Patent Publication Number: US-2023151362-A1

Title: Methods and means for efficient dkipping of exon 45 in duchenne muscular dystrophy pre-mrna

Description:
RELATED APPLICATIONS 
     This application is a continuation of U.S. Application No. 16/584,115, filed Sep. 26, 2019, which is a continuation of U.S. Application No. 16/283,458, filed Feb. 22, 2019, which is a continuation of U.S. Application No. 16/229,821, filed Dec. 21, 2018, which is a continuation of U.S. Application No. 15/390,836, filed Dec. 27, 2016, which is a continuation of U.S. Application No. 14/542,183, filed Nov. 14, 2014, now U.S. Pat. No. 9,528,109, issued Dec. 27, 2016, which is a continuation of U.S. Application No. 14/200,251, filed Mar. 7, 2014, which is a continuation of U.S. Application No. 14/134,971, filed Dec. 19, 2013, which is a continuation of U.S. Application No. 14/097,210, filed Dec. 4, 2013, which is a continuation of U.S. Application No. 13/094,548, filed Apr. 26, 2011, now U.S. Pat. No. 9,926,557, issued Mar. 27, 2018, which is a continuation of International Application No. PCT/NL2009/005006, filed Jan. 13, 2009, which is a continuation in part of International Application No. PCT/NL2008/050673, filed Oct. 27, 2008. 
     This application is a continuation of U.S. Application No. 16/584,115, filed Sep. 26, 2019, which is a continuation of U.S. Application No. 16/283,458, filed Feb. 22, 2019, which is a continuation of U.S. Application No. 16/229,534, filed Dec. 21, 2018, which is a continuation of U.S. Application No. 15/390,836, filed Dec. 27, 2016, which is a continuation of U.S. Application No. 14/542,183, filed Nov. 14, 2014, now U.S. Pat. No. 9,528,109, issued Dec. 27, 2016, which is a continuation of U.S. Application No. 14/200,251, filed Mar. 7, 2014, which is a continuation of U.S. Application No. 14/134,971, filed Dec. 19, 2013, which is a continuation of U.S. Application No. 14/097,210, filed Dec. 4, 2013, which is a continuation of U.S. Application No. 13/094,548, filed Apr. 26, 2011, now U.S. Pat. No. 9,926,557, issued Mar. 27, 2018, which is a continuation of International Application No. PCT/NL2009/005006, filed Jan. 13, 2009, which is a continuation in part of International Application No. PCT/NL2008/050673, filed Oct. 27, 2008. 
     The contents of each of the above-referenced applications are incorporated by reference herein in their entirety. 
    
    
     INCORPORATION BY REFERENCE OF SEQUENCE LISTING XML 
     This application contains a Sequence Listing XML titled “0105_06_US1CN11_SL.xml”, created on Nov. 10, 2022, having a file size of 303,034 bytes. The contents of this xml file are incorporated by reference herein in their entirety. 
     FIELD 
     The invention relates to the field of genetics, more specifically human genetics. The invention in particular relates to the modulation of splicing of the human Duchenne Muscular Dystrophy pre-mRNA. 
     BACKGROUND OF THE INVENTION 
     Myopathies are disorders that result in functional impairment of muscles. Muscular dystrophy (MD) refers to genetic diseases that are characterized by progressive weakness and degeneration of skeletal muscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. They are recessive disorders and because the gene responsible for DMD and BMD resides on the X-chromosome, mutations mainly affect males with an incidence of about 1 in 3500 boys. 
     DMD and BMD are caused by genetic defects in the DMD gene encoding dystrophin, a muscle protein that is required for interactions between the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction. DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and DMD patients often die before the age of thirty due to respiratory- or heart failure. In contrast, BMD patients often remain ambulatory until later in life, and have near normal life expectancies. DMD mutations in the DMD gene are 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. 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). 
     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 (AartsmaRus 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. 
     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). 
     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 
     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. 
     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. 
     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. 
     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. 
     Within the context of the invention inducing and/or promoting skipping of an exon as indicated herein means that at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more (muscle) cells of a treated patient will not contain said exon. This is preferably assessed by PCR as described in the examples. 
     Preferably, a method of the invention by inducing 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. 
     Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional dystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional dystrophin mRNA. Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the protein level (by immunofluorescence and western blot analyses) and preferably means that a detectable amount of a functional dystrophin protein is detectable by immunofluorescence or western blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein. 
     As defined herein, a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 1. A functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a 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 DOC complex may be visualized by either co-immunoprecipitation using total protein extracts or immunofluorescence analysis of cross-sections, from a muscle biopsy, as known to the skilled person. 
     Individuals or patients suffering from Duchenne muscular dystrophy typically have a mutation in the 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 function al dystrophin is a dystrophin of an individual having BMD: typically said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). The central rod-shaped domain of wild type dystrophin comprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). For example, a central rod-shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC. 
     A method of the invention may alleviate one or more characteristics of a 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. 
     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. 
     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. 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. 
     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. 
     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. 
     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. 
     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 et al (Manzur AY et al, (2008), Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review), Wiley publishers, The Cochrane collaboration) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy 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. 
     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. 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. 
     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 s physiological salt solution. Particularly preferred for a method of the invention is the use of an excipient that will further enhance delivery of said molecule, oligonucleotide or functional equivalent thereof as defined herein, to a cell and into a cell, preferably a muscle cell. Preferred excipients are defined in the section entitled “pharmaceutical composition”. In vitro, we obtained very good results using polyethylenimine (PEI, ExGen  500 , MBI Fermentas) as shown in the example. 
     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 AA, Kaman WE, et al. Antisense induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet 2004; 74(1): 83-92, Aartsma-Rus A, Kaman WE, Weij R, den Dunnen JT, van Ommen GJ, van Deutekom JC. Exploring the frontiers of therapeutic exon skipping for Duchenne muscular dystrophy by double targeting within one or multiple exons. Mol Ther 2006; 14(3):401-7). In most cases double-exon skipping results in the exclusion of only the two targeted exons from the 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. 
     In case, more than one compounds are used in a method of the inventions, said compounds can be administered to an individual in any order. In one embodiment, said compounds are administered simultaneously (meaning that said compounds are administered within 10 hours, preferably within one hour). This is however not necessary. In another embodiment, said compounds are administered sequentially. 
     Molecule 
     In a second aspect, there is provided a molecule for use in a method as described in the previous section entitled “Method”. This molecule preferably comprises or consists of an oligonucleotide. Said oligonucleotide is preferably an antisense oligonucleotide (AON) or antisense oligoribonucleotide. 
     It was found by the present investigators that especially exon 45 is specifically skipped at a high frequency sing 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. 
     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. A continuous stretch of at least 21, 25, 30, 35, 40, 45, 50 nucleotides within exon 45 is preferably selected from the sequence:  
     
       
         
           
               
            
               
                 5′- CCAGGAUGGCAUUGGGCAGCGGCAAACUGUUGUCAGA 
               
               
                 ACAUUGAAUGCAACUGGGGAAGAAAUAAUUCAGCAAUC-3′ 
               
               
                 (SEQ ID NO: 2). 
               
