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

Abstract

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

Claims

1. A molecule that binds to a continuous stretch of at least 21 nucleotides within exon 45 of DMD pre-mRNA.

2. A molecule according to claim 1, whereby said molecule binds to a continuous stretch of at least 25 nucleotides within said exon.

3. A molecule according to claim 1, whereby said molecule comprises an antisense oligonucleotide of between 21 and 30 bases.

4. A molecule according to claim 1, whereby said molecule comprises an antisense oligonucleotide of 25 bases.

5. A molecule according to claim 1, whereby said molecule binds to a continuous stretch of at least 21 nucleotides within the following nucleotide sequence: TABLE-US-00003 (SEQIDNO:2) 5-CCAGGAUGGCAUUGGGCAGCGGCAAACUGUUGUCAGAACAUUGAAUGC AACUGGGGAAGAAAUAAUUCAGCAAUC.

6. A molecule according to claim 1, comprising or consisting of an antisense nucleotide sequence selected from the ones depicted in Table 1, except SEQ ID NO:68.

7. A molecule according to claim 6, comprising or consisting of the antisense nucleotide sequence selected from 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.

8. A molecule according to claim 7, comprising or consisting of the antisense nucleotide sequence from SEQ ID NO: 3.

9. A molecule according to claim 1, comprising a 2-O-alkyl phosphorothioate antisense oligonucleotide.

10. A molecule according to claim 9, comprising a 2-O-methyl phosphorothioate ribose.

11. A viral-based vector, comprising an expression cassette that drives expression of the molecule as defined in claim 1.

12. A pharmaceutical composition comprising a molecule as defined in claim 1, a pharmaceutical acceptable carrier, and optionally combined with a molecule which is able to induce or promote skipping of exon 7, 44, 46, 51, 53, 59, 67 (as depicted in table 2) of the DMD pre-mRNA of a patient.

13. (canceled)

14. A method for inducing 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 with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon.

15. A method according to claim 14, wherein the patient is provided with a functional dystrophin protein and/or wherein the production of an aberrant dystrophin protein in said patient is decreased, wherein the level of said functional dystrophin is assessed by comparison to the level of said dystrophin in said patient at the onset of the method.

Description

LEGENDS TO THE FIGURE

[0085] 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).

[0086] 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.

[0087] 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

[0088] 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

[0089] Myotube cultures derived from a healthy individual (human control) were obtained as described previously (Aartsma-Rus et al. Hum Mol Genet 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 0 to 500 nM of each AON. The transfection reagent polyethylenimine (PEI, ExGen500 MBI Fermentas) was used according to manufacturer's instructions, with 2 l PEI per g AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking exon 45. PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the Agilent DNA 1000 LabChip Kit and the Agilent 2100 bioanalyzer (Agilent Technologies, USA).

Results

[0090] A series of AONs targeting sequences within SEQ ID NO:2 within exon 45 were designed and tested in normal myotube cultures, by transfection and subsequent RT-PCR and sequence analysis of isolated RNA. PS220 (SEQ ID NO: 3) reproducibly induced highest levels of exon 45 skipping, when compared to PS221-PS225 (FIG. 1). High levels of exon 45 skipping of up to 75% were already obtained at 400 nM PS220 (FIG. 2). In a direct comparison, PS220 (a 25-mer) was reproducibly more efficient in inducing exon 45 skipping than its shorter 17-mer counterpart AON 45-5 (SEQ ID NO: 68; previously published as h45AON5 (Aartsma-Rus et al. Am J Hum Genet 2004; 74: 83-92)), at both AON concentrations of 200 nM and 500 nM and with 63% versus 3% respectively at 500 nM (FIG. 3). This result is probably due to the fact that the extended length of PS220, in fact completely overlapping AON 45-5, increases the free energy of the AON-target complex such that the efficiency of inducing exon 45 skipping is also increased.

