCAUUCAAUGUUCUGA (PS224) SEQ ID NO 8 GAUUGCUGAAUUAUUUCUUCC (PS225) SEQ ID NO 9 GAUUGCUGAAUUAUUUCUUCCCCAG SEQ ID NO 10 AUUGCUGAAUUAUUUCUUCCCCAGU SEQ ID NO 11 UUGCUGAAUUAUUUCUUCCCCAGUU SEQ ID NO 12 UGCUGAAUUAUUUCUUCCCCAGUUG SEQ ID NO 13 GCUGAAUUAUUUCUUCCCCAGUUGC SEQ ID NO 14 CUGAAUUAUUUCUUCCCCAGUUGCA SEQ ID NO 15 UGAAUUAUUUCUUCCCCAGUUGCAU SEQ ID NO 16 GAAUUAUUUCUUCCCCAGUUGCAUU SEQ ID NO 17 AAUUAUUUCUUCCCCAGUUGCAUUC SEQ ID NO 18 AUUAUUUCUUCCCCAGUUGCAUUCA SEQ ID NO 19 UUAUUUCUUCCCCAGUUGCAUUCAA SEQ ID NO 20 UAUUUCUUCCCCAGUUGCAUUCAAU SEQ ID NO 21 AUUUCUUCCCCAGUUGCAUUCAAUG SEQ ID NO 22 UUUCUUCCCCAGUUGCAUUCAAUGU SEQ ID NO 23 UUCUUCCCCAGUUGCAUUCAAUGUU SEQ ID NO 24 UCUUCCCCAGUUGCAUUCAAUGUUC SEQ ID NO 25 CUUCCCCAGUUGCAUUCAAUGUUCU SEQ ID NO 26 UUCCCCAGUUGCAUUCAAUGUUCUG SEQ ID NO 27 UCCCCAGUUGCAUUCAAUGUUCUGA SEQ ID NO 28 CCCCAGUUGCAUUCAAUGUUCUGAC SEQ ID NO 29 CCCAGUUGCAUUCAAUGUUCUGACA SEQ ID NO 30 CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID NO 31 CAGUUGCAUUCAAUGUUCUGACAAC SEQ ID NO 32 AGUUGCAUUCAAUGUUCUGACAACA SEQ ID NO 33 UCC UGU AGA AUA CUG GCA UC SEQ ID NO 34 UGC AGA CCU CCU GCC ACC GCA GAU UCA SEQ ID NO 35 UUGCAGACCUCCUGCCACCGCAGAUUCAG GCUUC SEQ ID NO 36 GUUGCAUUCAAUGUUCUGACAACAG SEQ ID NO 37 UUGCAUUCAAUGUUCUGACAACAGU SEQ ID NO 38 UGCAUUCAAUGUUCUGACAACAGUU SEQ ID NO 39 GCAUUCAAUGUUCUGACAACAGUUU SEQ ID NO 40 CAUUCAAUGUUCUGACAACAGUUUG SEQ ID NO 41 AUUCAAUGUUCUGACAACAGUUUGC SEQ ID NO 42 UCAAUGUUCUGACAACAGUUUGCCG SEQ ID NO 43 CAAUGUUCUGACAACAGUUUGCCGC SEQ ID NO 44 AAUGUUCUGACAACAGUUUGCCGCU SEQ ID NO 45 AUGUUCUGACAACAGUUUGCCGCUG SEQ ID NO 46 UGUUCUGACAACAGUUUGCCGCUGC SEQ ID NO 47 GUUCUGACAACAGUUUGCCGCUGCC SEQ ID NO 48 UUCUGACAACAGUUUGCCGCUGCCC SEQ ID NO 49 UCUGACAACAGUUUGCCGCUGCCCA SEQ ID NO 50 CUGACAACAGUUUGCCGCUGCCCAA SEQ ID NO 51 UGACAACAGUUUGCCGCUGCCCAAU SEQ ID NO 52 GACAACAGUUUGCCGCUGCCCAAUG SEQ ID NO 53 ACAACAGUUUGCCGCUGCCCAAUGC SEQ ID NO 54 CAACAGUUUGCCGCUGCCCAAUGCC SEQ ID NO 55 AACAGUUUGCCGCUGCCCAAUGCCA SEQ ID NO 56 ACAGUUUGCCGCUGCCCAAUGCCAU SEQ ID NO 57 CAGUUUGCCGCUGCCCAAUGCCAUC SEQ ID NO 58 AGUUUGCCGCUGCCCAAUGCCAUCC SEQ ID NO 59 GUUUGCCGCUGCCCAAUGCCAUCCU SEQ ID NO 60 UUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 61 UUGCCGCUGCCCAAUGCCAUCCUGG SEQ ID NO 62 UGCCGCUGCCCAAUGCCAUCCUGGA SEQ ID NO 63 GCCGCUGCCCAAUGCCAUCCUGGAG SEQ ID NO 64 CCGCUGCCCAAUGCCAUCCUGGAGU SEQ ID NO 65 CGCUGCCCAAUGCCAUCCUGGAGUU SEQ ID NO 66 UGU UUU UGA GGA UUG CUG AA SEQ ID NO 67 UGUUCUGACAACAGUUUGCCGCUGCCCAAUGC CAUCCUGG SEQ ID NO 68 GCCCAAUGCCAUCCUGG (45-5)