            
           
         
       
     
     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 oligonucleotide 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. 
     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 US Pat. 6875736, 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. 
     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. 
     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 2 1 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, 4 7 nucleotides, 48 nucleotides, 49 nucleotides or 50 nucleotides. 
     A most preferred molecule of the invention comprises a nucleotide-based sequence of 25 nucleotides. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. 15 (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566-568). 
     A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage. 
     In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a substitution of 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. 
     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. 
     It is understood by a skilled person that it is not necessary for all positions in an antisense oligonucleotide to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single antisense oligonucleotide or even at a single position within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents. 
     A preferred antisense oligonucleotide according to the invention comprises a 2′-O-alkyl phosphorothioate antisense oligonucleotide, such as 2′—O—methyl modified ribose (RNA), 2′—O—ethyl modified ribose, 2′—O—propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives. 
     A most preferred antisense oligonucleotide according to the invention comprises a 2′—O—methyl phosphorothioate ribose. 
     A functional equivalent of a molecule of the invention may be defined as an oligonucleotide as defined herein wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is inducing exon 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 DOC 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. 
     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. 
     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. 
     A preferred antisense oligonucleotide comprises a peptide-linked PMO. 
     A preferred antisense oligonucleotide comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an antisense oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an antisense oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells. Therefore, in one embodiment it is preferred to use a combination of antisense oligonucleotides comprising different nucleotide analogs or equivalents for modulating skipping of exon 45 of the human DMD pre-mRNA. 
     A cell can be provided with a molecule capable of interfering with essential sequences that result in highly efficient skipping of exon 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™ 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500(MBIFermentas)), or derivatives thereof. 
     One preferred antisense oligonucleotide expression system is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-based vectors have been developed that can be used for prolonged expression of small antisense nucleotide sequences for highly efficient skipping of exon 45 of the DMD pre-mRNA. 
     A preferred AAV-based vector comprises an expression cassette that is driven by a polymerase III-promoter (Pol III). A preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA promoter. 
     The invention therefore also provides a viral-based vector, comprising a Pol III-promoter driven expression cassette for expression of one or more antisense sequences of the invention for inducing skipping of exon 45 of the human DMD pre-mRNA. 
     Pharmaceutical Composition 
     If required, a molecule or a vector expressing an antisense oligonucleotide of the invention can be incorporated into a pharmaceutically active mixture or composition by adding a pharmaceutically acceptable carrier. 
     Therefore, in a further aspect, the invention provides a composition, preferably a pharmaceutical composition comprising a molecule comprising an antisense oligonucleotide according to the invention, and/or a viral-based vector expressing the antisense sequence(s) according to the invention and a pharmaceutically acceptable carrier. 
     A preferred pharmaceutical composition comprises a molecule as defined herein and/or a vector as defined herein, and a pharmaceutical acceptable carrier or excipient, optionally combined with a molecule and/or a vector which is able to modulate skipping of exon 7, 44, 46, 51, 53, 59, 67 of the DMD pre-mRNA. 
     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, lipofectinTM, 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 allows further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity. 
     Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt)and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles. 
     Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver a molecule or a compound as defined herein, preferably an oligonucleotide across cell membranes into cells. 
     In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate a compound as defined herein, preferably an oligonucleotide as colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of a compound as defined herein, preferably an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver a compound as defined herein, preferably an oligonucleotide for use in the current invention to deliver said compound for the treatment of Duchenne Muscular Dystrophy in humans. 
     In addition, a compound as defined herein, preferably an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake into the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide (-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake into cells and/or the intracellular release of a compound as defined herein, preferably an oligonucleotide from vesicles, e.g. endosomes or lysosomes. 
     Therefore, in a preferred embodiment, a compound as defined herein, preferably an oligonucleotide are formulated in a medicament which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising a compound as defined herein, preferably an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. It is to be understood that a molecule or compound or oligonucleotide may not be formulated in one single composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each compound. 
     In a preferred embodiment, an in vitro concentration of a molecule or an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 µM is used. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM. molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If several molecules or oligonucleotides are used, these concentrations may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further. 
     More preferably, a compound preferably an oligonucleotide 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 
     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. 
     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. 
     In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a molecule or a viral based vector or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”. 
     Each embodiment as identified herein may be combined together unless otherwise indicated. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. 
     The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. 
    
    
     
       LEGENDS TO THE FIGURE 
         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). 
         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. 
         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 
     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 
     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 MEI Fermentas) was used according to manufacturer’s instructions, with 2 µl PEI per µg 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 
     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 1
           
               
               
             
               
                 AONs in exon 45 
               
               
                 SEQ ID NO: 
               
             
            