TABLE-US-00001 TABLE1 AONsinexon45 SEQIDNO3 UUUGCCGCUGCCCAAUGCCAUCCUG (PS220) SEQIDNO4 AUUCAAUGUUCUGACAACAGUUUGC (PS221) SEQIDNO5 CCAGUUGCAUUCAAUGUUCUGACAA (PS222) SEQIDNO6 CAGUUGCAUUCAAUGUUCUGAC (PS223) SEQIDNO7 AGUUGCAUUCAAUGUUCUGA (PS224) SEQIDNO8 GAUUGCUGAAUUAUUUCUUCC (PS225) SEQIDNO9 GAUUGCUGAAUUAUUUCUUCCCCAG SEQIDNO10 AUUGCUGAAUUAUUUCUUCCCCAGU SEQIDNO11 UUGCUGAAUUAUUUCUUCCCCAGUU SEQIDNO12 UGCUGAAUUAUUUCUUCCCCAGUUG SEQIDNO13 GCUGAAUUAUUUCUUCCCCAGUUGC SEQIDNO14 CUGAAUUAUUUCUUCCCCAGUUGCA SEQIDNO15 UGAAUUAUUUCUUCCCCAGUUGCAU SEQIDNO16 GAAUUAUUUCUUCCCCAGUUGCAUU SEQIDNO17 AAUUAUUUCUUCCCCAGUUGCAUUC SEQIDNO18 AUUAUUUCUUCCCCAGUUGCAUUCA SEQIDNO19 UUAUUUCUUCCCCAGUUGCAUUCAA SEQIDNO20 UAUUUCUUCCCCAGUUGCAUUCAAU SEQIDNO21 AUUUCUUCCCCAGUUGCAUUCAAUG SEQIDNO22 UUUCUUCCCCAGUUGCAUUCAAUGU SEQIDNO23 UUCUUCCCCAGUUGCAUUCAAUGUU SEQIDNO24 UCUUCCCCAGUUGCAUUCAAUGUUC SEQIDNO25 CUUCCCCAGUUGCAUUCAAUGUUCU SEQIDNO26 UUCCCCAGUUGCAUUCAAUGUUCUG SEQIDNO27 UCCCCAGUUGCAUUCAAUGUUCUGA SEQIDNO28 CCCCAGUUGCAUUCAAUGUUCUGAC SEQIDNO29 CCCAGUUGCAUUCAAUGUUCUGACA SEQIDNO30 CCAGUUGCAUUCAAUGUUCUGACAA SEQIDNO31 CAGUUGCAUUCAAUGUUCUGACAAC SEQIDNO32 AGUUGCAUUCAAUGUUCUGACAACA SEQIDNO33 UCCUGUAGAAUACUGGCAUC SEQIDNO34 UGCAGACCUCCUGCCACCGCAGAUU CA SEQIDNO35 UUGCAGACCUCCUGCCACCGCAGAU UCAGGCUUC SEQIDNO36 GUUGCAUUCAAUGUUCUGACAACAG SEQIDNO37 UUGCAUUCAAUGUUCUGACAACAGU SEQIDNO38 UGCAUUCAAUGUUCUGACAACAGUU SEQIDNO39 GCAUUCAAUGUUCUGACAACAGUUU SEQIDNO40 CAUUCAAUGUUCUGACAACAGUUUG SEQIDNO41 AUUCAAUGUUCUGACAACAGUUUGC SEQIDNO42 UCAAUGUUCUGACAACAGUUUGCCG SEQIDNO43 CAAUGUUCUGACAACAGUUUGCCGC SEQIDNO44 AAUGUUCUGACAACAGUUUGCCGCU SEQIDNO45 AUGUUCUGACAACAGUUUGCCGCUG SEQIDNO46 UGUUCUGACAACAGUUUGCCGCUGC SEQIDNO47 GUUCUGACAACAGUUUGCCGCUGCC SEQIDNO48 UUCUGACAACAGUUUGCCGCUGCCC SEQIDNO49 UCUGACAACAGUUUGCCGCUGCCCA SEQIDNO50 CUGACAACAGUUUGCCGCUGCCCAA SEQIDNO51 UGACAACAGUUUGCCGCUGCCCAAU SEQIDNO52 GACAACAGUUUGCCGCUGCCCAAUG SEQIDNO53 ACAACAGUUUGCCGCUGCCCAAUGC SEQIDNO54 CAACAGUUUGCCGCUGCCCAAUGCC SEQIDNO55 AACAGUUUGCCGCUGCCCAAUGCCA SEQIDNO56 ACAGUUUGCCGCUGCCCAAUGCCAU SEQIDNO57 CAGUUUGCCGCUGCCCAAUGCCAUC SEQIDNO58 AGUUUGCCGCUGCCCAAUGCCAUCC SEQIDNO59 GUUUGCCGCUGCCCAAUGCCAUCCU SEQIDNO60 UUUGCCGCUGCCCAAUGCCAUCCUG SEQIDNO61 UUGCCGCUGCCCAAUGCCAUCCUGG SEQIDNO62 UGCCGCUGCCCAAUGCCAUCCUGGA SEQIDNO63 GCCGCUGCCCAAUGCCAUCCUGGAG SEQIDNO64 CCGCUGCCCAAUGCCAUCCUGGAGU SEQIDNO65 CGCUGCCCAAUGCCAUCCUGGAGUU SEQIDNO66 UGUUUUUGAGGAUUGCUGAA SEQIDNO67 UGUUCUGACAACAGUUUGCCGCUGC CCAAUGCCAUCCUGG SEQIDNO68 GCCCAAUGCCAUCCUGG (45-5)

