Exon Exchange Approach to Repair Duchenne Dystrophin Transcripts
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{"title"=>"Exon exchange approach to repair duchenne dystrophin transcripts", "type"=>"journal", "authors"=>[{"first_name"=>"Stéphanie", "last_name"=>"Lorain", "scopus_author_id"=>"6507347710"}, {"first_name"=>"Cécile", "last_name"=>"Peccate", "scopus_author_id"=>"8148842400"}, {"first_name"=>"Maëva", "last_name"=>"le Hir", "scopus_author_id"=>"36458665400"}, {"first_name"=>"Luis", "last_name"=>"Garcia", "scopus_author_id"=>"35321077100"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-77956509696", "sgr"=>"77956509696", "issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\n1932-6203 (Linking)", "pmid"=>"20531943", "doi"=>"10.1371/journal.pone.0010894", "pui"=>"359515681"}, "id"=>"525f6718-c372-3483-a6cb-f6e69a4d68f6", "abstract"=>"BACKGROUND: Trans-splicing strategies for mRNA repair involve engineered transcripts designed to anneal target mRNAs in order to interfere with their natural splicing, giving rise to mRNA chimeras where endogenous mutated exons have been replaced by exogenous replacement sequences. A number of trans-splicing molecules have already been proposed for replacing either the 5' or the 3' part of transcripts to be repaired. Here, we show the feasibility of RNA surgery by using a double trans-splicing approach allowing the specific substitution of a given mutated exon.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: As a target we used a minigene encoding a fragment of the mdx dystrophin gene enclosing the mutated exon (exon 23). This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains. We obtained accurate and efficient replacement of exon 23 in the mRNA target. Adding up a downstream intronic splice enhancer DISE in the exon exchange molecule enhanced drastically its efficiency up to 25-45% of repair depending on the construction in use.\\n\\nCONCLUSIONS/SIGNIFICANCE: These results demonstrate the possibility to fix up mutated exons, refurbish deleted exons and introduce protein motifs, while keeping natural untranslated sequences, which are essential for mRNA stability and translation regulation. Conversely to the well-known exon skipping, exon exchange has the advantage to be compatible with almost any type of mutations and more generally to a wide range of genetic conditions. In particular, it allows addressing disorders caused by dominant mutations.", "link"=>"http://www.mendeley.com/research/exon-exchange-approach-repair-duchenne-dystrophin-transcripts", "reader_count"=>36, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>10, "Student > Ph. D. Student"=>6, "Student > Postgraduate"=>3, "Student > Master"=>4, "Student > Bachelor"=>5, "Lecturer"=>1, "Professor"=>5}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>10, "Student > Ph. D. Student"=>6, "Student > Postgraduate"=>3, "Student > Master"=>4, "Student > Bachelor"=>5, "Lecturer"=>1, "Professor"=>5}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>25, "Medicine and Dentistry"=>5, "Neuroscience"=>1, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Neuroscience"=>{"Neuroscience"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>25}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Unspecified"=>{"Unspecified"=>1}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"Sweden"=>1, "France"=>1, "Germany"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/847451"], "description"=>"<p>(A) The exon exchange molecule (ExCh) AS-E24-AS' comprises the same elements as the TS molecule (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010894#pone-0010894-g001\" target=\"_blank\">Fig. 1A</a>) followed by a 5′ donor splice site (5′SS) and a second antisense sequence (AS') of 150 nt complementary to intron 23. The sequence of 5′SS is given in bold and is illustrated by a black ball. ExCh constructs were made with five different AS' antisense sequences, AS4 to AS8. Arrows indicate the positions of the forward A and reverse C PCR primers in the minigene. (B) Expected transcripts generated by cis-splicing (E23 inclusion and skipping) and exon exchange, and predicted sizes of the corresponding PCR amplification products detected using the RT-PCR strategy illustrated in (A). (C) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with dystrophin minigene and constructions pSMD2 (Ctrl), pSMD2-U7-SD23-BP22 (U7), the TS constructions pSMD2-AS1-E24 (AS1), pSMD2-AS2-E24 (AS2) and ExCh molecules pSMD2-AS-E24-AS' containing AS1 or AS2 and AS4 to AS8. AS2-2XAS4, ExCh plasmid pSMD2-AS2-E24-2XAS4 containing two AS4 copies; H20: PCR negative control. Representative results from two independent transfection experiments. (D) Accurate E22-E24 and E24-E24 junctions were confirmed by sequencing of the 408bp product.</p>", "links"=>[], "tags"=>["dystrophin", "minigene"], "article_id"=>517912, "categories"=>["Molecular Biology", "Genetics"], "users"=>["Stéphanie Lorain", "Cécile Peccate", "Maëva Le Hir", "Luis García"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010894.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Exon_replacement_approach_on_dystrophin_minigene_transcripts_/517912", "title"=>"Exon replacement approach on dystrophin minigene transcripts.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-28 02:11:52"}
