Antisense PMO Found in Dystrophic Dog Model Was Effective in Cells from Exon 7-Deleted DMD Patient
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Mendeley | Further Information

{"title"=>"Antisense PMO found in Dystrophic Dog model was effective in cells from exon 7-deleted DMD patient", "type"=>"journal", "authors"=>[{"first_name"=>"Takashi", "last_name"=>"Saito", "scopus_author_id"=>"36063820300"}, {"first_name"=>"Akinori", "last_name"=>"Nakamura", "scopus_author_id"=>"35412454500"}, {"first_name"=>"Yoshitsugu", "last_name"=>"Aoki", "scopus_author_id"=>"36456762400"}, {"first_name"=>"Toshifumi", "last_name"=>"Yokota", "scopus_author_id"=>"7402558385"}, {"first_name"=>"Takashi", "last_name"=>"Okada", "scopus_author_id"=>"57196324941"}, {"first_name"=>"Makiko", "last_name"=>"Osawa", "scopus_author_id"=>"7202375550"}, {"first_name"=>"Shin'ichi", "last_name"=>"Takeda", "scopus_author_id"=>"14054950700"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-77957913755", "pmid"=>"20805873", "sgr"=>"77957913755", "doi"=>"10.1371/journal.pone.0012239", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "issn"=>"19326203", "pui"=>"359761529"}, "id"=>"ed3b2eea-0faa-341c-8182-3cfcb523fad6", "abstract"=>"BACKGROUND: Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J)) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: We converted fibroblasts of CXMD(J) and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J) and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species.\\n\\nCONCLUSION/SIGNIFICANCE: Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.", "link"=>"http://www.mendeley.com/research/antisense-pmo-found-dystrophic-dog-model-effective-cells-exon-7deleted-dmd-patient", "reader_count"=>28, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Researcher"=>4, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>4, "Student > Master"=>7, "Other"=>2, "Student > Bachelor"=>4, "Professor"=>3, "Lecturer > Senior Lecturer"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Researcher"=>4, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>4, "Student > Master"=>7, "Other"=>2, "Student > Bachelor"=>4, "Professor"=>3, "Lecturer > Senior Lecturer"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>5, "Agricultural and Biological Sciences"=>10, "Medicine and Dentistry"=>7, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Social Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>7}, "Social Sciences"=>{"Social Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>10}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}, "Unspecified"=>{"Unspecified"=>3}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"United Kingdom"=>1}, "group_count"=>3}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/835277"], "description"=>"<p>Distances from dystrophin exon 6 to exon 8 are indicated based on the GenBank reference sequences of <i>Canis familiaris</i> chromosome X genomic contig, whole genome shotgun sequence (NW_879562.1) and <i>Homo sapiens</i> 211000035840903 genomic scaffold, whole genome shotgun sequence (CH471074.1).</p>", "links"=>[], "tags"=>["dystrophin", "exons", "11"], "article_id"=>505641, "categories"=>["Molecular Biology", "Cell Biology", "Neuroscience"], "users"=>["Takashi Saito", "Akinori Nakamura", "Yoshitsugu Aoki", "Toshifumi Yokota", "Takashi Okada", "Makiko Osawa", "Shin'ichi Takeda"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012239.g005", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Location_of_dystrophin_exons_5_to_11_in_the_genome_/505641", "title"=>"Location of dystrophin exons 5 to 11 in the genome.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-08-18 01:34:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/835201"], "description"=>"<p>(A) Schematic diagram of the three- and four-antisense PMO cocktails. For DMD 8772, Ex6B was replaced with hEx6B. In the four-antisense PMO cocktail, one additional sequence (Ex8G, Ex8I, or Ex8K) was added to the three-antisense PMO cocktail. (B) RT-PCR of dystrophin mRNA isolated from MyoD-transduced fibroblasts after treatment with the three- and four-antisense PMO cocktails. In-frame exon skipping products were 99 bp in dog and 221 bp and 92 bp in human. (C) Representative immunostaining and immunoblotting analysis of MyoD-transduced fibroblasts treated with antisense PMO cocktails. The nuclei were counterstained with DAPI. Scale bar: 100 µm. Expected molecular weights of truncated human dystrophin with exons 6–8 and exons 6–9 skipped are 18.3 kDa and 23.1 kDa, respectively, smaller than the full-length dystrophin.</p>", "links"=>[], "tags"=>["exon", "skipping", "dystrophin", "dmd", "8772-derived"], "article_id"=>505561, "categories"=>["Molecular Biology", "Cell Biology", "Neuroscience"], "users"=>["Takashi Saito", "Akinori Nakamura", "Yoshitsugu Aoki", "Toshifumi Yokota", "Takashi Okada", "Makiko Osawa", "Shin'ichi Takeda"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012239.g004", "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Multi_exon_skipping_and_recovery_of_dystrophin_in_CXMD_J_and_DMD_8772_derived_cells_/505561", "title"=>"Multi exon skipping and recovery of dystrophin in CXMD<sub>J</sub> and DMD 8772-derived cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-08-18 01:32:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/834979"], "description"=>"<p>(A) Schematic diagram of the retroviral expression vector. (B) Histograms showing GFP fluorescence intensity compared with cell numbers of normal human and DMD 8772 MyoD-GFP-transduced fibroblasts. Both cells were analyzed five days after retroviral transfection. (C) Immunostaining of MyoD-transduced of dog and human fibroblasts after 10 and 15 days of myogenic differentiation, respectively. MyHC, myosin heavy chain. The nuclei were counter-stained with DAPI. Scale bar: 100 µm. (D) The time course of dystrophin expression in dog and human MyoD-transduced fibroblasts by qRT-PCR and immunoblotting analysis. The mRNA levels were normalized to <i>GAPDH</i> and expressed relative to the amount of the lowest one in each group. For immunoblotting, 5 µg of total protein was loaded into each lane. Error bars indicate standard deviation. (E) Determination of dystrophin mRNA expression in each cell type from DMD 8772 by qRT-PCR. MyoD-transduced fibroblasts were assayed 15 days after differentiation. Normalization and relative expression are the same as (D).