TABLE-US-00002 TABLE2 AONsinexons51,53,7,44,46,59,and67 DMDGeneExon51 SEQIDNO69 AGAGCAGGUACCUCCAACAUCAAGG SEQIDNO70 GAGCAGGUACCUCCAACAUCAAGGA SEQIDNO71 AGCAGGUACCUCCAACAUCAAGGAA SEQIDNO72 GCAGGUACCUCCAACAUCAAGGAAG SEQIDNO73 CAGGUACCUCCAACAUCAAGGAAGA SEQIDNO74 AGGUACCUCCAACAUCAAGGAAGAU SEQIDNO75 GGUACCUCCAACAUCAAGGAAGAUG SEQIDNO76 GUACCUCCAACAUCAAGGAAGAUGG SEQIDNO77 UACCUCCAACAUCAAGGAAGAUGGC SEQIDNO78 ACCUCCAACAUCAAGGAAGAUGGCA SEQIDNO79 CCUCCAACAUCAAGGAAGAUGGCAU SEQIDNO80 CUCCAACAUCAAGGAAGAUGGCAUU SEQIDNO81 CUCCAACAUCAAGGAAGAUGGCAUU UCUAG SEQIDNO82 UCCAACAUCAAGGAAGAUGGCAUUU SEQIDNO83 CCAACAUCAAGGAAGAUGGCAUUUC SEQIDNO84 CAACAUCAAGGAAGAUGGCAUUUCU SEQIDNO85 AACAUCAAGGAAGAUGGCAUUUCUA SEQIDNO86 ACAUCAAGGAAGAUGGCAUUUCUAG SEQIDNO87 ACAUCAAGGAAGAUGGCAUUUCUAG UUUGG SEQIDNO88 ACAUCAAGGAAGAUGGCAUUUCUAG SEQIDNO89 CAUCAAGGAAGAUGGCAUUUCUAGU SEQIDNO90 AUCAAGGAAGAUGGCAUUUCUAGUU SEQIDNO91 UCAAGGAAGAUGGCAUUUCUAGUUU SEQIDNO92 UCAAGGAAGAUGGCAUUUCU SEQIDNO93 CAAGGAAGAUGGCAUUUCUAGUUUG SEQIDNO94 AAGGAAGAUGGCAUUUCUAGUUUGG SEQIDNO95 AGGAAGAUGGCAUUUCUAGUUUGGA SEQIDNO96 GGAAGAUGGCAUUUCUAGUUUGGAG SEQIDNO97 GAAGAUGGCAUUUCUAGUUUGGAGA SEQIDNO98 AAGAUGGCAUUUCUAGUUUGGAGAU SEQIDNO99 AGAUGGCAUUUCUAGUUUGGAGAUG SEQIDNO100 GAUGGCAUUUCUAGUUUGGAGAUGG SEQIDNO101 AUGGCAUUUCUAGUUUGGAGAUGGC SEQIDNO102 UGGCAUUUCUAGUUUGGAGAUGGCA SEQIDNO103 GGCAUUUCUAGUUUGGAGAUGGCAG SEQIDNO104 GCAUUUCUAGUUUGGAGAUGGCAGU SEQIDNO105 CAUUUCUAGUUUGGAGAUGGCAGUU SEQIDNO106 AUUUCUAGUUUGGAGAUGGCAGUUU SEQIDNO107 UUUCUAGUUUGGAGAUGGCAGUUUC SEQIDNO108 UUCUAGUUUGGAGAUGGCAGUUUCC DMDGeneExon53 