               
                 3 (PS220) 
                 UUUGCCGCUGCCCAAUGCCAUCCUG 
               
               
                 4 (PS221) 
                 AUUCAAUGUUCUGACAACAGUUUGC 
               
               
                 5 (PS222) 
                 CCAGUUGCAUUCAAUGUUCUGACAA 
               
               
                 6 (PS223) 
                 CCAGUUGCAUUCAAUGUUCUGACAA 
               
               
                 7 (PS224) 
                 AGUUGCAUUCAAUGUUCUGA 
               
               
                 8 (PS225) 
                 GAUUGCUGAAUUAUUUCUUCC 
               
               
                 9 
                 GAUUGCUGAAUUAUUUCUUCCCCAG 
               
               
                 10 
                 AUUGCUGAAUUAUUUCUUCCCCAGU 
               
               
                 11 
                 UUGCUGAAUUAUUUCUUCCCCAGUU 
               
               
                 12 
                 UGCUGAAUUAUUUCUUCCCCAGUUG 
               
               
                 13 
                 GCUGAAUUAUUUCUUCCCCAGUUGC 
               
               
                 14 
                 CUGAAUUAUUUCUUCCCCAGUUGCA 
               
               
                 15 
                 UGAAUUAUUUCUUCCCCAGUUGCAU 
               
               
                 16 
                 GAAUUAUUUCUUCCCCAGUUGCAUU 
               
               
                 17 
                 AAUUAUUUCUUCCCCAGUUGCAUUC 
               
               
                 18 
                 AUUAUUUCUUCCCCAGUUGCAUUCA 
               
               
                 19 
                 UUAUUUCUUCCCCAGUUGCAUUCAA 
               
               
                 20 
                 UAUUUCUUCCCCAGUUGCAUUCAAU 
               
               
                 21 
                 AUUUCUUCCCCAGUUGCAUUCAAUG 
               
               
                 22 
                 UUUCUUCCCCAGUUGCAUUCAAUGU 
               
               
                 23 
                 UUCUUCCCCAGUUGCAUUCAAUGUU 
               
               
                 24 
                 UCUUCCCCAGUUGCAUUCAAUGUUC 
               
               
                 25 
                 CUUCCCCAGUUGCAUUCAAUGUUCU 
               
               
                 26 
                 UUCCCCAGUUGCAUUCAAUGUUCUG 
               
               
                 27 
                 UCCCCAGUUGCAUUCAAUGUUCUGA 
               
               
                 28 
                 CCCCAGUUGCAUUCAAUGUUCUGAC 
               
               
                 29 
                 CCCAGUUGCAUUCAAUGUUCUGACA 
               
               
                 30 
                 CCAGUUGCAUUCAAUGUUCUGACAA 
               
               
                 31 
                 CAGUUGCAUUCAAUGUUCUGACAAC 
               
               
                 32 
                 AGUUGCAUUCAAUGUUCUGACAACA 
               
               
                 33 
                 UCC UGU AGA AUA CUG GCA UC 
               
               
                 34 
                 UGC AGA CCU CCU GCC ACC GCA GAU UCA 
               
               
                 35 
                 UUGCAGACCUCCUGCCACCGCAGAUUC AGGCUUC 
               
               
                 36 
                 GUUGCAUUCAAUGUUCUGACAACAG 
               
               
                 37 
                 UUGCAUUCAAUGUUCUGACAACAGU 
               
               
                 38 
                 UGCAUUCAAUGUUCUGACAACAGUU 
               
               
                 39 
                 GCAUUCAAUGUUCUGACAACAGUUU 
               
               
                 40 
                 CAUUCAAUGUUCUGACAACAGUUUG 
               
               
                 41 
                 AUUCAAUGUUCUGACAACAGUUUGC 
               
               
                 42 
                 UCAAUGUUCUGACAACAGUUUGCCG 
               
               
                 43 
                 CAAUGUUCUGACAACAGUUUGCCGC 
               
               
                 44 
                 AAUGUUCUGACAACAGUUUGCCGCU 
               
               
                 45 
                 AUGUUCUGACAACAGUUUGCCGCUG 
               
               
                 46 
                 UGUUCUGACAACAGUUUGCCGCUGC 
               
               
                 47 
                 GUUCUGACAACAGUUUGCCGCUGCC 
               
               
                 48 
                 UUCUGACAACAGUUUGCCGCUGCCC 
               
               
                 49 
                 UCUGACAACAGU UUGCCGCUGCCCA 
               
               
                 50 
                 CUGACAACAGUUUGCCGCUGCCCAA 
               
               
                 51 
                 UGACAACAGUUUGCCGCUGCCCAAU 
               
               
                 52 
                 GACAACAGUUUGCCGCUGCCCAAUG 
               
               
                 53 
                 ACAACAGUUUGCCGCUGCCCAAUGC 
               
               
                 54 
                 CAACAGUUUGCCGCUGCCCAAUGCC 
               
               
                 55 
                 AACAGUUUGCCGCUGCCCAAUGCCA 
               
               
                 56 
                 ACAGUUUGCCGCUGCCCAAUGCCAU 
               
               
                 57 
                 CAGUUUGCCGCUGCCCAAUGCCAUC 
               
               
                 58 
                 AGUUUGCCGCUGCCCAAUGCCAUCC 
               
               
                 59 
                 GUUUGCCGCUGCCCAAUGCCAUCCU 
               
               
                 60 
                 UUUGCCGCUGCCCAAUGCCAUCCUG 
               
               
                 61 
                 UUGCCGCUGCCCAAUGCCAUCCUGG 
               
               
                 62 
                 UGCCGCUGCCCAAUGCCAUCCUGGA 
               
               
                 63 
                 GCCGCUGCCCAAUGCCAUCCUGGAG 
               
               
                 64 
                 CCGCUGCCCAAUGCCAUCCUGGAGU 
               
               
                 65 
                 CGCUGCCCAAUGCCAUCCUGGAGUU 
               
               
                 66 
                 UGU UUU UGA GGA UUG CUG AA 
               
               
                 67 
                 UGUUCUGACAACAGUUUGCCGCUGCCCAAUGCCAUCCUGG 
               
               
                 68 (45-5) 
                 GCCCAAUGCCAUCCUGG 
               
            
           
         
       
     
     
       
         
          TABLE 2
           
               
               
             
               
                 AONs in exons 51, 53, 7, 44, 46, 59, and 67 
               
               
                 SEQ ID NO: 
               
               
                 DMD Gene Exon 51 
               
             
            
               
                 69 
                 AGAGCAGGUACCUCCAACAUCAAGG 
               
               
                 70 
                 GAGCAGGUACCUCCAACAUCAAGGA 
               
               
                 71 
                 AGCAGGUACCUCCAACAUCAAGGAA 
               
               
                 72 
                 GCAGGUACCUCCAACAUCAAGGAAG 
               
               
                 73 
                 CAGGUACCUCCAACAUCAAGGAAGA 
               
               
                 74 
                 AGGUACCUCCAACAUCAAGGAAGAU 
               
               
                 75 
                 GGUACCUCCAACAUCAAGGAAGAUG 
               
               
                 76 
                 GUACCUCCAACAUCAAGGAAGAUGG 
               
               
                 77 
                 UACCUCCAACAUCAAGGAAGAUGGC 
               
               
                 78 
                 ACCUCCAACALTCAAGGAAGATJGGCA 
               
               
                 79 
                 CCUCCAACAUCAAGGAAGAUGGCAU 
               
               
                 80 
                 CUCCAACAUCAAGGAAGAUGGCAUU 
               
               
                 81 
                 CUCCAACAUCAAGGAAGAUGGCAUUUCUAG 
               
               
                 82 
                 UCCAACAUCAAGGAAGAUGGCAUUU 
               
               
                 83 
                 CCAACAUCAAGGAAGAUGGCAUUUC 
               
               
                 84 
                 CAACAUCAAGGAAGAUGGCAUUUCU 
               
               
                 85 
                 AACAUCAAGGAAGAUGGCAUUUCUA 
               
               
                 86 
                 ACAUCAAGGAAGAUGGCAUUUCUAG 
               
               
                 87 
                 ACAUCAAGGAAGAUGGCAUUUCUAGUUTTGG 
               
               
                 88 
                 ACAUCAAGGAAGAUGGCAUUUCUAG 
               
               
                 89 
                 CAUCAAGGAAGAUGGCAUUUCUAGU 
               
               
                 90 
                 AUCAAGGAAGAUGGCAUUUCUAGUU 
               
               
                 91 
                 UCAAGGAAGAUGGCAUUUCUAGUUU 
               
               
                 92 
                 UCAAGGAAGAUGGCAUUUCU 
               
               
                 93 
                 CAAGGAAGAUGGCAUUUCUAGUUUG 
               
               
                 94 
                 AAGGAAGATJGGCALTUUTCUAGULTUGG 
               
               
                 95 
                 AGGAAGAUGGCAUUUCUAGUUUGGA 
               
               
                 96 
                 GGAAGAUGGCAUUUCUAGUUUGGAG 
               
               
                 97 
                 GAAGAUGGCAUUUCUAGUUUGGAGA 
               
               
                 98 
                 AAGAUGGCAUUUCUAGUUUGGAGAU 
               
               
                 99 
                 AGAUGGCAUUUCUAGUUUGGAGAUG 
               
               
                 100 
                 GAUGGCAUUUCUAGUUUGGAGAUGG 
               
               
                 101 
                 AUGGCAUUUCUAGUUUGGAGAUGGC 
               
               
                 102 
                 UGGCAUUUCUAGUUUGGAGAUGGCA 
               
               
                 103 
                 GGCAUUUCUAGUUUGGAGAUGGCAG 
               
               
                 104 
                 GCAUUUCUAGUUUGGAGAUGGCAGU 
               
               
                 105 
                 CAUUUCUAGUUUGGAGAUGGCAGUU 
               
               
                 106 
                 AUUUCUAGUUUGGAGAUGGCAGUUU 
               
               
                 107 
                 UUUCUAGUUUGGAGAUGGCAGUUUC 
               
               
                 108 
                 UUCUAGUUUGGAGAUGGCAGUUUCC 
               
            
           
           
               
               
             
               
                 DMD Gene Exon 53 
               
             
            
               
                 109 
                 CCAUUGUGUUGAAUCCUUUAACAUU 
               
               
                 110 
                 CCALTUGUGULTGAAUCCUULTAAC 
               
               
                 111 
                 AUUGUGUUGAAUCCUUUAAC 
               
               
                 112 
                 CCUGUCCUAAGACCUGCUCA 
               
               
                 113 
                 CUUUUGGAUUGCAUCUACUGUAUAG 
               
               
                 114 
                 CAUUCAACUGUUGCCUCCGGUUCUG 
               
               
                 115 
                 CUGUUGCCUCCGGUUCUGAAGGUG 
               
               
                 116 
                 CAUUCAACUGUUGCCUCCGGUUCUGAAGGUG 
               
               
                 117 
                 CUGAAGGUGUUCUUGUACUUCAUCC 
               
               
                 118 
                 UGUAUAGGGACCCUCCUUCCAUGACUC 
               
               
                 119 
                 AUCCCACUGAUUCUGAAUUC 
               
               
                 120 
                 UUGGCUCUGGCCUGUCCUAAGA 
               
               
                 121 
                 AAGACCUGCUCAGCUUCUUCCUUAGCUUCCAGCCA 
               
            
           
           
               
               
             
               
                 DMD Gene Exon 7 
               
             
            
               
                 122 
                 UGCAUGUUCCAGUCGUUGUGUGG 
               
               
                 123 
                 CACUAUUCCAGUCAAAUAGGUCUGG 
               
               
                 124 
                 AUUUACCAACCUUCAGGAUCGAGUA 
               
               
                 125 
                 GGCCUAAAACACAUACACAUA 
               
            
           
           
               
               
             
               
                 DMD Gene Exon 44 
               
             
            
