Exon-skipping events in candidates for clinical trials of morpholino

Background:  Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most...

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Veröffentlicht in:Pediatrics international 2011-08, Vol.53 (4), p.524-529
Hauptverfasser: Nakano, Shiho, Ozasa, Shiro, Yoshioka, Kowashi, Fujii, Isao, Mitsui, Kouichi, Nomura, Keiko, Kosuge, Hirofumi, Endo, Fumio, Matsukura, Makoto, Kimura, Shigemi
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container_end_page 529
container_issue 4
container_start_page 524
container_title Pediatrics international
container_volume 53
creator Nakano, Shiho
Ozasa, Shiro
Yoshioka, Kowashi
Fujii, Isao
Mitsui, Kouichi
Nomura, Keiko
Kosuge, Hirofumi
Endo, Fumio
Matsukura, Makoto
Kimura, Shigemi
description Background:  Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in‐frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino‐mediated exon skipping, which changes out‐of‐frame deletions to in‐frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon‐skipping event in cells of DMD patients before the clinical trial. Methods:  Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon‐skipping event was analyzed on reverse transcription–polymerase chain reaction. Results:  Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51‐skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon‐skipping patterns, which are total or partial exon 51‐skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out‐of‐frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51‐skipping event. Conclusion:  An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.
doi_str_mv 10.1111/j.1442-200X.2011.03330.x
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The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in‐frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino‐mediated exon skipping, which changes out‐of‐frame deletions to in‐frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon‐skipping event in cells of DMD patients before the clinical trial. Methods:  Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon‐skipping event was analyzed on reverse transcription–polymerase chain reaction. Results:  Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51‐skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon‐skipping patterns, which are total or partial exon 51‐skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out‐of‐frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51‐skipping event. Conclusion:  An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.</description><identifier>ISSN: 1328-8067</identifier><identifier>EISSN: 1442-200X</identifier><identifier>DOI: 10.1111/j.1442-200X.2011.03330.x</identifier><identifier>PMID: 21342350</identifier><language>eng</language><publisher>Melbourne, Australia: Blackwell Publishing Asia</publisher><subject>Antisense ; Becker's muscular dystrophy ; Bone mineral density ; Clinical trials ; Clinical Trials as Topic ; Clonal deletion ; DNA - genetics ; Duchenne muscular dystrophy ; Duchenne's muscular dystrophy ; Dystrophin ; Enhancers ; Exon skipping ; Exons ; fibroblast ; Fibroblasts ; Gene deletion ; Genetic Therapy ; Genetics ; Humans ; In Vitro Techniques ; Infection ; Language ; Molecular biology ; morpholino ; Morpholinos - therapeutic use ; Muscular dystrophy ; Muscular Dystrophy, Duchenne - genetics ; Muscular Dystrophy, Duchenne - therapy ; Oligonucleotides, Antisense - therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sequence Deletion ; Splicing ; Therapy ; Thionucleotides - therapeutic use ; Transfection</subject><ispartof>Pediatrics international, 2011-08, Vol.53 (4), p.524-529</ispartof><rights>2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society</rights><rights>2011 The Authors. 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The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in‐frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino‐mediated exon skipping, which changes out‐of‐frame deletions to in‐frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon‐skipping event in cells of DMD patients before the clinical trial. Methods:  Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon‐skipping event was analyzed on reverse transcription–polymerase chain reaction. 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The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in‐frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino‐mediated exon skipping, which changes out‐of‐frame deletions to in‐frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon‐skipping event in cells of DMD patients before the clinical trial. Methods:  Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon‐skipping event was analyzed on reverse transcription–polymerase chain reaction. Results:  Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51‐skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon‐skipping patterns, which are total or partial exon 51‐skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out‐of‐frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51‐skipping event. Conclusion:  An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.</abstract><cop>Melbourne, Australia</cop><pub>Blackwell Publishing Asia</pub><pmid>21342350</pmid><doi>10.1111/j.1442-200X.2011.03330.x</doi><tpages>6</tpages></addata></record>
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subjects Antisense
Becker's muscular dystrophy
Bone mineral density
Clinical trials
Clinical Trials as Topic
Clonal deletion
DNA - genetics
Duchenne muscular dystrophy
Duchenne's muscular dystrophy
Dystrophin
Enhancers
Exon skipping
Exons
fibroblast
Fibroblasts
Gene deletion
Genetic Therapy
Genetics
Humans
In Vitro Techniques
Infection
Language
Molecular biology
morpholino
Morpholinos - therapeutic use
Muscular dystrophy
Muscular Dystrophy, Duchenne - genetics
Muscular Dystrophy, Duchenne - therapy
Oligonucleotides, Antisense - therapeutic use
Reverse Transcriptase Polymerase Chain Reaction
Sequence Analysis, DNA
Sequence Deletion
Splicing
Therapy
Thionucleotides - therapeutic use
Transfection
title Exon-skipping events in candidates for clinical trials of morpholino
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