  • {"files"=>["https://ndownloader.figshare.com/files/847697"], "description"=>"<p>(A) Expected amplicons from PCR1 with E23-F & E23-R primers and (B) from PCR2 with E24-F & E24-R primers. (C) Histogram showing numbers of parental and exchanged transcripts evaluated by absolute quantitative real-time RT-PCR. PCR1 corresponds to the parental transcripts whereas PCR2 to the repaired transcripts. PCR1+PCR2 corresponds to the totality of the dystrophin minigene transcripts. (D) ExChange efficiency estimated by calculating the ratio between levels obtained for repaired transcripts (PCR2) and whole dystrophin minigene transcripts (PCR1+PCR2). Representative results from two independent transfection experiments.</p>", "links"=>[], "tags"=>["dystrophin", "exon", "induced", "as4", "containing", "exch"], "article_id"=>518150, "categories"=>["Molecular Biology", "Genetics"], "users"=>["Stéphanie Lorain", "Cécile Peccate", "Maëva Le Hir", "Luis García"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010894.g004", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Efficiency_of_dystrophin_exon_exchange_induced_by_AS4_containing_ExCh_molecules_/518150", "title"=>"Efficiency of dystrophin exon exchange induced by AS4 containing ExCh molecules.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-28 02:15:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/847855"], "description"=>"<p>(A) The ExCh AS2-E23-DISE-AS4 molecule comprises the same elements as AS2-E24-DISE-AS4, except E23 instead of E24. E23 is the wild-type murine dystrophin E23 carrying a HindIII restriction site absent in the <i>mdx</i> minigene E23. (B) Expected transcripts generated by cis-splicing and E23 exchange, and predicted size of the corresponding RT-PCR products using primers A and C. (C) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with dystrophin minigene and constructions pSMD2 (Ctrl) and ExCh plasmid pSMD2-AS2-E23-DISE-AS4; H20: PCR negative control. Representative results from two independent transfection experiments. (D) Cloning results of the RT-PCR amplicons obtained in (C) with AS2-E23-DISE-AS4 molecule. The clone numbers corresponding to parental and repaired inserts are described.</p>", "links"=>[], "tags"=>["23", "correction", "exon"], "article_id"=>518312, "categories"=>["Molecular Biology", "Genetics"], "users"=>["Stéphanie Lorain", "Cécile Peccate", "Maëva Le Hir", "Luis García"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010894.g006", "stats"=>{"downloads"=>1, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Exon_23_correction_by_exon_replacement_/518312", "title"=>"Exon 23 correction by exon replacement.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-28 02:18:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/847614"], "description"=>"<p>(A) Exon exchange molecules AS-E24-AS' with intronic splice enhancers ISE or DISE sequences. (B) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with dystrophin minigene and constructs pSMD2 (Ctrl), pSMD2-U7-SD23-BP22 (U7) and the following ExCh plasmids with AS4 or AS8: pSMD2-AS2-E24-AS' (-), pSMD2-AS2-ISE-E24-AS' (ISE), pSMD2-AS2-E24-DISE-AS' (DISE) and pSMD2-AS2-E24-2XAS' (2XAS'). H20: PCR negative control.</p>", "links"=>[], "tags"=>["intronic", "splice", "enhancer", "sequences", "exon"], "article_id"=>518075, "categories"=>["Molecular Biology", "Genetics"], "users"=>["Stéphanie Lorain", "Cécile Peccate", "Maëva Le Hir", "Luis García"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010894.g003", "stats"=>{"downloads"=>1, "page_views"=>34, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_intronic_splice_enhancer_sequences_on_exon_replacement_efficiency_/518075", "title"=>"Effect of intronic splice enhancer sequences on exon replacement efficiency.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-28 02:14:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/421575", "https://ndownloader.figshare.com/files/421610", "https://ndownloader.figshare.com/files/421653", "https://ndownloader.figshare.com/files/421708"], "description"=>"<div><h3>Background</h3><p>Trans-splicing strategies for mRNA repair involve engineered transcripts designed to anneal target mRNAs in order to interfere with their natural splicing, giving rise to mRNA chimeras where endogenous mutated exons have been replaced by exogenous replacement sequences. A number of trans-splicing molecules have already been proposed for replacing either the 5′ or the 3′ part of transcripts to be repaired. Here, we show the feasibility of RNA surgery by using a double trans-splicing approach allowing the specific substitution of a given mutated exon.</p><h3>Methodology/Principal Findings</h3><p>As a target we used a minigene encoding a fragment of the <em>mdx</em> dystrophin gene enclosing the mutated exon (exon 23). This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains. We obtained accurate and efficient replacement of exon 23 in the mRNA target. Adding up a downstream intronic splice enhancer DISE in the exon exchange molecule enhanced drastically its efficiency up to 25–45% of repair depending on the construction in use.