               
                 126 
                 UCAGCUUCUGUUAGCCACUG 
               
               
                 127 
                 UUCAGCUUCUGUUAGCCACU 
               
               
                 128 
                 UUCAGCUUCUGUUAGCCACUG 
               
               
                 129 
                 UCAGCUUCUGUUAGCCACUGA 
               
               
                 130 
                 UUCAGCUUCUGUUAGCCACUGA 
               
               
                 131 
                 UCAGCLTUCUGULJAGCCACUGA 
               
               
                 132 
                 UUCAGCUUCUGUUAGCCACUGA 
               
               
                 133 
                 CAGCUUCUGUUAGCCACUGAU 
               
               
                 134 
                 UUCAGCUUCUGUUAGCCACUGAU 
               
               
                 135 
                 UCAGCUUCUGUUAGCCACUGAUU 
               
               
                 136 
                 UUCAGCUUCUGUUAGCCACUGAUU 
               
               
                 137 
                 UCAGCLTUCUGULJAGCCACUGATJUA 
               
               
                 138 
                 UUCAGCUUCUGUUAGCCACUGAUA 
               
               
                 139 
                 UCAGCUUCUGUUAGCCACUGAUU AA 
               
               
                 140 
                 UUCAGCUUCUGUUAGCCACUGAUUAA 
               
               
                 141 
                 UCAGCUUCUGUUAGCCACUGAUUAAA 
               
               
                 142 
                 UUCAGCUUCUGUUAGCCACUGAUUAAA 
               
               
                 143 
                 CAGCUUCUGUUAGCCACUG 
               
               
                 144 
                 CAGCUUCUGUUAGCCACUGAU 
               
               
                 145 
                 AGCUUCUGUUAGCCACUGAUU 
               
               
                 146 
                 CAGCUUCUGUUAGCCACUGAUU 
               
               
                 147 
                 AGCUUCUGUUAGCCACUGAUUA 
               
               
                 148 
                 CAGCUUCUGUUAGCCACUGAUUA 
               
               
                 149 
                 AGCUUCUGUUAGCCACUGAUUAA 
               
               
                 150 
                 CAGCUUCUGUUAGCCACUGAUUAA 
               
               
                 151 
                 AGCUUCUGUUAGCCACUGAUUAAA 
               
               
                 152 
                 CAGCUUCUGUUAGCCACUGAUUAAA 
               
               
                 153 
                 AGCUUCUGUUAGCCACUGAUUAAA 
               
               
                 154 
                 AGCUUCUGUUAGCCACUGATT 
               
               
                 155 
                 GCUUCUGUUAGCCACUGAUU 
               
               
                 156 
                 AGCUUCUGUUAGCCACUGAUU 
               
               
                 157 
                 GCUUCUGUUAGCCACUGAUUA 
               
               
                 158 
                 AGCUUCUGUUAGCCACUGAUUA 
               
               
                 159 
                 GCUUCUGUUAGCCACUGAUUAA 
               
               
                 160 
                 AGCUUCUGUUAGCCACUGAUUAA 
               
               
                 161 
                 GCUUCUGLTUAGCCACUGAUUAAA 
               
               
                 162 
                 AGCUUCUGUUAGCCACUGAUUAAA 
               
               
                 163 
                 GCUUCUGUUAGCCACUGAUUAAA 
               
               
                 164 
                 CCAUUUGUAUUUAGCAUGUUCCC 
               
               
                 165 
                 AGAUACCAUUUGUAUUUAGC 
               
               
                 166 
                 GCCAUUUCUCAACAGAUCU 
               
               
                 167 
                 GCCAUUUCUCAACAGAUCUGUCA 
               
               
                 168 
                 AUUCUCAGGAAUUUGUGUCUUUC 
               
               
                 169 
                 UCUCAGGAAUUUGUGUCUUUC 
               
               
                 170 
                 GUUCAGCUUCUGUUAGCC 
               
               
                 171 
                 CUGAUUAAAUAUCUTTUAUAUC 
               
               
                 17 
                 GCCGCCAUUUCUCAACAG 
               
               
                 173 
                 GUAUUUAGCAUGUUCCCA 
               
               
                 174 
                 CAGGAAUUUGUGUCUUUC 
               
            
           
           
               
               
             
               
                 DMD Gene Exon 46 
               
             
            