</p>", "links"=>[], "tags"=>["fibroblasts", "dystrophin"], "article_id"=>505349, "categories"=>["Molecular Biology", "Cell Biology", "Neuroscience"], "users"=>["Takashi Saito", "Akinori Nakamura", "Yoshitsugu Aoki", "Toshifumi Yokota", "Takashi Okada", "Makiko Osawa", "Shin'ichi Takeda"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012239.g002", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Myogenic_conversion_of_fibroblasts_and_dystrophin_expression_/505349", "title"=>"Myogenic conversion of fibroblasts and dystrophin expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-08-18 01:29:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/415813", "https://ndownloader.figshare.com/files/415820", "https://ndownloader.figshare.com/files/415823", "https://ndownloader.figshare.com/files/415834"], "description"=>"<div><h3>Background</h3><p>Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD<sub>J</sub>) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells.</p><h3>Methodology/Principal Findings</h3><p>We converted fibroblasts of CXMD<sub>J</sub> and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells <em>in vitro</em>. In the CXMD<sub>J</sub> and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species.</p><h3>Conclusion/Significance</h3><p>Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.</p></div>", "links"=>[], "tags"=>["antisense", "pmo", "dystrophic", "was", "cells", "exon", "7-deleted", "dmd"], "article_id"=>142102, "categories"=>["Molecular Biology", "Cell Biology", "Neuroscience"], "users"=>["Takashi Saito", "Akinori Nakamura", "Yoshitsugu Aoki", "Toshifumi Yokota", "Takashi Okada", "Makiko Osawa", "Shin'ichi Takeda"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0012239.s001", "https://dx.doi.org/10.1371/journal.pone.0012239.s002", "https://dx.doi.org/10.1371/journal.pone.0012239.s003", "https://dx.doi.org/10.1371/journal.pone.0012239.s004"], "stats"=>{"downloads"=>2, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Antisense_PMO_Found_in_Dystrophic_Dog_Model_Was_Effective_in_Cells_from_Exon_7_Deleted_DMD_Patient/142102", "title"=>"Antisense PMO Found in Dystrophic Dog Model Was Effective in Cells from Exon 7-Deleted DMD Patient", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2010-08-18 00:35:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/835077"], "description"=>"<p>(A) Exonic splicing enhancer motifs predicted <i>in silico</i> based on human sequence (small coloured boxes) and positions of antisense PMOs (green and blue rectangular areas). The horizontal axis represents base positions in each exon from 5′ to 3′, and the vertical axis represents relative predicted values of the motifs. PESE: putative exonic splicing enhancer. ESE: exonic splicing enhancer. Base mismatches between dog and human (black bar) are indicated in the exon (grey box). RT-PCR of dystrophin mRNA of MyoD-transduced CXMD<sub>J</sub> fibroblasts treated with (B) a mixture of Ex6A and Ex6B and (C) only Ex8A or mixtures containing Ex8A.</p>", "links"=>[], "tags"=>["antisense", "pmo", "targeting", "exons"], "article_id"=>505441, "categories"=>["Molecular Biology", "Cell Biology", "Neuroscience"], "users"=>["Takashi Saito", "Akinori Nakamura", "Yoshitsugu Aoki", "Toshifumi Yokota", "Takashi Okada", "Makiko Osawa", "Shin'ichi Takeda"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012239.g003", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Design_of_antisense_PMO_sequence_targeting_exons_6_and_8_/505441", "title"=>"Design of antisense PMO sequence targeting exons 6 and 8.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-08-18 01:30:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/834900"], "description"=>"<p>(A) Splice-site mutation of a splice acceptor site in intron 6 (asterisk) excludes exon 7 from dog dystrophin mRNA. Frame-shift deletion of dystrophin exon 7 in DMD 8772 was diagnosed by MLPA analysis. Skipping of exon 9 is a frequent splice variant. Both ends of the schematic box of the exon represent a phase of the codon (see detail, Yokota et al. 2009). (B) RT-PCR and sequence analysis of dystrophin mRNA using normal and DMD 8772 lymphocytes. Double bands due to a splicing variant of exon 9 were observed. (C) Breakpoint analysis of DMD 8772 revealed a 50.4 kb deletion from intron 6 to intron 7, and the insertion of 13 bases of unknown origin.</p>", "links"=>[], "tags"=>["dmd"], "article_id"=>505266, "categories"=>["Molecular Biology", "Cell Biology", "Neuroscience"], "users"=>["Takashi Saito", "Akinori Nakamura", "Yoshitsugu Aoki", "Toshifumi Yokota", "Takashi Okada", "Makiko Osawa", "Shin'ichi Takeda"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012239.g001", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mutation_analysis_of_DMD_8772_/505266", "title"=>"Mutation analysis of DMD 8772.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-08-18 01:27:46"}

PMC Usage Stats | Further Information

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