SEQIDNO109 CCAUUGUGUUGAAUCCUUUAACAUU SEQIDNO110 CCAUUGUGUUGAAUCCUUUAAC SEQIDNO111 AUUGUGUUGAAUCCUUUAAC SEQIDNO112 CCUGUCCUAAGACCUGCUCA SEQIDNO113 CUUUUGGAUUGCAUCUACUGUAUAG SEQIDNO114 CAUUCAACUGUUGCCUCCGGUUCUG SEQIDNO115 CUGUUGCCUCCGGUUCUGAAGGUG SEQIDNO116 CAUUCAACUGUUGCCUCCGGUUCUG AAGGUG SEQIDNO117 CUGAAGGUGUUCUUGUACUUCAUCC SEQIDNO118 UGUAUAGGGACCCUCCUUCCAUGACUC SEQIDNO119 AUCCCACUGAUUCUGAAUUC SEQIDNO120 UUGGCUCUGGCCUGUCCUAAGA SEQIDNO121 AAGACCUGCUCAGCUUCUUCCUUAG CUUCCAGCCA DMDGeneExon7 SEQIDNO122 UGCAUGUUCCAGUCGUUGUGUGG SEQIDNO123 CACUAUUCCAGUCAAAUAGGUCUGG SEQIDNO124 AUUUACCAACCUUCAGGAUCGAGUA SEQIDNO125 GGCCUAAAACACAUACACAUA DMDGeneExon44 SEQIDNO126 UCAGCUUCUGUUAGCCACUG SEQIDNO127 UUCAGCUUCUGUUAGCCACU SEQIDNO128 UUCAGCUUCUGUUAGCCACUG SEQIDNO129 UCAGCUUCUGUUAGCCACUGA SEQIDNO130 UUCAGCUUCUGUUAGCCACUGA SEQIDNO131 UCAGCUUCUGUUAGCCACUGA SEQIDNO132 UUCAGCUUCUGUUAGCCACUGA SEQIDNO133 UCAGCUUCUGUUAGCCACUGAU SEQIDNO134 UUCAGCUUCUGUUAGCCACUGAU SEQIDNO135 UCAGCUUCUGUUAGCCACUGAUU SEQIDNO136 UUCAGCUUCUGUUAGCCACUGAUU SEQIDNO137 UCAGCUUCUGUUAGCCACUGAUUA SEQIDNO138 UUCAGCUUCUGUUAGCCACUGAUA SEQIDNO139 UCAGCUUCUGUUAGCCACUGAUUAA SEQIDNO140 UUCAGCUUCUGUUAGCCACUGAUUAA SEQIDNO141 UCAGCUUCUGUUAGCCACUGAUUAAA SEQIDNO142 UUCAGCUUCUGUUAGCCACUGAUUAAA SEQIDNO143 CAGCUUCUGUUAGCCACCG SEQIDNO144 CAGCUUCUGUUAGCCACCGAU SEQIDNO145 AGCUCCUGUCAGCCACUGACU SEQIDNO146 CAGCUUCUGUUAGCCACUGAUU SEQIDNO147 AGCUCCUGUUAGCCACUGACUA SEQIDNO148 CAGCUUCUGUUAGCCACCGAUUA SEQIDNO149 AGCUCCUGUUAGCCACUGACUAA SEQIDNO150 CAGCUUCUGUUAGCCACCGAUUAA SEQIDNO151 AGCUUCUGUCAGCCACUGAUUAAA