               
                 175 
                 GCUUUUCUUUUAGUUGCUGCUCUUU 
               
               
                 176 
                 CUUUUCUUUUAGUUGCUGCUCUUUU 
               
               
                 177 
                 UUUUCUUUUAGUUGCUGCUCUUUUC 
               
               
                 178 
                 UUUCUUUUAGUUGCUGCUCUUUUCC 
               
               
                 179 
                 UUCUUUUAGUUGCUGCUCUUUUCCA 
               
               
                 180 
                 UCUUUUAGUUGCUGCUCUUUUCCAG 
               
               
                 181 
                 CUUUUAGUUGCUGCUCUUUUCCAGG 
               
               
                 182 
                 UUUUAGUUGCUGCUCUUUUCCAGGU 
               
               
                 183 
                 UUUAGUUGCUGCUCUUUUCCAGGUU 
               
               
                 184 
                 UUAGUUGCUGCUCUUUUCCAGGUUC 
               
               
                 185 
                 UAGUUGCUGCUCUUUUCCAGGUUCA 
               
               
                 186 
                 AGUUGCUGCUCUUUUCCAGGUUCAA 
               
               
                 187 
                 GUUGCUGCUCUUUUCCAGGUUCAAG 
               
               
                 188 
                 UUGCUGCUCUUUUCCAGGUUCAAGU 
               
               
                 189 
                 UGCUGCUCUUUUCCAGGUUCAAGUG 
               
               
                 190 
                 GCUGCUCUUUUCCAGGUUCAAGUGG 
               
               
                 191 
                 CUGCUCUUUUCCAGGUUCAAGUGGG 
               
               
                 192 
                 UGCUCUUUUCCAGGUUCAAGUGGGA 
               
               
                 193 
                 GCUCUUUUCCAGGUUCAAGUGGGAC 
               
               
                 194 
                 CUCLTUUTUCCAGGUU-CAAGUGGGAUA 
               
               
                 195 
                 UCUUUUCCAGGUUCAAGUGGGAUAC 
               
               
                 196 
                 CUUUUCCAGGUUCAAGUGGGAUACU 
               
               
                 197 
                 UUUUCCAGGUUCAAGUGGGAUACUA 
               
               
                 198 
                 UUUCCAGGUUCAAGUGGGAUACUAG 
               
               
                 199 
                 UUCCAGGUUCAAGUGGGAUACUAGC 
               
               
                 200 
                 UCCAGGUUCAAGUGGGAUACUAGCA 
               
               
                 201 
                 CCAGGUlTCAAGUGGGAUACUAGCAA 
               
               
                 202 
                 CAGGUUCAAGUGGGAUACUAGCAAU 
               
               
                 203 
                 AGGUUCAAGUGGGAUACUAGCAAUG 
               
               
                 204 
                 GGUUCAAGUGGGAUACUAGCAAUGU 
               
               
                 205 
                 GUUCAAGUGGGAUACUAGCAAUGUU 
               
               
                 206 
                 UUCAAGUGGGAUACUAGCAAUGUUA 
               
               
                 207 
                 UCAAGUGGGAUACUAGCAAUGUUAU 
               
               
                 208 
                 CAAGUGGGAUACUAGCAAUGUUAUC 
               
               
                 209 
                 AAGUGGGAUACUAGCAAUGULJAUCU 
               
               
                 210 
                 AGUGGGAUACUAGCAAUGUUAUCUG 
               
               
                 211 
                 GUGGGAUACUAGCAAUGUUAUCUGC 
               
               
                 212 
                 UGGGAUACUAGCAAUGUUAUCUGCU 
               
               
                 213 
                 GGGAUACUAGCAAUGUUAUCUGCUU 
               
               
                 214 
                 GGAUACUAGCAAUGUUAUCUGCUUC 
               
               
                 215 
                 GAUACUAGCAAUGUUAUCUGCUUCC 
               
               
                 216 
                 AUACUAGCAAUGUUAUCUGCUUCCU 
               
               
                 217 
                 UACUAGCAALTGLTUAUCUGCULTCCUC 
               
               
                 218 
                 ACUAGCAAUGUUAUCUGCUUCCUCC 
               
               
                 219 
                 CUAGCAAUGUUAUCUGCUUCCUCCA 
               
               
                 220 
                 UAGCAAUGUUAUCUGCUUCCUCCAA 
               
               
                 221 
                 AGCAAUGUUAUCUGCUUCCUCCAAC 
               
               
                 222 
                 GCAAUGUUAUCUGCUUCCUCCAACC 
               
               
                 223 
                 CAAUGUUAUCUGCUUCCUCCAACCA 
               
               
                 224 
                 AAUGUUAUCUGCUUCCUCCAACCAU 
               
               
                 225 
                 AUGUUAUCUGCUUCCUCCAACCAUA 
               
               
                 226 
                 UGUUAUCUGCUUCCUCCAACCAUAA 
               
               
                 227 
                 GUUAUCUGCUUCCUCCAACCAUAAA 
               
               
                 228 
                 GCUGCUCUUUUCCAGGUUC 
               
               
                 229 
                 UCUUUUCCAGGUUCAAGUGG 
               
               
                 230 
                 AGGUUCAAGUGGGAUACUA 
               
            
           
           
               
               
             
               
                 DMD Gene Exon 59 
               
             
            
               
                 231 
                 CAAUUUUUCCCACUCAGUAUU 
               
               
                 232 
                 UUGAAGUUCCUGGAGUCUU 
               
               
                 233 
                 UCCUCAGGAGGCAGCUCUAAAU 
               
            
           
           
               
               
             
               
                 DMD Gene Exon 67 
               
             
            
               
                 234 
                 GCGCUGGUCACAAAAUCCUGUUGAAC 
               
               
                 235 
                 CACUUGCUUGAAAAGGUCUACAAAGGA 
               
               
                 236 
                 GGUGAAUAACUUACAAAtlUUGGAAGC 
               
            
           
         
       
     
     
       
         
           
               
            
               
                  SEQ ID NO: 1: Met Leu Trp Trp Glu Glu Val Glu Asp Cys Tyr Glu Arg Glu Asp Val 
               
               
                 Gln Lys Lys Thr Phe Thr Lys Trp Val Asn Ala Gln Phe Ser Lys Phe Gly Lys Gln His Ile Glu 
               
               
                 Asn Leu Phe Ser Asp Leu Gln Asp Gly Arg Arg Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln 
               
               
                 Lys Leu Pro Lys Glu Lys Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Lys Ala Leu Arg 
               
               
                 Val Leu Gln Asn Asn Asn Val Asp Leu Val Asn Ile Gly Ser Thr Asp Ile Val Asp Gly Asn His 
               
               
                 Lys Leu Thr Leu Gly Leu Ile Trp Asn Ile Ile Leu His Trp Gln Val Lys Asn Val Met Lys Asn Ile 
               
               
                 Met Ala Gly Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu Ser Trp Val Arg Gln Ser Thr Arg 
               
               
                 Asn Tyr Pro Gln Val Asn Val Ile Asn Phe Thr Thr Ser Trp Ser Asp Gly Leu Ala Leu Asn Ala 
               
               
                 Leu Ile His Ser His Arg Pro Asp Leu Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala Thr Gln 
               
               
                 Arg Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly Ile Glu Lys Leu Leu Asp Pro Glu 
               
               
                 Asp Val Asp Thr Thr Tyr Pro Asp Lys Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu 
               
               
                 Pro Gln Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu Met Leu Pro Arg Pro Pro Lys Val Thr Lys 
               
               
                 Glu Glu His Phe Gln Leu His His Gln Met His Tyr Ser Gln Gln Ile Thr Val Ser Leu Ala Gln Gly 
               
               
                 Tyr Glu Arg Thr Ser Ser Pro Lys Pro Arg Phe Lys Ser Tyr Ala Tyr Thr Gln Ala Ala Tyr Val Thr 
               
               
                 Thr Ser Asp Pro Thr Arg Ser Pro Phe Pro Ser Gln His Leu Glu Ala Pro Glu Asp Lys Ser Phe Gly 
               
               
                 Ser Ser Leu Met Glu Ser Glu Val Asn Leu Asp Arg Tyr Gln Thr Ala Leu Glu Glu Val Leu Ser 
               
               
                 Trp Leu Leu Ser Ala Glu Asp Thr Leu Gln Ala Gln Gly Glu Ile Ser Asn Asp Val Glu Val Val 
               
               
                 Lys Asp Gln Phe His Thr His Glu Gly Tyr Met Met Asp Leu Thr Ala His Gln Gly Arg Val Gly 
               
               
                 Asn Ile Leu Gln Leu Gly Ser Lys Leu Ile Gly Thr Gly Lys Leu Ser Glu Asp Glu Glu Thr Glu 
               
               
                 Val Gln Glu Gln Met Asn Leu Leu Asn Ser Arg Trp Glu Cys Leu Arg Val Ala Ser Met Glu Lys 
               
               
                 Gln Ser Asn Leu His Arg Val Leu Met Asp Leu Gln Asn Gln Lys Leu Lys Glu Leu Asn Asp Trp 
               
               
                 Leu Thr Lys Thr Glu Glu Arg Thr Arg Lys Met Glu Glu Glu Pro Leu Gly Pro Asp Leu Glu Asp 
               
               
                 Leu Lys Arg Gln Val Gln Gln His Lys Val Leu Gln Glu Asp Leu Glu Gln Glu Gln Val Arg Val 
               
               
                 Asn Ser Leu Thr His Met Val Val Val Val Asp Glu Ser Ser Gly Asp His Ala Thr Ala Ala Leu 
               
               
                 Glu Glu Gln Leu Lys Val Leu Gly Asp Arg Trp Ala Asn Ile Cys Arg Trp Thr Glu Asp Arg Trp 
               
               
                 Val Leu Leu Gln Asp Ile Leu Leu Lys Trp Gln Arg Leu Thr Glu Glu Gln Cys Leu Phe Ser Ala 
               
               
                 Trp Leu Ser Glu Lys Glu Asp Ala Val Asn Lys Ile His Thr Thr Gly Phe Lys Asp Gln Asn Glu 
               
               
                 Met Leu Ser Ser Leu Gln Lys Leu Ala Val Leu Lys Ala Asp Leu Glu Lys Lys Lys Gln Ser Met 
               
               
                 Gly Lys Leu Tyr Ser Leu Lys Gin Asp Leu Leu Ser Thr Leu Lys Asn Lys Ser Val Thr Gln Lys 
               
               
                 Thr Glu Ala Trp Leu Asp Asn Phe Ala Arg Cys Trp Asp Asn Leu Val Gln Lys Leu Glu Lys Ser 
               
               
                 Thr Ala Gln Ile Ser Gln Ala Val Thr Thr Thr Gln Pro Ser Leu Thr Gln Thr Thr Val Met Glu Thr 
               
               
                 Val Thr Thr Val Thr Thr Arg Glu Gln Ile Leu Val Lys His Ala Gln Glu Glu Leu Pro Pro Pro Pro 
               
               
                 Pro Gln Lys Lys Arg Gln Ile Thr Val Asp Ser Glu Ile Arg Lys Arg Leu Asp Val Asp Ile Thr Glu 
               
               
                 Leu His Ser Trp Ile Thr Arg Ser Glu Ala Val Leu Gln Ser Pro Glu Phe Ala Ile Phe Arg Lys Glu 
               
               
                 Gly Asn Phe Ser Asp Leu Lys Glu Lys Val Asn Ala Ile Glu Arg Glu Lys Ala Glu Lys Phe Arg 
               