TABLE-US-00003 TABLE 2 AONs in exons 51, 53, 7, 44, 46, 59, and 67 DMD Gene Exon 51 SEQ ID NO 69 AGAGCAGGUACCUCCAACAUCAAGG SEQ ID NO 70 GAGCAGGUACCUCCAACAUCAAGGA SEQ ID NO 71 AGCAGGUACCUCCAACAUCAAGGAA SEQ ID NO 72 GCAGGUACCUCCAACAUCAAGGAAG SEQ ID NO 73 CAGGUACCUUCCAACAUCAAGGAAGA SEQ ID NO 74 AGGUACCUCCAACAUCAAGGAAGAU SEQ ID NO 75 GGUACCUCCAACAUCAAGGAAGAUG SEQ ID NO 76 GUACCUCCAACAUCAAGGAAGAUGG SEQ ID NO 77 UACCUCCAACAUCAAGGAAGAUGGC SEQ ID NO 78 ACCUCCAACAUCAAGGAAGAUGGCA SEQ ID NO 79 CCUCCAACAUCAAGGAAGAUGGCAU SEQ ID NO 80 CUCCAACAUCAAGGAAGAUGGCAUU SEQ ID NO 81 CUCCAACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO 82 UCCAACAUCAAGGAAGAUGGCAUUU SEQ ID NO 83 CCAACAUCAAGGAAGAUGGCAUUUC SEQ ID NO 84 CAACAUCAAGGAAGAUGGCAUUUCU SEQ ID NO 85 AACAUCAAGGAAGAUGGCAUUUCUA SEQ ID NO 86 ACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO 87 ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG SEQ ID NO 88 ACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO 89 CAUCAAGGAAGAUGGCAUUUCUAGU SEQ ID NO 90 AUCAAGGAAGAUGGCAUUUCUAGUU SEQ ID NO 91 UCAAGGAAGAUGGCAUUUCUAGUUU SEQ ID NO 92 UCAAGGAAGAUGGCAUUUCU SEQ ID NO 93 CAAGGAAGAUGGCAUUUCUAGUUUG SEQ ID NO 94 AAGGAAGAUGGCAUUUCUAGUUUGG SEQ ID NO 95 AGGAAGAUGGCAUUUCUAGUUUGGA SEQ ID NO 96 GGAAGAUGGCAUUUCUAGUUUGGAG SEQ ID NO 97 GAAGAUGGCAUUUCUAGUUUGGAGA SEQ ID NO 98 AAGAUGGCAUUUCUAGUUUGGAGAU SEQ ID NO 99 AGAUGGCAUUUCUAGUUUGGAGAUG SEQ ID NO 100 GAUGGCAUUUCUAGUUUGGAGAUGG SEQ ID NO 101 AUGGCAUUUCUAGUUUGGAGAUGGC SEQ ID NO 102 UGGCAUUUCUAGUUUGGAGAUGGCA SEQ ID NO 103 GGCAUUUCUAGUUUGGAGAUGGCAG SEQ ID NO 104 GCAUUUCUAGUUUGGAGAUGGCAGU SEQ ID NO 105 CAUUUCUAGUUUGGAGAUGGCAGUU SEQ ID NO 106 AUUUCUAGUUUGGAGAUGGCAGUUU SEQ ID NO 107 UUUCUAGUUUGGAGAUGGCAGUUUC SEQ ID NO 108 UUCUAGUUUGGAGAUGGCAGUUUCC DMD Gene Exon 53 SEQ ID NO 109 CCAUUGUGUUGAAUCCUUUAACAUU SEQ ID NO 110 CCAUUGUGUUGAAUCCUUUAAC SEQ ID NO 111 AUUGUGUUGAAUCCUUUAAC SEQ ID NO 112 CCUGUCCUAAGACCUGCUCA SEQ ID NO 113 CUUUUGGAUUGCAUCUACUGUAUAG SEQ ID NO 114 CAUUCAACUGUUGCCUCCGGUUCUG SEQ ID NO 115 CUGUUGCCUCCGGUUCUGAAGGUG SEQ ID NO 116 CAUUCAACUGUUGCCUCCGGUUCUGAAGGUG SEQ ID NO 117 CUGAAGGUGUUCUUGUACUUCAUCC SEQ ID NO 118 UGUAUAGGGACCCUCCUUCCAUGACUC SEQ ID NO 119 AUCCCACUGAUUCUGAAUUC SEQ ID NO 120 UUGGCUCUGGCCUGUCCUAAGA SEQ ID NO 121 AAGACCUGCUCAGCUUCUUUCCUUAGCUUCCAGCCA DMD Gene Exon 7 SEQ ID NO 122 UGCAUGUUCCAGUCGUUGUGUGG SEQ ID NO 123 CACUAUUCCAGUCAAAUAGGUCUGG SEQ ID NO 124 AUUUACCAACCUUCAGGAUCGAGUA SEQ ID NO 125 GGCCUAAAACACAUACACAUA DMD Gene Exon 44 SEQ ID NO 126 UCAGCUUCUGUUAGCCACUG SEQ ID NO 127 UUCAGCUUCUGUUAGCCACU SEQ ID NO 128 UUCAGCUUCUGUUAGCCACUG SEQ ID NO 129 UCAGCUUCUGUUAGCCACUGA SEQ ID NO 130 UUCAGCUUCUGUUAGCCACUGA SEQ ID NO 131 UCAGCUUCUGUUAGCCACUGA SEQ ID NO 132 UUCAGCUUCUGUUAGCCACUGA SEQ ID NO 133 UCAGCUUCUGUUAGCCACUGAU SEQ ID NO 134 UUCAGCUUCUGUUAGCCACUGAU SEQ ID NO 135 UCAGCUUCUGUUAGCCACUGAUU SEQ ID NO 136 UUCAGCUUCUGUUAGCCACUGAUU SEQ ID NO 137 UCAGCUUCUGUUAGCCACUGAUUA SEQ ID NO 138 UUCAGCUUCUGUUAGCCACUGAUA SEQ ID NO 139 UCAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 140 UUCAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 141 UCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 142 UUCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 143 CAGCUUCUGUUAGCCACUG SEQ ID NO 144 CAGCUUCUGUUAGCCACUGAU SEQ ID NO 145 AGCUUCUGUUAGCCACUGAUU SEQ ID NO 146 CAGCUUCUGUUAGCCACUGAUU SEQ ID NO 147 AGCUUCUGUUAGCCACUGAUUA SEQ ID NO 148 CAGCUUCUGUUAGCCACUGAUUA SEQ ID NO 149 AGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 150 CAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 