SEQIDNO152 CAGCUUCUGUUAGCCACCGAUUAAA SEQIDNO153 AGCUUCUGUCAGCCACUGAUUAAA SEQIDNO154 AGCUUCUGUUAGCCACUGAU SEQIDNO155 GCUUCUGUUAGCCACUGAUU SEQIDNO156 AGCUUCUGUCAGCCACUGAUU SEQIDNO157 GCUUCUGUUAGCCACUGAUUA SEQIDNO158 AGCUUCUGUCAGCCACUGAUUA SEQIDNO159 GCUUCUGUUAGCCACUGAUUAA SEQIDNO160 AGCUUCUGUCAGCCACUGAUUAA SEQIDNO161 GCUUCUGUUAGCCACUGAUUAAA SEQIDNO162 AGCUUCUGUCAGCCACUGAUUAAA SEQIDNO163 GCUUCUGUUAGCCACUGAUUAAA SEQIDNO164 CCAUUUGUACUUAGCAUGUUCCC SEQIDNO165 AGAUACCAUCUGUAUUCAGC SEQIDNO166 GCCAUUUCUCAACAGAUCU SEQIDNO167 GCCAUUUCUCAACAGAUCUGUCA SEQIDNO168 AUUCUCAGGAAUUUGUGUCUUUC SEQIDNO169 UCUCAGGAAUUUGUGUCUUUC SEQIDNO170 GUUCAGCUUCUGUUAGCC SEQIDNO171 CUGAUUAAAUAUCUUUAUAUC SEQIDNO172 GCCGCCAUUUCUCAACAG SEQIDNO173 GUAUUUAGCAUGUUCCCA SEQIDNO174 CAGGAAUUUGUGUCUUCC DMDGeneExon46 SEQIDNO175 GCUUUUCUUUUAGUUGCUGCUCUUU SEQIDNO176 CUUUUCUUUUAGUUGCUGCUCUUUU SEQIDNO177 UUUUCUUUUAGUUGCUGCUCUUUUC SEQIDNO178 UUUCUUUUAGUUGCUGCUCUUUUCC SEQIDNO179 UUCUUUUAGUUGCUGCUCUUUUCCA SEQIDNO180 UCUUUUAGUUGCUGCUCUUUUCCAG SEQIDNO181 CUUUUAGUUGCUGCUCUUUUCCAGG SEQIDNO182 UUUUAGUUGCUGCUCUUUUCCAGGU SEQIDNO183 UUUAGUUGCUGCUCUUUUCCAGGUU SEQIDNO184 UUAGUUGCUGCUCUUUUCCAGGUUC SEQIDNO185 UAGUUGCUGCUCUUUUCCAGGUUCA SEQIDNO186 AGUUGCUGCUCUUUUCCAGGUUCAA SEQIDNO187 GUUGCUGCUCUUUUCCAGGUUCAAG SEQIDNO188 UUGCUGCUCUUUUCCAGGUUCAAGU SEQIDNO189 UGCUGCUCUUUUCCAGGUUCAAGUG SEQIDNO190 GCUGCUCUUUUCCAGGUUCAAGUGG SEQIDNO191 CUGCUCUUUUCCAGGUUCAAGUGGG SEQIDNO192 UGCUCUUUUCCAGGUUCAAGUGGGA SEQIDNO193 GCUCUUUUCCAGGUUCAAGUGGGAC SEQIDNO194 CUCUUUUCCAGGUUCAAGUGGGAUA SEQIDNO195 