               
                 Lys Leu Ciln Asp Ala Ser Arg Ser Ala Gln Ala Leu Val Glu Gln Met Val Asn Glu Gly Val Asn 
               
               
                 Ala Asp Ser Ile Lys Gln Ala Ser Glu Gln Leu Asn Ser Arg Trp Ile Glu Phe Cys Gln Leu Leu Ser 
               
               
                 Glu Arg Leu Asn Trp Leu Glu Tyr Gln Asn Asn Ile Ile Ala Phe Tyr Asn Ciln Leu Ciln Gln Leu 
               
               
                 Glu Gln Met Thr Thr Thr Ala Glu Asn Trp Leu Lys Ile Gln Pro Thr Thr Pro Ser Glu Pro Thr Ala 
               
               
                 Ile Lys Ser Gln Leu Lys Ile Cys Lys Asp Glu Val Asn Arg Leu Ser Gly Leu Gln Pro Gln Ile Glu 
               
               
                 Arg Leu Lys Ile Gln Ser Ile Ala Leu Lys Glu Lys Gly Gln Gly Pro Met Phe Leu Asp Ala Asp 
               
               
                 Phe Val Ala Phe Thr Asn His Phe Lys Gln Val Phe Ser Asp Val Gln Ala Arg Glu Lys Glu Leu 
               
               
                 Gln Thr Ile Phe Asp Thr Leu Pro Pro Met Arg Tyr Gln Glu Thr Met Ser Ala Ile Arg Thr Trp Val 
               
               
                 Gln Gln Ser Glu Thr Lys Leu Ser Ile Pro Gln Leu Ser Val Thr Asp Tyr Glu Ile Met Glu Gln Arg 
               
               
                 Leu Gly Glu Leu Gln Ala Leu Gln Ser Ser Leu Gln Glu Gln Gln Ser Gly Leu Tyr Tyr Leu Ser 
               
               
                 Thr Thr Val Lys Glu Met Ser Lys Lys Ala Pro Ser Glu Ile Ser Arg Lys Tyr Gln Ser Glu Phe 
               
               
                 Glu Glu Ile Glu Gly Arg Trp Lys Lys Leu Ser Ser Gln Leu Val Glu His Cys Gln Lys Leu Glu 
               
               
                 Glu Gln Met Asn Lys Leu Arg Lys Ile Gln Asn His Ile Gln Thr Leu Lys Lys Trp Met Ala Glu 
               
               
                 Val Asp Val Phe Leu Lys Glu Glu Trp Pro Ala Leu Gly Asp Ser Glu Ile Leu Lys Lys Gln Leu 
               
               
                 Lys Gln Cys Arg Leu Leu Val Ser Asp Ile Gln Thr Ile Gln Pro Ser Leu Asn Ser Val Asn Glu 
               
               
                 Gly Gly Gln Lys Ile Lys Asn Glu Ala Glu Pro Glu Phe Ala Ser Arg Leu Glu Thr Glu Leu Lys 
               
               
                 Glu Leu Asn Thr Gln Trp Asp His Met Cys Gln Gln Val Tyr Ala Arg Lys Glu Ala Leu Lys 
               
               
                 Gly Gly Leu Glu Lys Thr Val Ser Leu Gln Lys Asp Leu Ser Glu Met His Glu Trp Met Thr 
               
               
                 Gln Ala Glu Glu Glu Tyr Leu Glu Arg Asp Phe Glu Tyr Lys Thr Pro Asp Glu Leu Gln Lys 
               
               
                 Ala Val Glu Glu Met Lys Arg Ala Lys Glu Glu Ala Gln Gln Lys Glu Ala Lys Val Lys Leu 
               
               
                 Leu Thr Glu Ser Val Asn Ser Val Ile Ala Gln Ala Pro Pro Val Ala Gln Glu Ala Leu Lys Lys 
               
               
                 Glu Leu Glu Thr Leu Thr Thr Asn Tyr Gln Trp Leu Cys Thr Arg Leu Asn Gly Lys Cys Lys 
               
               
                 Thr Leu Glu Glu Val Trp Ala Cys Trp His Glu Leu Leu Ser Tyr Leu Glu Lys Ala Asn Lys 
               
               
                 Trp Leu Asn Glu Val Glu Phe Lys Leu Lys Thr Thr Glu Asn Ile Pro Gly Gly Ala Glu Glu Ile 
               
               
                 Ser Glu Val Leu Asp Ser Leu Glu Asn Leu Met Arg His Ser Glu Asp Asn Pro Asn Gln Ile Arg 
               
               
                 Ile Leu Ala Gln Thr Leu Thr Asp Gly Gly Val Met Asp Glu Leu Ile Asn Glu Glu Leu Glu Thr 
               
               
                 Phe Asn Ser Arg Trp Arg Glu Leu His Glu Glu Ala Val Arg Arg Gln Lys Leu Leu Glu Gln 
               
               
                 Ser Ile Gln Ser Ala Gln Glu Thr Glu Lys Ser Leu His Leu Ile Gln Glu Ser Leu Thr Phe Ile Asp 
               
               
                 Lys Gln Leu Ala Ala Tyr Ile Ala Asp Lys Val Asp Ala Ala Gln Met Pro Gln Glu Ala Gln Lys 
               
               
                 Ile Gln Ser Asp Leu Thr Ser His Glu Ile Ser Leu Glu Glu Met Lys Lys His Asn Gln Gly Lys 
               
               
                 Glu Ala Ala Gln Arg Val Leu Ser Gln Ile Asp Val Ala Gln Lys Lys Leu Gln Asp Val Ser Met 
               
               
                 Lys Phe Arg Leu Phe Gln Lys Pro Ala Asn Phe Glu Gln Arg Leu Gln Glu Ser Lys Met Ile Leu 
               
               
                 Asp Glu Val Lys Met His Leu Pro Ala Leu Glu Thr Lys Ser Val Glu Gln Glu Val Val Gln Ser 
               
               
                 Gln Leu Asn His Cys Val Asn Leu Tyr Lys Ser Leu Ser Glu Val Lys Ser Glu Val Glu Met Val 
               
               
                 Ile Lys Thr Gly Arg Gln Ile Val Gln Lys Lys Gln Thr Glu Asn Pro Lys Glu Leu Asp Glu Arg 
               
               
                 Val Thr Ala Leu Lys Leu His Tyr Asn Glu Leu Gly Ala Lys Val Thr Glu Arg Lys Gln Gln 
               
               
                 Leu Glu Lys Cys Leu Lys Leu Ser Arg Lys Met Arg Lys Glu Met Asn Val Leu Thr Glu Trp 
               
               
                 Leu Ala Ala Thr Asp Met Glu Leu Thr Lys Arg Ser Ala Val Glu Gly Met Pro Ser Asn Leu 
               
               
                 Asp Ser Glu Val Ala Trp Gly Lys Ala Thr Gln Lys Glu Ile Glu Lys Gln Lys Val His Leu Lys 
               
               
                 Ser Ile Thr Glu Val Gly Glu Ala Leu Lys Thr Val Leu Gly Lys Lys Glu Thr Leu Val Glu Asp 
               
               
                 Lys Leu Ser Leu Leu Asn Ser Asn Trp Ile Ala Val Thr Ser Arg Ala Glu Glu Trp Leu Asn Leu 
               
               
                 Leu Leu Glu Tyr Gln Lys His Met Glu Thr Phe Asp Gln Asn Val Asp His Ile Thr Lys Trp Ile 
               
               
                 Ile Gln Ala Asp Thr Leu Leu Asp Glu Ser Glu Lys Lys Lys Pro Gln Gln Lys Glu Asp Val Leu 
               
               
                 Lys Arg Leu Lys Ala Glu Leu Asn Asp Ile Arg Pro Lys Val Asp Ser Thr Arg Asp Gln Ala Ala 
               