151 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 152 CAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 153 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 154 AGCUUCUGUUAGCCACUGAU SEQ ID NO 155 GCUUCUGUUAGCCACUGAUU SEQ ID NO 156 AGCUUCUGUUAGCCACUGAUU SEQ ID NO 157 GCUUCUGUUAGCCACUGAUUA SEQ ID NO 158 AGCUUCUGUUAGCCACUGAUUA SEQ ID NO 159 GCUUCUGUUAGCCACUGAUUAA SEQ ID NO 160 AGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 161 GCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 162 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 163 GCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 164 CCAUUUGUAUUUAGCAUGUUCCC SEQ ID NO 165 AGAUACCAUUUGUAUUUAGC SEQ ID NO 166 GCCAUUUCUCAACAGAUCU SEQ ID NO 167 GCCAUUUCUCAACAGAUCUGUCA SEQ ID NO 168 AUUCUCAGGAAUUUGUGUCUUUC SEQ ID NO 169 UCUCAGGAAUUUGUGUCUUUC SEQ ID NO 170 GUUCAGCUUCUGUUAGCC SEQ ID NO 171 CUGAUUAAAUAUCUUUAUAU C SEQ ID NO 172 GCCGCCAUUUCUCAACAG SEQ ID NO 173 GUAUUAGCAUGUUCCCA SEQ ID NO 174 CAGGAAUUUGUGUCUUUC DMD Gene Exon 46 SEQ ID NO 175 GCUUUUCUUUUAGUUGCUGCUCUUU SEQ ID NO 176 CUUUUCUUUUAGUUGCUGCUCUUUU SEQ ID NO 177 UUUUCUUUUAGUUGCUGCUCUUUUC SEQ ID NO 178 UUUCUUUUAGUUGCUGCUCUUUUCC SEQ ID NO 179 UUCUUUUAGUUGCUGCUCUUUUCCA SEQ ID NO 180 UCUUUUAGUUGCUGCUCUUUUCCAG SEQ ID NO 181 CUUUUAGUUGCUGCUCUUUUCCAGG SEQ ID NO 182 UUUUAGUUGCUGCUCUUUUCCAGGU SEQ ID NO 183 UUUAGUUGCUGCUCUUUUCCAGGUU SEQ ID NO 184 UUAGUUGCUGCUCUUUUCCAGGUUC SEQ ID NO 185 UAGUUGCUGCUCUUUUCCAGGUUCA SEQ ID NO 186 AGUUGCUGCUCUUUUCCAGGUUCAA SEQ ID NO 187 GUUGCUGCUCUUUUCCAGGUUCAAG SEQ ID NO 188 UUGCUGCUCUUUUCCAGGUUCAAGU SEQ ID NO 189 UGCUGCUCUUUUCCAGGUUCAAGUG SEQ ID NO 190 GCUGCUCUUUUCCAGGUUCAAGUGG SEQ ID NO 191 CUGCUCUUUUCCAGGUUCAAGUGGG SEQ ID NO 192 UGCUCUUUUCCAGGUUCAAGUGGGA SEQ ID NO 193 GCUCUUUUCCAGGUUCAAGUGGGAC SEQ ID NO 194 CUCUUUUCCAGGUUCAAGUGGGAUA SEQ ID NO 195 UCUUUUCCAGGUUCAAGUGGGAUAC SEQ ID NO 196 CUUUUCCAGGUUCAAGUGGGAUACU SEQ ID NO 197 UUUUCCAGGUUCAAGUGGGAUACUA SEQ ID NO 198 UUUCCAGGUUCAAGUGGGAUACUAG SEQ ID NO 199 UUCCAGGUUCAAGUGGGAUACUAGC SEQ ID NO 200 UCCAGGUUCAAGUGGGAUACUAGCA SEQ ID NO 201 CCAGGUUCAAGUGGGAUACUAGCAA SEQ ID NO 202 CAGGUUCAAGUGGGAUACUAGCAAU SEQ ID NO 203 AGGUUCAAGUGGGAUACUAGCAAUG SEQ ID NO 204 GGUUCAAGUGGGAUACUAGCAAUGU SEQ ID NO 205 GUUCAAGUGGGAUACUAGCAAUGUU SEQ ID NO 206 UUCAAGUGGGAUACUAGCAAUGUUA SEQ ID NO 207 UCAAGUGGGAUACUAGCAAUGUUAU SEQ ID NO 208 CAAGUGGGAUACUAGCAAUGUUAUC SEQ ID NO 209 AAGUGGGAUACUAGCAAUGUUAUCU SEQ ID NO 210 AGUGGGAUACUAGCAAUGUUAUCUG SEQ ID NO 211 GUGGGAUACUAGCAAUGUUAUCUGC SEQ ID NO 212 UGGGAUACUAGCAAUGUUAUCUGCU SEQ ID NO 213 GGGAUACUAGCAAUGUUAUCUGCUU SEQ ID NO 214 GGAUACCAGCAAUGUUAUCUGCUUC SEQ ID NO 215 GAUACUAGCAAUGUUAUCUGCUUCC SEQ ID NO 216 AUACUAGCAAUGUUAUCUGCUUCCU SEQ ID NO 217 UACUAGCAAUGUUAUCUGCUUCCUC SEQ ID NO 218 ACUAGCAAUGUUAUCUGCUUCCUCC SEQ ID NO 219 CUAGCAAUGUUAUCUGCUUCCUCCA SEQ ID NO 220 UAGCAAUGUUAUCUGCUUCCUCCAA SEQ ID NO 221 AGCAAUGUUAUCUGCUUCCUCCAAC SEQ ID NO 222 GCAAUGUUAUCUGCUUCCUCCAACC SEQ ID NO 223 CAAUGUUAUCUGCUUCCUCCAACCA SEQ ID NO 224 AAUGUUAUCUGCUUCCUCCAACCAU SEQ ID NO 225 AUGUUAUCUGCUUCCUCCAACCAUA SEQ ID NO 226 UGUUAUCUGCUUCCUCCAACCAUAA SEQ ID NO 227 GUUAUCUGCUUCCUCCAACCAUAAA SEQ ID NO 228 GCUGCUCUUUUCCAGGUUC SEQ ID NO 229 UCUUUUCCAGGUUCAAGUGG SEQ ID NO 230 AGGUUCAAGUGGGAUACUA DMD Gene Exon 59 SEQ ID NO 231 CAAUUUUUCCCACUCAGUAUU SEQ ID NO 232 UUGAAGUUCCUGGAGUCUU SEQ ID NO 233 UCCUCAGGAGGCAGCUCUAAAU DMD Gene Exon 67 SEQ ID NO 234 GCGCUGGUCACAAAAUCCUGUUGAAC SEQ ID NO 235 CACUUGCUUGAAAAGGUCUACAAAGGA SEQ ID NO 236 GGUGAAUAACUUACAAAUUUGGAAGC