UCUUUUCCAGGUUCAAGUGGGAUAC SEQIDNO196 CUUUUCCAGGUUCAAGUGGGAUACU SEQIDNO197 UUUUCCAGGUUCAAGUGGGAUACUA SEQIDNO198 UUUCCAGGUUCAAGUGGGAUACUAG SEQIDNO199 UUCCAGGUUCAAGUGGGAUACUAGC SEQIDNO200 UCCAGGUUCAAGUGGGAUACUAGCA SEQIDNO201 CCAGGUUCAAGUGGGAUACUAGCAA SEQIDNO202 CAGGUUCAAGUGGGAUACUAGCAAU SEQIDNO203 AGGUUCAAGUGGGAUACUAGCAAUG SEQIDNO204 GGUUCAAGUGGGAUACUAGCAAUGU SEQIDNO205 GUUCAAGUGGGAUACUAGCAAUGUU SEQIDNO206 UUCAAGUGGGAUACUAGCAAUGUUA SEQIDNO207 UCAAGUGGGAUACUAGCAAUGUUAU SEQIDNO208 CAAGUGGGAUACUAGCAAUGUUAUC SEQIDNO209 AAGUGGGAUACUAGCAAUGUUAUCU SEQIDNO210 AGUGGGAUACUAGCAAUGUUAUCUG SEQIDNO211 GUGGGAUACUAGCAAUGUUAUCUGC SEQIDNO212 UGGGAUACUAGCAAUGUUAUCUGCU SEQIDNO213 GGGAUACUAGCAAUGUUAUCUGCUU SEQIDNO214 GGAUACUAGCAAUGUUAUCUGCUUC SEQIDNO215 GAUACUAGCAAUGUUAUCUGCUUCC SEQIDNO216 AUACUAGCAAUGUUAUCUGCUUCCU SEQIDNO217 UACUAGCAAUGUUAUCUGCUUCCUC SEQIDNO218 ACUAGCAAUGUUAUCUGCUUCCUCC SEQIDNO219 CUAGCAAUGUUAUCUGCUUCCUCCA SEQIDNO220 UAGCAAUGUUAUCUGCUUCCUCCAA SEQIDNO221 AGCAAUGUUAUCUGCUUCCUCCAAC SEQIDNO222 GCAAUGUUAUCUGCUUCCUCCAACC SEQIDNO223 CAAUGUUAUCUGCUUCCUCCAACCA SEQIDNO224 AAUGUUAUCUGCUUCCUCCAACCAU SEQIDNO225 AUGUUAUCUGCUUCCUCCAACCAUA SEQIDNO226 UGUUAUCUGCUUCCUCCAACCAUAA SEQIDNO227 GUUAUCUGCUUCCUCCAACCAUAAA SEQIDNO228 GCUGCUCUUUUCCAGGUUC SEQIDNO229 UCUUUUCCAGGUUCAAGUGG SEQIDNO230 AGGUUCAAGUGGGAUACUA DMDGeneExon59 SEQIDNO231 CAAUUUUUCCCACUCAGUAUU SEQIDNO232 UUGAAGUUCCUGGAGUCUU SEQIDNO233 UCCUCAGGAGGCAGCUCUAAAU DMDGeneExon67 SEQIDNO234 GCGCUGGUCACAAAAUCCUGUUGAAC SEQIDNO235 CACUUGCUUGAAAAGGUCUACAAAGGA SEQIDNO236 GGUGAAUAACUUACAAAUUUGGAAGC