               
                 Asn Leu Met Ala Asn Arg Gly Asp His Cys Arg Lys Leu Val Glu Pro Gln Ile Ser Glu Leu 
               
               
                 Asn His Arg Phe Ala Ala Ile Ser His Arg Ile Lys Thr Gly Lys Ala Ser Ile Pro Leu Lys Glu 
               
               
                 Leu Glu Gln Phe Asn Ser Asp Ile Gln Lys Leu Leu Glu Pro Leu Glu Ala Glu Ile Gln Gln Gly 
               
               
                 Val Asn Leu Lys Glu Glu Asp Phe Asn Lys Asp Met Asn Glu Asp Asn Glu Gly Thr Val Lys 
               
               
                 Glu Leu Leu Gln Arg Gly Asp Asn Leu Gln Gln Arg Ile Thr Asp Glu Arg Lys Arg Glu Glu 
               
               
                 Ile Lys Ile Lys Gln Gln Leu Leu Gln Thr Lys His Asn Ala Leu Lys Asp Leu Arg Ser Gln Arg 
               
               
                 Arg Lys Lys Ala Leu Glu Ile Ser His Gln Trp Tyr Gln Tyr Lys Arg Gln Ala Asp Asp Leu Leu 
               
               
                 Lys Cys Leu Asp Asp Ile Glu Lys Lys Leu Ala Ser Leu Pro Glu Pro Arg Asp Glu Arg Lys Ile 
               
               
                 Lys Glu Ile Asp Arg Glu Leu Gln Lys Lys Lys Glu Glu Leu Asn Ala Val Arg Arg Gln Ala 
               
               
                 Glu Gly Leu Ser Glu Asp Gly Ala Ala Met Ala Val Glu Pro Thr Gln Ile Gln Leu Ser Lys Arg 
               
               
                 Trp Arg Glu Ile Glu Ser Lys Phe Ala Gln Phe Arg Arg Leu Asn Phe Ala Gln Ile His Thr Val 
               
               
                 Arg Glu Glu Thr Met Met Val Met Thr Glu Asp Met Pro Leu Glu Ile Ser Tyr Val Pro Ser Thr 
               
               
                 Tyr Leu Thr Glu Ile Thr His Val Ser Gln Ala Leu Leu Glu Val Glu Gln Leu Leu Asn Ala Pro 
               
               
                 Asp Leu Cys Ala Lys Asp Phe Glu Asp Leu Phe Lys Gln Glu Glu Ser Leu Lys Asn Ile Lys 
               
               
                 Asp Ser Leu Gln Gln Ser Ser Gly Arg Ile Asp Ile Ile His Ser Lys Lys Thr Ala Ala Leu Gln Ser 
               
               
                 Ala Thr Pro Val Glu Arg Val Lys Leu Gln Glu Ala Leu Ser Gln Leu Asp Phe Gln Trp Glu Lys 
               
               
                 Val Asn Lys Met Tyr Lys Asp Arg Gln Gly Arg Phe Asp Arg Ser Val Glu Lys Trp Arg Arg 
               
               
                 Phe His Tyr Asp Ile Lys Ile Phe Asn Gln Trp Leu Thr Glu Ala Glu Gln Phe Leu Arg Lys Thr 
               
               
                 Gln Ile Pro Glu Asn Trp Glu His Ala Lys Tyr Lys Trp Tyr Leu Lys Glu Leu Gln Asp Gly Ile 
               
               
                 Gly Gln Arg Gln Thr Val Val Arg Thr Leu Asn Ala Thr Gly Glu Glu Ile Ile Gln Gln Ser Ser 
               
               
                 Lys Thr Asp Ala Ser Ile Leu Gln Glu Lys Leu Gly Ser Leu Asn Leu Arg Trp Gln Glu Val 
               
               
                 Cys Lys Gln Leu Ser Asp Arg Lys Lys Arg Leu Glu Glu Gln Lys Asn Ile Leu Ser Glu Phe 
               
               
                 Gln Arg Asp Leu Asn Glu Phe Val Leu Trp Leu Glu Glu Ala Asp Asn Ile Ala Ser Ile Pro Leu 
               
               
                 Glu Pro Gly Lys Glu Gln Gln Leu Lys Glu Lys Leu Glu Gln Val Lys Leu Leu Val Glu Glu 
               
               
                 Leu Pro Leu Arg Gln Gly Ile Leu Lys Gln Leu Asn Glu Thr Gly Gly Pro Val Leu Val Ser Ala 
               
               
                 Pro Ile Ser Pro Glu Glu Gln Asp Lys Leu Glu Asn Lys Leu Lys Gln Thr Asn Leu Gln Trp Ile 
               
               
                 Lys Val Ser Arg Ala Leu Pro Glu Lys Gln Gly Glu Ile Glu Ala Gln Ile Lys Asp Leu Gly Gln 
               
               
                 Leu Glu Lys Lys Leu Glu Asp Leu Glu Glu Gln Leu Asn His Leu Leu Leu Trp Leu Ser Pro 
               
               
                 Ile Arg Asn Gln Leu Glu Ile Tyr Asn Gln Pro Asn Gln Glu Gly Pro Phe Asp Val Gln Glu Thr 
               
               
                 Glu Ile Ala Val Gln Ala Lys Gln Pro Asp Val Glu Glu Ile Leu Ser Lys Gly Gln His Leu Tyr 
               
               
                 Lys Glu Lys Pro Ala Thr Gln Pro Val Lys Arg Lys Leu Glu Asp Leu Ser Ser Glu Trp Lys Ala 
               
               
                 Val Asn Arg Leu Leu Gln Glu Leu Arg Ala Lys Gln Pro Asp Leu Ala Pro Gly Leu Thr Thr Ile 
               
               
                 Gly Ala Ser Pro Thr Gln Thr Val Thr Leu Val Thr Gln Pro Val Val Thr Lys Glu Thr Ala Ile 
               
               
                 Ser Lys Leu Glu Met Pro Ser Ser Leu Met Leu Glu Val Pro Ala Leu Ala Asp Phe Asn Arg 
               
               
                 Ala Trp Thr Glu Leu Thr Asp Trp Leu Ser Leu Leu Asp Gln Val Ile Lys Ser Gln Arg Val Met 
               
               
                 Val Gly Asp Leu Glu Asp Ile Asn Glu Met Ile Ile Lys Gln Lys Ala Thr Met Gln Asp Leu Glu 
               
               
                 Gln Arg Arg Pro Gln Leu Glu Glu Leu Ile Thr Ala Ala Gln Asn Leu Lys Asn Lys Thr Ser Asn 
               
               
                 Gln Glu Ala Arg Thr Ile Ile Thr Asp Arg Ile Glu Arg Ile Gln Asn Gln Trp Asp Glu Val Gln 
               
               
                 Glu His Leu Gln Asn Arg Arg Gln Gln Leu Asn Glu Met Leu Lys Asp Ser Thr Gln Trp Leu 
               
               
                 Glu Ala Lys Glu Glu Ala Glu Gln Val Leu Gly Gln Ala Arg Ala Lys Leu Glu Ser Trp Lys Glu 
               
               
                 Gly Pro Tyr Thr Val Asp Ala Ile Gln Lys Lys Ile Thr Glu Thr Lys Gln Leu Ala Lys Asp Leu 
               
               
                 Arg Gln Trp Gln Thr Asn Val Asp Val Ala Asn Asp Leu Ala Leu Lys Leu Leu Arg Asp Tyr 
               
               
                 Ser Ala Asp Asp Thr Arg Lys Val His Met Ile Thr Glu Asn Ile Asn Ala Ser Trp Arg Ser Ile 
               
               
                 His Lys Arg Val Ser Glu Arg Glu Ala Ala Leu Glu Glu Thr His Arg Leu Leu Gln Gln Phe Pro 
               
               
                 Leu Asp Leu Glu Lys Phe Leu Ala Trp Leu Thr Glu Ala Glu Thr Thr Ala Asn Val Leu Gln 
               