Sequence CWU 1

1

23613685PRThomo 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 275RNAArtificial Sequenceexon 2ccaggauggc auugggcagc ggcaaacugu ugucagaaca uugaaugcaa cuggggaaga 60aauaauucag caauc 75325RNAArtificial Sequenceoligonucleotide 3uuugccgcug cccaaugcca uccug 25425RNAArtificial Sequenceoligonucleotide 4auucaauguu cugacaacag uuugc 25525RNAArtificial Sequenceoligonucleotide 5ccaguugcau ucaauguucu gacaa 25622RNAArtificial Sequenceoligonucleotide 6caguugcauu caauguucug ac 22720RNAArtificial Sequenceoligonucleotide 7aguugcauuc aauguucuga 20821RNAArtificial Sequenceoligonucleotide 8gauugcugaa uuauuucuuc c 21925RNAArtificial Sequenceoligonucleotide 9gauugcugaa uuauuucuuc cccag 251025RNAArtificial Sequenceoligonucleotide 10auugcugaau uauuucuucc ccagu 251125RNAArtificial Sequenceoligonucleotide 11uugcugaauu auuucuuccc caguu 251225RNAArtificial Sequenceoligonucleotide 12ugcugaauua uuucuucccc aguug 251325RNAArtificial Sequenceoligonucleotide 13gcugaauuau uucuucccca guugc 251425RNAArtificial Sequenceoligonucleotide 14cugaauuauu ucuuccccag uugca 251525RNAArtificial Sequenceoligonucleotide 15ugaauuauuu cuuccccagu ugcau 251625RNAArtificial Sequenceoligonucleotide 16gaauuauuuc uuccccaguu gcauu 251725RNAArtificial Sequenceoligonucleotide 17aauuauuucu uccccaguug cauuc 251825RNAArtificial Sequenceoligonucleotide 18auuauuucuu ccccaguugc auuca 251925RNAArtificial Sequenceoligonucleotide 19uuauuucuuc cccaguugca uucaa 252025RNAArtificial Sequenceoligonucleotide 20uauuucuucc ccaguugcau ucaau 252125RNAArtificial Sequenceoligonucleotide 21auuucuuccc caguugcauu caaug 252225RNAArtificial Sequenceoligonucleotide 22uuucuucccc aguugcauuc aaugu 252325RNAArtificial Sequenceoligonucleotide 23uucuucccca guugcauuca auguu 252425RNAArtificial Sequenceoligonucleotide 24ucuuccccag uugcauucaa uguuc 252525RNAArtificial Sequenceoligonucleotide 25cuuccccagu ugcauucaau guucu 252625RNAArtificial Sequenceoligonucleotide 26uuccccaguu gcauucaaug uucug 252725RNAArtificial Sequenceoligonucleotide 27uccccaguug cauucaaugu ucuga 252825RNAArtificial Sequenceoligonucleotide 28ccccaguugc auucaauguu cugac 252925RNAArtificial Sequenceoligonucleotide 29cccaguugca uucaauguuc ugaca 253025RNAArtificial Sequenceoligonucleotide 30ccaguugcau ucaauguucu gacaa 253125RNAArtificial Sequenceoligonucleotide 31caguugcauu caauguucug acaac 253225RNAArtificial Sequenceoligonucleotide 32aguugcauuc aauguucuga caaca 253320RNAArtificial Sequenceoligonucleotide 33uccuguagaa uacuggcauc 203427RNAArtificial Sequenceoligonucleotide 34ugcagaccuc cugccaccgc agauuca 273534RNAArtificial Sequenceoligonucleotide 35uugcagaccu ccugccaccg cagauucagg cuuc 343625RNAArtificial Sequenceoligonucleotide 36guugcauuca auguucugac aacag 253725RNAArtificial Sequenceoligonucleotide 37uugcauucaa uguucugaca acagu 253825RNAArtificial Sequenceoligonucleotide 38ugcauucaau guucugacaa caguu 253925RNAArtificial Sequenceoligonucleotide 39gcauucaaug uucugacaac aguuu 254025RNAArtificial Sequenceoligonucleotide 40cauucaaugu ucugacaaca guuug 254125RNAArtificial Sequenceoligonucleotide 41auucaauguu cugacaacag uuugc 254225RNAArtificial Sequenceoligonucleotide 42ucaauguucu gacaacaguu ugccg 254325RNAArtificial Sequenceoligonucleotide 43caauguucug acaacaguuu gccgc 254425RNAArtificial Sequenceoligonucleotide 44aauguucuga caacaguuug ccgcu 254525RNAArtificial Sequenceoligonucleotide 45auguucugac aacaguuugc cgcug 254625RNAArtificial Sequenceoligonucleotide 46uguucugaca acaguuugcc gcugc 254725RNAArtificial Sequenceoligonucleotide 47guucugacaa caguuugccg cugcc 254825RNAArtificial Sequenceoligonucleotide 48uucugacaac aguuugccgc ugccc 254925RNAArtificial Sequenceoligonucleotide 49ucugacaaca guuugccgcu gccca 255025RNAArtificial Sequenceoligonucleotide 50cugacaacag uuugccgcug cccaa 255125RNAArtificial Sequenceoligonucleotide 51ugacaacagu uugccgcugc ccaau 255225RNAArtificial Sequenceoligonucleotide 52gacaacaguu ugccgcugcc caaug