</p><h3>Conclusions/Significance</h3><p>These results demonstrate the possibility to fix up mutated exons, refurbish deleted exons and introduce protein motifs, while keeping natural untranslated sequences, which are essential for mRNA stability and translation regulation. Conversely to the well-known exon skipping, exon exchange has the advantage to be compatible with almost any type of mutations and more generally to a wide range of genetic conditions. In particular, it allows addressing disorders caused by dominant mutations.</p></div>", "links"=>[], "tags"=>["exon", "duchenne", "dystrophin", "transcripts"], "article_id"=>143260, "categories"=>["Molecular Biology", "Genetics"], "users"=>["Stéphanie Lorain", "Cécile Peccate", "Maëva Le Hir", "Luis García"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0010894.s001", "https://dx.doi.org/10.1371/journal.pone.0010894.s002", "https://dx.doi.org/10.1371/journal.pone.0010894.s003", "https://dx.doi.org/10.1371/journal.pone.0010894.s004"], "stats"=>{"downloads"=>5, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Exon_Exchange_Approach_to_Repair_Duchenne_Dystrophin_Transcripts/143260", "title"=>"Exon Exchange Approach to Repair Duchenne Dystrophin Transcripts", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2010-05-28 00:54:20"}
  • {"files"=>["https://ndownloader.figshare.com/files/847774"], "description"=>"<p>(A) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with 250 ng of dystrophin minigene and 100, 200, 400 and 750 ng of pSMD2-AS2-E24-DISE-AS4 plasmids. (B) Histogram showing numbers of parental and exchanged transcripts evaluated by absolute quantitative real-time RT-PCR illustrated in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010894#pone-0010894-g004\" target=\"_blank\">Fig. 4A</a>. PCR1 corresponds to the parental transcripts whereas PCR2 to the repaired transcripts. PCR1+PCR2 corresponds to the totality of the dystrophin minigene transcripts. (C) ExChange efficiency of the dosing study estimated by calculating the ratio between levels obtained for repaired transcripts (PCR2) and whole dystrophin minigene transcripts (PCR1+PCR2). Representative results from two independent transfection experiments.</p>", "links"=>[], "tags"=>["as2-e24-dise-as4"], "article_id"=>518237, "categories"=>["Molecular Biology", "Genetics"], "users"=>["Stéphanie Lorain", "Cécile Peccate", "Maëva Le Hir", "Luis García"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010894.g005", "stats"=>{"downloads"=>5, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dosing_study_of_AS2_E24_DISE_AS4_molecule_/518237", "title"=>"Dosing study of AS2-E24-DISE-AS4 molecule.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-28 02:17:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/847284"], "description"=>"<p>(A) The murine dystrophin minigene consists of exons 22 to 24 (boxes E22 to E24) with natural introns (lines). The splice sequences are illustrated by black balls: 5′ splice sites (5′SS), branching points (BP), polypyrimidine tracts (PPT) and 3′ acceptor sites (3′SS). The cross represents the nonsense <i>mdx</i> mutation in E23. The trans-splicing (TS) molecule AS-E24 comprises a 150 nt antisense sequence (AS) complementary to intron 22 as well as a spacer, a strong conserved yeast branch point sequence, a polypyrimidine tract, a 3′ acceptor site and E24. The sequence of the spacer, BP, PPT and 3′SS is given with the BP, PPT and 3′SS in bold and illustrated by black balls. The E24 sequence from minigene (white box) and TS molecule (black box) are identical, only the downstream sequence differs in length. TS constructs were made with three different antisense sequences, AS1 to AS3. Arrows indicate the positions of the forward A and reverse B PCR primers in the minigene and the TS molecule. (B) Expected transcripts generated by cis-splicing (E23 inclusion and skipping) and trans-splicing, and the predicted sizes of the corresponding PCR amplification products detected using the RT-PCR strategy illustrated in (A). (C) RT-PCR analysis using PCR primers A and B of NIH3T3 cells cotransfected with dystrophin minigene and constructions pSMD2 (Ctrl), pSMD2-U7-SD23-BP22 (U7), pSMD2-E24 (AS-), pSMD2-AS1-E24 (AS1), pSMD2-AS2-E24 (AS2) and pSMD2-AS3-E24 (AS3). RT- AS2: samples containing dystrophin minigene and pSMD2-AS2-E24 without reverse transcription; H2O: PCR negative control. Representative results from three independent transfection experiments. (D) An exact E22-E24 junction was confirmed by sequencing of the 304bp product.</p>", "links"=>[], "tags"=>["molecular biology", "genetics and genomics/gene therapy", "molecular biology/rna splicing"], "article_id"=>517743, "categories"=>["Molecular Biology", "Genetics"], "users"=>["Stéphanie Lorain", "Cécile Peccate", "Maëva Le Hir", "Luis García"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0010894.g001", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Trans_splicing_strategy_for_3_8242_replacement_/517743", "title"=>"Trans-splicing strategy for 3′ replacement.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-05-28 02:09:03"}

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Relative Metric

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