               
                 Asp Ala Thr Arg Lys Glu Arg Leu Leu Glu Asp Ser Lys Gly Val Lys Glu Leu Met Lys Gln 
               
               
                 Trp Gln Asp Leu Gln Gly Glu Ile Glu Ala His Thr Asp Val Tyr His Asn Leu Asp Glu Asn Ser 
               
               
                 Gln Lys Ile Leu Arg Ser Leu Glu Gly Ser Asp Asp Ala Val Leu Leu Gln Arg Arg Leu Asp 
               
               
                 Asn Met Asn Phe Lys Trp Ser Glu Leu Arg Lys Lys Ser Leu Asn Ile Arg Ser His Leu Glu Ala 
               
               
                 Ser Ser Asp Gln Trp Lys Arg Leu His Leu Ser Leu Gln Glu Leu Leu Val Trp Leu Gln Leu 
               
               
                 Lys Asp Asp Glu Leu Ser Arg Gln Ala Pro Ile Gly Gly Asp Phe Pro Ala Val Gln Lys Gln Asn 
               
               
                 Asp Val His Arg Ala Phe Lys Arg Glu Leu Lys Thr Lys Glu Pro Val Ile Met Ser Thr Leu Glu 
               
               
                 Thr Val Arg Ile Phe Leu Thr Glu Gln Pro Leu Glu Gly Leu Glu Lys Leu Tyr Gln Glu Pro Arg 
               
               
                 Glu Leu Pro Pro Glu Glu Arg Ala Gln Asn Val Thr Arg Leu Leu Arg Lys Gln Ala Glu Glu 
               
               
                 Val Asn Thr Glu Trp Glu Lys Leu Asn Leu His Ser Ala Asp Trp Gln Arg Lys Ile Asp Glu Thr 
               
               
                 Leu Glu Arg Leu Gln Glu Leu Gln Glu Ala Thr Asp Glu Leu Asp Leu Lys Leu Arg Gln Ala 
               
               
                 Glu Val Ile Lys Gly Ser Trp Gln Pro Val Gly Asp Leu Leu Ile Asp Ser Leu Gln Asp His Leu 
               
               
                 Glu Lys Val Lys Ala Leu Arg Gly Glu Ile Ala Pro Leu Lys Glu Asn Val Ser His Val Asn Asp 
               
               
                 Leu Ala Arg Gln Leu Thr Thr Leu Gly Ile Gln Leu Ser Pro Tyr Asn Leu Ser Thr Leu Glu Asp 
               
               
                 Leu Asn Thr Arg Trp Lys Leu Leu Gln Val Ala Val Glu Asp Arg Val Arg Gln Leu His Glu 
               
               
                 Ala His Arg Asp Phe Gly Pro Ala Ser Gln His Phe Leu Ser Thr Ser Val Gln Gly Pro Trp Glu 
               
               
                 Arg Ala Ile Ser Pro Asn Lys Val Pro Tyr Tyr Ile Asn His Glu Thr Gln Thr Thr Cys Trp Asp 
               
               
                 His Pro Lys Met Thr Glu Leu Tyr Gln Ser Leu Ala Asp Leu Asn Asn Val Arg Phe Ser Ala 
               
               
                 Tyr Arg Thr Ala Met Lys Leu Arg Arg Leu Gln Lys Ala Leu Cys Leu Asp Leu Leu Ser Leu 
               
               
                 Ser Ala Ala Cys Asp Ala Leu Asp Gln His Asn Leu Lys Gln Asn Asp Gln Pro Met Asp Ile 
               
               
                 Leu Gln Ile Ile Asn Cys Leu Thr Thr Ile Tyr Asp Arg Leu Glu Gln Glu His Asn Asn Leu Val 
               
               
                 Asn Val Pro Leu Cys Val Asp Met Cys Leu Asn Trp Leu Leu Asn Val Tyr Asp Thr Gly Arg 
               
               
                 Thr Gly Arg Ile Arg Val Leu Ser Phe Lys Thr Gly Ile Ile Ser Leu Cys Lys Ala His Leu Glu 
               
               
                 Asp Lys Tyr Arg Tyr Leu Phe Lys Gln Val Ala Ser Ser Thr Gly Phe Cys Asp Gln Arg Arg 
               
               
                 Leu Gly Leu Leu Leu His Asp Ser Ile Gln Ile Pro Arg Gln Leu Gly Glu Val Ala Ser Phe Gly 
               
               
                 Gly Ser Asn Ile Glu Pro Ser Val Arg Ser Cys Phe Gln Phe Ala Asn Asn Lys Pro Glu Ile Glu 
               
               
                 Ala Ala Leu Phe Leu Asp Trp Met Arg Leu Glu Pro Gin Ser Met Val Trp Leu Pro Val Leu 
               
               
                 His Arg Val Ala Ala Ala Glu Thr Ala Lys His Gln Ala Lys Cys Asn Ile Cys Lys Glu Cys Pro 
               
               
                 Ile Ile Gly Phe Arg Tyr Arg Ser Leu Lys His Phe Asn Tyr Asp Ile Cys Gln Ser Cys Phe Phe 
               
               
                 Ser Gly Arg Val Ala Lys Gly His Lys Met His Tyr Pro Met Val Glu Tyr Cys Thr Pro Thr Thr 
               
               
                 Ser Gly Glu Asp Val Arg Asp Phe Ala Lys Val Leu Lys Asn Lys Phe Arg Thr Lys Arg Tyr 
               
               
                 Phe Ala Lys His Pro Arg Met Gly Tyr Leu Pro Val Gln Thr Val Leu Glu Gly Asp Asn Met 
               
               
                 Glu Thr Pro Val Thr Leu Ile Asn Phe Trp Pro Val Asp Ser Ala Pro Ala Ser Ser Pro Gln Leu 
               
               
                 Ser His Asp Asp Thr His Ser Arg Ile Glu His Tyr Ala Ser Arg Leu Ala Glu Met Glu Asn Ser 
               
               
                 Asn Gly Ser Tyr Leu Asn Asp Ser Ile Ser Pro Asn Glu Ser Ile Asp Asp Glu His Leu Leu Ile 
               
               
                 Gln His Tyr Cys Gln Ser Leu Asn Gln Asp Ser Pro Leu Ser Gln Pro Arg Ser Pro Ala Gln Ile 
               
               
                 Leu Ile Ser Leu Glu Ser Glu Glu Arg Gly Glu Leu Glu Arg Ile Leu Ala Asp Leu Glu Glu Glu 
               
               
                 Asn Arg Asn Leu Gln Ala Glu Tyr Asp Arg Leu Lys Gln Gln His Glu His Lys Gly Leu Ser 
               
               
                 Pro Leu Pro Ser Pro Pro Glu Met Met Pro Thr Ser Pro Gln Ser Pro Arg Asp Ala Glu Leu Ile 
               
               
                 Ala Glu Ala Lys Leu Leu Arg Gln His Lys Gly Arg Leu Glu Ala Arg Met Gln Ile Leu Glu 
               
               
                 Asp His Asn Lys Gln Leu Glu Ser Gln Leu His Arg Leu Arg Gln Leu Leu Glu Gln Pro Glnthi 
               
               
                 Ala Glu Ala Lys Val Asn Gly Thr Thr Val Ser Ser Pro Ser Thr Ser Leu Gln Arg Ser Asp Ser 
               
               
                 Ser Gln Pro Met Leu Leu Arg Val Val Gly Ser Gln Thr Ser Asp Ser Met Gly Glu Glu Asp Leu 
               
               
                 Leu Ser Pro Pro Gln Asp Thr Ser Thr Gly Leu Glu Glu Val Met Glu Gln Leu Asn Asn Ser Phe 
               
               
                 Pro Ser Ser Arg Gly Arg Asn Thr Pro Gly Lys Pro Met Arg Glu Asp Thr Met