255325RNAArtificial Sequenceoligonucleotide 53acaacaguuu gccgcugccc aaugc 255425RNAArtificial Sequenceoligonucleotide 54caacaguuug ccgcugccca augcc 255525RNAArtificial Sequenceoligonucleotide 55aacaguuugc cgcugcccaa ugcca 255625RNAArtificial Sequenceoligonucleotide 56acaguuugcc gcugcccaau gccau 255725RNAArtificial Sequenceoligonucleotide 57caguuugccg cugcccaaug ccauc 255825RNAArtificial Sequenceoligonucleotide 58aguuugccgc ugcccaaugc caucc 255925RNAArtificial Sequenceoligonucleotide 59guuugccgcu gcccaaugcc auccu 256025RNAArtificial Sequenceoligonucleotide 60uuugccgcug cccaaugcca uccug 256125RNAArtificial Sequenceoligonucleotide 61uugccgcugc ccaaugccau ccugg 256225RNAArtificial Sequenceoligonucleotide 62ugccgcugcc caaugccauc cugga 256325RNAArtificial Sequenceoligonucleotide 63gccgcugccc aaugccaucc uggag 256425RNAArtificial Sequenceoligonucleotide 64ccgcugccca augccauccu ggagu 256525RNAArtificial Sequenceoligonucleotide 65cgcugcccaa ugccauccug gaguu 256620RNAArtificial Sequenceoligoncleotide 66uguuuuugag gauugcugaa 206740RNAArtificial Sequenceoligonucleotide 67uguucugaca acaguuugcc gcugcccaau gccauccugg 406817RNAArtificial Sequenceoligonucleotide 68gcccaaugcc auccugg 176925RNAArtificial Sequenceoligonucleotide 69agagcaggua ccuccaacau caagg 257025RNAArtificial Sequenceoligonucleotide 70gagcagguac cuccaacauc aagga 257125RNAArtificial Sequenceoligonucleotide 71agcagguacc uccaacauca aggaa 257225RNAArtificial Sequenceoligonucleotide 72gcagguaccu ccaacaucaa ggaag 257325RNAArtificial Sequenceoligonucleotide 73cagguaccuc caacaucaag gaaga 257425RNAArtificial Sequenceoligonucleotide 74agguaccucc aacaucaagg aagau 257525RNAArtificial Sequenceoligonucleotide 75gguaccucca acaucaagga agaug 257625RNAArtificial Sequenceoligonucleotide 76guaccuccaa caucaaggaa gaugg 257725RNAArtificial Sequenceoligonucleotide 77uaccuccaac aucaaggaag auggc 257825RNAArtificial Sequenceoligonucleotide 78accuccaaca ucaaggaaga uggca 257925RNAArtificial Sequenceoligonucleotide 79ccuccaacau caaggaagau ggcau 258025RNAArtificial Sequenceoligonucleotide 80cuccaacauc aaggaagaug gcauu 258130RNAArtificial Sequenceoligonucleotide 81cuccaacauc aaggaagaug gcauuucuag 308225RNAArtificial Sequenceoligonucleotide 82uccaacauca aggaagaugg cauuu 258325RNAArtificial Sequenceoligonucleotide 83ccaacaucaa ggaagauggc auuuc 258425RNAArtificial Sequenceoligonucleotide 84caacaucaag gaagauggca uuucu 258525RNAArtificial Sequenceoligonucleotide 85aacaucaagg aagauggcau uucua 258625RNAArtificial Sequenceoligonucleotide 86acaucaagga agauggcauu ucuag 258730RNAArtificial Sequenceoligonucleotide 87acaucaagga agauggcauu ucuaguuugg 308825RNAArtificial Sequenceoligonucleotide 88acaucaagga agauggcauu ucuag 258925RNAArtificial Sequenceoligonucleotide 89caucaaggaa gauggcauuu cuagu 259025RNAArtificial Sequenceoligonucleotide 90aucaaggaag auggcauuuc uaguu 259125RNAArtificial Sequenceoligonucleotide 91ucaaggaaga uggcauuucu aguuu 259220RNAArtificial Sequenceoligonucleotide 92ucaaggaaga uggcauuucu 209325RNAArtificial Sequenceoligonucleotide 93caaggaagau ggcauuucua guuug 259425RNAArtificial Sequenceoligonucleotide 94aaggaagaug gcauuucuag uuugg 259525RNAArtificial Sequenceoligonucleotide 95aggaagaugg cauuucuagu uugga 259625RNAArtificial Sequenceoligonucleotide 96ggaagauggc auuucuaguu uggag 259725RNAArtificial Sequenceoligonucleotide 97gaagauggca uuucuaguuu ggaga 259825RNAArtificial Sequenceoligonucleotide 98aagauggcau uucuaguuug gagau 259925RNAArtificial Sequenceoligonucleotide 99agauggcauu ucuaguuugg agaug 2510025RNAArtificial Sequenceoligonucleotide 100gauggcauuu cuaguuugga gaugg 2510125RNAArtificial Sequenceoligonucleotide 101auggcauuuc uaguuuggag auggc 2510225RNAArtificial Sequenceoligonucleotide 102uggcauuucu aguuuggaga uggca 2510325RNAArtificial Sequenceoligonucleotide 103ggcauuucua guuuggagau ggcag 2510425RNAArtificial Sequenceoligonucleotide 104gcauuucuag uuuggagaug gcagu 2510525RNAArtificial Sequenceoligonucleotide 105cauuucuagu uuggagaugg caguu 2510625RNAArtificial Sequenceoligonucleotide 106auuucuaguu uggagauggc aguuu 2510725RNAArtificial Sequenceoligonucleotide 107uuucuaguuu ggagauggca guuuc 2510825RNAArtificial Sequenceoligonucleotide 108uucuaguuug gagauggcag uuucc 2510925RNAArtificial Sequenceoligonucleotide 109ccauuguguu gaauccuuua acauu 2511022RNAArtificial Sequenceoligonucleotide 110ccauuguguu gaauccuuua ac 2211120RNAArtificial Sequenceoligonucleotide 111auuguguuga auccuuuaac 2011220RNAArtificial Sequenceoligonucleotide 112ccuguccuaa gaccugcuca 2011325RNAArtificial Sequenceoligonucleotide 113cuuuuggauu gcaucuacug uauag 2511425RNAArtificial Sequenceoligonucleotide 114cauucaacug uugccuccgg uucug 2511524RNAArtificial Sequenceoligonucleotide 115cuguugccuc cgguucugaa ggug 2411631RNAArtificial Sequenceoligonucleotide 116cauucaacug uugccuccgg uucugaaggu g 3111725RNAArtificial Sequenceoligonucleotide 117cugaaggugu ucuuguacuu caucc 2511827RNAArtificial Sequenceoligonucleotide 118uguauaggga cccuccuucc augacuc 2711920RNAArtificial Sequenceoligonucleotide 119aucccacuga uucugaauuc 2012022RNAArtificial Sequenceoligonucleotide 120uuggcucugg ccuguccuaa ga 2212135RNAArtificial Sequenceoligonucleotide 121aagaccugcu cagcuucuuc cuuagcuucc agcca 3512223RNAArtificial Sequenceoligonucleotide 122ugcauguucc agucguugug ugg 2312325RNAArtificial Sequenceoligonucleotide 123cacuauucca gucaaauagg ucugg 2512425RNAArtificial Sequenceoligonucleotide 124auuuaccaac cuucaggauc gagua 2512521RNAArtificial Sequenceoligonucleotide 125ggccuaaaac acauacacau a 2112620RNAArtificial Sequenceoligonucleotide 126ucagcuucug uuagccacug 2012720RNAArtificial Sequenceoligonucleotide 127uucagcuucu guuagccacu 2012821RNAArtificial Sequenceoligonucleotide 128uucagcuucu guuagccacu g 2112921RNAArtificial Sequenceoligonucleotide 129ucagcuucug uuagccacug a 2113022RNAArtificial Sequenceoligonucleotide 130uucagcuucu guuagccacu ga 2213121RNAArtificial Sequenceoligonucleotide 131ucagcuucug uuagccacug a 2113222RNAArtificial Sequenceoligonucleotide 132uucagcuucu guuagccacu ga 2213322RNAArtificial Sequenceoligonucleotide 133ucagcuucug uuagccacug au 2213423RNAArtificial Sequenceoligonucleotide 134uucagcuucu guuagccacu gau 2313523RNAArtificial Sequenceoligonucleotide 135ucagcuucug uuagccacug auu 2313624RNAArtificial Sequenceoligonucleotide 136uucagcuucu guuagccacu gauu 2413724RNAArtificial Sequenceoligonucleotide 137ucagcuucug uuagccacug auua 2413824RNAArtificial Sequenceoligonucleotide 138uucagcuucu guuagccacu gaua 2413925RNAArtificial Sequenceoligonucleotide 139ucagcuucug uuagccacug auuaa 2514026RNAArtificial Sequenceoligonucleotide 140uucagcuucu guuagccacu gauuaa 2614126RNAArtificial Sequenceoligonucleotide 141ucagcuucug uuagccacug auuaaa 2614227RNAArtificial Sequenceoligonucleotide 142uucagcuucu guuagccacu gauuaaa 2714319RNAArtificial Sequenceoligonucleotide 143cagcuucugu uagccacug 1914421RNAArtificial Sequenceoligonucleotide 144cagcuucugu uagccacuga u 2114521RNAArtificial Sequenceoligonucleotide 145agcuucuguu agccacugau u 2114622RNAArtificial Sequenceoligonucleotide 146cagcuucugu uagccacuga uu 2214722RNAArtificial Sequenceoligonucleotide 147agcuucuguu agccacugau ua 2214823RNAArtificial Sequenceoligonculeotide 148cagcuucugu uagccacuga uua 2314923RNAArtificial Sequenceoligonucleotide 149agcuucuguu agccacugau uaa 2315024RNAArtificial Sequenceoligonucleotide 150cagcuucugu uagccacuga uuaa 2415124RNAArtificial Sequenceoligonucleotide 151agcuucuguu agccacugau uaaa 2415225RNAArtificial Sequenceoligonucleotide 152cagcuucugu uagccacuga uuaaa 2515324RNAArtificial Sequenceoligonucleotide 153agcuucuguu agccacugau uaaa 2415420RNAArtificial Sequenceoligonucleotide 154agcuucuguu agccacugau 2015520RNAArtificial Sequenceoligonucleotide 155gcuucuguua gccacugauu 2015621RNAArtificial Sequenceoligonucleotide 156agcuucuguu agccacugau u 2115721RNAArtificial Sequenceoligonucleotide 157gcuucuguua gccacugauu a 2115822RNAArtificial Sequenceoligonculeotide 158agcuucuguu agccacugau ua 2215922RNAArtificial Sequenceoligonucleotide 159gcuucuguua gccacugauu aa 2216023RNAArtificial Sequenceoligonucleotide 160agcuucuguu agccacugau uaa 2316123RNAArtificial Sequenceoligonucleotide 161gcuucuguua gccacugauu aaa 2316224RNAArtificial Sequenceoligonucleotide 162agcuucuguu agccacugau uaaa 2416323RNAArtificial Sequenceoligonucleotide 163gcuucuguua gccacugauu aaa 2316423RNAArtificial Sequenceoligonucleotide 164ccauuuguau uuagcauguu ccc 2316520RNAArtificial Sequenceoligonucleotide 165agauaccauu uguauuuagc 2016619RNAArtificial Sequenceoligonucleotide 166gccauuucuc aacagaucu 1916723RNAArtificial Sequenceoligonucleotide 167gccauuucuc aacagaucug uca 2316823RNAArtificial Sequenceoligonucleotide 168auucucagga auuugugucu uuc 2316921RNAArtificial Sequenceoligonucleotide 169ucucaggaau uugugucuuu c 2117018RNAArtificial Sequenceoligonucleotide 170guucagcuuc uguuagcc 1817121RNAArtificial Sequenceoligonucleotide 171cugauuaaau aucuuuauau c 2117218RNAArtificial Sequenceoligonucleotide 172gccgccauuu cucaacag 1817318RNAArtificial Sequenceoligonucleotide 173guauuuagca uguuccca 1817418RNAArtificial Sequenceoligonucleotide 174caggaauuug ugucuuuc 1817525RNAArtificial Sequenceoligonucleotide 175gcuuuucuuu uaguugcugc ucuuu 2517625RNAArtificial Sequenceoligonucleotide 176cuuuucuuuu aguugcugcu cuuuu 2517725RNAArtificial Sequenceoligonucleotide 177uuuucuuuua guugcugcuc uuuuc 2517825RNAArtificial Sequenceoligonucleotide 178uuucuuuuag uugcugcucu uuucc 2517925RNAArtificial Sequenceoligonucleotide 179uucuuuuagu ugcugcucuu uucca 2518025RNAArtificial Sequenceoligonucleotide 180ucuuuuaguu gcugcucuuu uccag 2518125RNAArtificial Sequenceoligonucleotide 181cuuuuaguug cugcucuuuu ccagg 2518225RNAArtificial Sequenceoligonucleotide 182uuuuaguugc ugcucuuuuc caggu 2518325RNAArtificial Sequenceoligonucleotide 183uuuaguugcu gcucuuuucc agguu 2518425RNAArtificial Sequenceoligonucleotide 184uuaguugcug cucuuuucca gguuc 2518525RNAArtificial Sequenceoligonucleotide 185uaguugcugc ucuuuuccag guuca 2518625RNAArtificial Sequenceoligonucleotide 186aguugcugcu cuuuuccagg uucaa 2518725RNAArtificial Sequenceoligonucleotide 187guugcugcuc uuuuccaggu ucaag 2518825RNAArtificial Sequenceoligonucleotide 188uugcugcucu uuuccagguu caagu 2518925RNAArtificial Sequenceoligonucleotide 189ugcugcucuu uuccagguuc aagug 2519025RNAArtificial Sequenceoligonucleotide 190gcugcucuuu uccagguuca agugg 2519125RNAArtificial Sequenceoligonucleotide 191cugcucuuuu ccagguucaa guggg 2519225RNAArtificial Sequenceoligonucleotide 192ugcucuuuuc cagguucaag uggga 2519325RNAArtificial Sequenceoligonucleotide 193gcucuuuucc agguucaagu gggac 2519425RNAArtificial Sequenceoligonucleotide 194cucuuuucca gguucaagug ggaua 2519525RNAArtificial Sequenceoligonucleotide 195ucuuuuccag guucaagugg gauac 2519625RNAArtificial Sequenceoligonucleotide 196cuuuuccagg uucaaguggg auacu 2519725RNAArtificial Sequenceoligonucleotide 197uuuuccaggu ucaaguggga uacua 2519825RNAArtificial Sequenceoligonucleotide 198uuuccagguu caagugggau acuag 2519925RNAArtificial Sequenceoligonucleotide 199uuccagguuc aagugggaua cuagc 2520025RNAArtificial Sequenceoligonucleotide 200uccagguuca agugggauac uagca 2520125RNAArtificial Sequenceoligonucleotide 201ccagguucaa gugggauacu agcaa 2520225RNAArtificial Sequenceoligonucleotide 202cagguucaag ugggauacua gcaau 2520325RNAArtificial Sequenceoligonucleotide 203agguucaagu gggauacuag

caaug 2520425RNAArtificial Sequenceoligonucleotide 204gguucaagug ggauacuagc aaugu 2520525RNAArtificial Sequenceoligonucleotide 205guucaagugg gauacuagca auguu 2520625RNAArtificial Sequenceoligonucleotide 206uucaaguggg auacuagcaa uguua 2520725RNAArtificial Sequenceoligonucleotide 207ucaaguggga uacuagcaau guuau 2520825RNAArtificial Sequenceoligonucleotide 208caagugggau acuagcaaug uuauc 2520925RNAArtificial Sequenceoligonucleotide 209aagugggaua cuagcaaugu uaucu 2521025RNAArtificial Sequenceoligonucleotide 210agugggauac uagcaauguu aucug 2521125RNAArtificial Sequenceoligonucleotide 211gugggauacu agcaauguua ucugc 2521225RNAArtificial Sequenceoligonucleotide 212ugggauacua gcaauguuau cugcu 2521325RNAArtificial Sequenceoligonucleotide 213gggauacuag caauguuauc ugcuu 2521425RNAArtificial Sequenceoligonucleotide 214ggauacuagc aauguuaucu gcuuc 2521525RNAArtificial Sequenceoligonucleotide 215gauacuagca auguuaucug cuucc 2521625RNAArtificial Sequenceoligonucleotide 216auacuagcaa uguuaucugc uuccu 2521725RNAArtificial Sequenceoligonucleotide 217uacuagcaau guuaucugcu uccuc 2521825RNAArtificial Sequenceoligonucleotide 218acuagcaaug uuaucugcuu ccucc 2521925RNAArtificial Sequenceoligonucleotide 219cuagcaaugu uaucugcuuc cucca 2522025RNAArtificial Sequenceoligonucleotide 220uagcaauguu aucugcuucc uccaa 2522125RNAArtificial Sequenceoligonucleotide 221agcaauguua ucugcuuccu ccaac 2522225RNAArtificial Sequenceoligonucleotide 222gcaauguuau cugcuuccuc caacc 2522325RNAArtificial Sequenceoligonucleotide 223caauguuauc ugcuuccucc aacca 2522425RNAArtificial Sequenceoligonucleotide 224aauguuaucu gcuuccucca accau 2522525RNAArtificial Sequenceoligonucleotide 225auguuaucug cuuccuccaa ccaua 2522625RNAArtificial Sequenceoligonucleotide 226uguuaucugc uuccuccaac cauaa 2522725RNAArtificial Sequenceoligonucleotide 227guuaucugcu uccuccaacc auaaa 2522819RNAArtificial Sequenceoligonucleotide 228gcugcucuuu uccagguuc 1922920RNAArtificial Sequenceoligonucleotide 229ucuuuuccag guucaagugg 2023019RNAArtificial Sequenceoligonucleotide 230agguucaagu gggauacua 1923121RNAArtificial Sequenceoligonucleotide 231caauuuuucc cacucaguau u 2123219RNAArtificial Sequenceoligonucleotide 232uugaaguucc uggagucuu 1923322RNAArtificial Sequenceoligonucleotide 233uccucaggag gcagcucuaa au 2223426RNAArtificial Sequenceoligonucleotide 234gcgcugguca caaaauccug uugaac 2623527RNAArtificial Sequenceoligonucleotide 235cacuugcuug aaaaggucua caaagga 2723626RNAArtificial Sequenceoligonucleotide 236ggugaauaac uuacaaauuu ggaagc 26

Claims

1. An isolated antisense oligonucleotide consisting of 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides, wherein said oligonucleotide is complementary along its entire length to a sequence in part of the human dystrophin exon 45 pre-mRNA, wherein said sequence is complementary to at least 22 nucleotides of a sequence consisting of 5'-UUUGCCGCUGCCCAAUGCCAUCCUG-3' (SEQ ID NO: 3).

2. A viral-based vector comprising an expression cassette comprising a nucleotide sequence encoding the oligonucleotide of claim 1.

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

4. The oligonucleotide of claim 1, wherein said oligonucleotide comprises a phosphorothioate internucleoside linkage and a 2'-O-alkyl substituted ribose moiety.

5. The oligonucleotide of claim 1, wherein said oligonucleotide induces skipping of exon 45.

6. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a nucleotide analogue, wherein the nucleotide analogue comprises a modified base, and/or a modified sugar moiety, and/or a modified internucleoside linkage.

7. The oligonucleotide of claim 6, wherein the nucleotide analogue comprises a modified base.

8. The oligonucleotide of claim 1, comprising a modified backbone.

9. The oligonucleotide of claim 6, wherein the modified sugar moiety is a ribose that is mono- or di-substituted at the 2', 3', and/or 5' position.

10. The oligonucleotide of claim 9, wherein the ribose is a 2'-O-substituted ribose.

11. The oligonucleotide of claim 10, wherein the ribose is a 2'-O methyl ribose.

12. The oligonucleotide of claim 6, wherein each sugar moiety of the oligonucleotide comprises a 2'-O-methyl substitution and each internucleoside linkage of said oligonucleotide comprises a phosphorothioate moiety.

13. An isolated antisense oligonucleotide consisting of 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides, wherein said oligonucleotide is complementary along its entire length to a sequence in part of the human dystrophin exon 45 pre-mRNA, wherein said sequence is complementary to at least 22 nucleotides of a sequence consisting of 5'UUUGCCGCUGCCCAAUGCCAUCCUG 3' (SEQ ID NO:3); wherein each sugar moiety of the oligonucleotide is 2'-O-methyl substituted and each of the internucleoside linkages present in the oligonucleotide comprises a phosphorothioate moiety.

14. The oligonucleotide of claim 8, wherein the modified backbone is selected from the group consisting of a morpholino backbone, a carbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl backbone, a thioformacetyl backbone, a methyleneformacetyl backbone, a riboacetyl backbone, an alkene containing backbone, a sulfamate backbone, a sulfonate backbone, a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazino backbone and an amide backbone.

15. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO), peptide nucleic acid, and/or locked nucleic acid.

16. The pharmaceutical composition of claim 3, further comprising a molecule which induces or promotes skipping of exon 7, 44, 46, 51, 53, 59, or 67 of dystrophin pre-mRNA of a patient.

17. A pharmaceutical composition comprising the antisense oligonucleotide of claim 13 and a pharmaceutically acceptable carrier.

18. The oligonucleotide of claim 1, wherein the oligonucleotide consists of 22, 23, 24, or 25 nucleotides.

19. The oligonucleotide of claim 1, wherein the oligonucleotide consists of 25, 26, 27, 28, or 29 nucleotides.

20. The oligonucleotide of claim 1, wherein the oligonucleotide consists of 25 nucleotides.

21. The oligonucleotide of claim 1, wherein the nucleotides of said oligonucleotide comprise purine and pyrimidine bases.

22. The oligonucleotide of claim 21, wherein the bases are selected from the group consisting of: adenine, cytosine, guanine, thymine and uracil.

23. The oligonucleotide of claim 13, wherein the oligonucleotide consists of 22, 23, 24, or 25 nucleotides.

24. The oligonucleotide of claim 13, wherein the oligonucleotide consists of 25, 26, 27, 28, or 29 nucleotides.

25. The oligonucleotide of claim 13, wherein the oligonucleotide consists of 25 nucleotides.

26. The oligonucleotide of claim 13, wherein the nucleotides of said oligonucleotide comprise purine and pyrimidine bases.

27. The oligonucleotide of claim 26, wherein the bases are selected from the group consisting of: adenine, cytosine, guanine, thymine and uracil.

28. The oligonucleotide of claim 19, wherein the oligonucleotide comprises the base sequence of 5'-UUUGCCGCUGCCCAAUGCCAUCCUG-3' (SEQ ID: NO: 3).

29. The oligonucleotide of claim 24, wherein the oligonucleotide comprises the base sequence of 5'-UUUGCCGCUGCCCAAUGCCAUCCUG-3' (SEQ ID: NO: 3).

30. The oligonucleotide of claim 5, wherein the oligonucleotide induces exon 45 skipping with an efficiency of at least 50%.

31. An oligomer for ameliorating DMD, the oligomer consisting of 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides, comprising at least 22 nucleotides of the base sequence of 5'-UUUGCCGCUGCCCAAUGCCAUCCUG-3' (SEQ ID: NO: 3); wherein the bases of the oligomer are selected from the group consisting of: adenine, cytosine, guanine, thymine and uracil; and wherein the molecule can bind to a target site to cause exon skipping in an exon of the dystrophin gene.

32. The oligomer of claim 31, wherein the oligomer consists of 22, 23, 24, or 25 nucleotides.

33. The oligomer of claim 31, wherein the oligomer consists of 25, 26, 27, 28, or 29 nucleotides.

34. The oligomer of claim 31, wherein the oligomer consists of 25 nucleotides.

35. An oligomer for alleviating DMD, the oligomer consisting of 25 nucleotides, and consisting of the sequence 5'-UUUGCCGCUGCCCAAUGCCAUCCUG-3' (SEQ ID: NO: 3); wherein the molecule can bind to a target site to cause exon skipping in an exon of the dystrophin gene.

36. An isolated antisense oligomer whose base sequence consists of the base sequence of 5'-UUUGCCGCUGCCCAAUGCCAUCCUG-3' (SEQ ID: NO: 3).

37. An isolated antisense oligomer consisting of 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides comprising at least 22 nucleotides of the base sequence of 5'-UUUGCCGCUGCCCAAUGCCAUCCUG-3' (SEQ ID: NO: 3).

38. The oligomer of claim 37, wherein the oligonucleotide consists of 22, 23, 24, or 25 nucleotides.

39. The oligomer of claim 37, wherein the oligonucleotide consists of 25, 26, 27, 28, or 29 nucleotides.

40. The oligomer of claim 37, wherein the oligomer consists of 25 nucleotides.

41. The oligonucleotide of claim 30, wherein, efficiency of exon skipping is determined using RT-PCR or sequence analysis.

42. The oligonucleotide of claim 6, wherein the nucleotide analogue comprises a modified internucleoside linkage.

43. The oligonucleotide of claim 42, wherein the modified internucleoside linkage is a phosphorothioate moiety.

44. An isolated antisense oligonucleotide consisting of 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides, wherein said oligonucleotide is complementary to at least 22 nucleotides of a sequence consisting of 5'UUUGCCGCUGCCCAAUGCCAUCCUG 3' (SEQ ID NO:3).

45. An isolated antisense oligonucleotide, consisting of 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides, wherein said oligonucleotide is complementary to at least 22 nucleotides of a sequence consisting of 5' UUUGCCGCUGCCCAAUGCCAUCCUG 3' (SEQ ID NO:3); wherein said oligonucleotide comprises at least one 2'-O-methyl substituted sugar moiety and at least one internucleoside linkage.

46. The oligonucleotide of claim 45, wherein each substituted sugar moiety of the oligonucleotide is 2'-O-methyl substituted.

47. The oligonucleotide of claim 45, wherein each internucleoside linkage of the oligonucleotide is a phosporothioate linkage.