RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect
Non-coding uridine-rich small nuclear RNAs (UsnRNAs) have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne muscular dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant adeno-associated virus (rAAV)...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2018-03-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253117303074 |
| _version_ | 1811221681895636992 |
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| author | Claire Domenger Marine Allais Virginie François Adrien Léger Emilie Lecomte Marie Montus Laurent Servais Thomas Voit Philippe Moullier Yann Audic Caroline Le Guiner |
| author_facet | Claire Domenger Marine Allais Virginie François Adrien Léger Emilie Lecomte Marie Montus Laurent Servais Thomas Voit Philippe Moullier Yann Audic Caroline Le Guiner |
| author_sort | Claire Domenger |
| collection | DOAJ |
| description | Non-coding uridine-rich small nuclear RNAs (UsnRNAs) have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne muscular dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant adeno-associated virus (rAAV)-U7snRNA vector to restore the reading frame of the DMD mRNA in the muscles of DMD dogs. We are now moving toward a phase I/II clinical trial with an rAAV-U7snRNA-E53, carrying an antisense sequence designed to hybridize exon 53 of the human DMD messenger. As observed for genome-editing tools, antisense sequences present a risk of off-target effects, reflecting partial hybridization onto unintended transcripts. To characterize the clinical antisense sequence, we studied its expression and explored the occurrence of its off-target effects in human in vitro models of skeletal muscle and liver. We presented a comprehensive methodology combining RNA sequencing and in silico filtering to analyze off-targets. We showed that U7snRNA-E53 induced the effective exon skipping of the DMD transcript without inducing the notable deregulation of transcripts in human cells, neither at gene expression nor at the mRNA splicing level. Altogether, these results suggest that the use of the rAAV-U7snRNA-E53 vector for exon skipping could be safe in eligible DMD patients. |
| first_indexed | 2024-04-12T08:03:45Z |
| format | Article |
| id | doaj.art-b56772ff2df9496ca4c2cf486504309a |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-12T08:03:45Z |
| publishDate | 2018-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-b56772ff2df9496ca4c2cf486504309a2022-12-22T03:41:14ZengElsevierMolecular Therapy: Nucleic Acids2162-25312018-03-011027729110.1016/j.omtn.2017.12.008RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target EffectClaire Domenger0Marine Allais1Virginie François2Adrien Léger3Emilie Lecomte4Marie Montus5Laurent Servais6Thomas Voit7Philippe Moullier8Yann Audic9Caroline Le Guiner10INSERM UMR 1089, Université de Nantes, CHU de Nantes, 44200 Nantes, France; Corresponding author: Claire Domenger, INSERM UMR 1089, IRS 2 Nantes Biotech, Université de Nantes 22, Boulevard Bénoni Goullin, 44200 Nantes, France.INSERM UMR 1089, Université de Nantes, CHU de Nantes, 44200 Nantes, FranceINSERM UMR 1089, Université de Nantes, CHU de Nantes, 44200 Nantes, FranceINSERM UMR 1089, Université de Nantes, CHU de Nantes, 44200 Nantes, FranceINSERM UMR 1089, Université de Nantes, CHU de Nantes, 44200 Nantes, FranceGénéthon, 91000 Evry, FranceInstitute I-Motion, Hôpital Armand Trousseau, 75012 Paris, FranceNIHR Biomedical Research Centre, UCL Institute of Child Health/Great Ormond Street Hospital NHS Trust, WC1N 1EH London, UKINSERM UMR 1089, Université de Nantes, CHU de Nantes, 44200 Nantes, FranceCNRS, UMR 6290 Institut Génétique et Développement de Rennes, Université de Rennes 1, 35000 Rennes, FranceINSERM UMR 1089, Université de Nantes, CHU de Nantes, 44200 Nantes, France; Corresponding author: Caroline Le Guiner, INSERM UMR 1089, IRS 2 Nantes Biotech, Université de Nantes 22, Boulevard Bénoni Goullin, 44200 Nantes, France.Non-coding uridine-rich small nuclear RNAs (UsnRNAs) have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne muscular dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant adeno-associated virus (rAAV)-U7snRNA vector to restore the reading frame of the DMD mRNA in the muscles of DMD dogs. We are now moving toward a phase I/II clinical trial with an rAAV-U7snRNA-E53, carrying an antisense sequence designed to hybridize exon 53 of the human DMD messenger. As observed for genome-editing tools, antisense sequences present a risk of off-target effects, reflecting partial hybridization onto unintended transcripts. To characterize the clinical antisense sequence, we studied its expression and explored the occurrence of its off-target effects in human in vitro models of skeletal muscle and liver. We presented a comprehensive methodology combining RNA sequencing and in silico filtering to analyze off-targets. We showed that U7snRNA-E53 induced the effective exon skipping of the DMD transcript without inducing the notable deregulation of transcripts in human cells, neither at gene expression nor at the mRNA splicing level. Altogether, these results suggest that the use of the rAAV-U7snRNA-E53 vector for exon skipping could be safe in eligible DMD patients.http://www.sciencedirect.com/science/article/pii/S2162253117303074exon skippingoff-targetRNA sequencingDuchenneU7snRNA |
| spellingShingle | Claire Domenger Marine Allais Virginie François Adrien Léger Emilie Lecomte Marie Montus Laurent Servais Thomas Voit Philippe Moullier Yann Audic Caroline Le Guiner RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect Molecular Therapy: Nucleic Acids exon skipping off-target RNA sequencing Duchenne U7snRNA |
| title | RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect |
| title_full | RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect |
| title_fullStr | RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect |
| title_full_unstemmed | RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect |
| title_short | RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect |
| title_sort | rna seq analysis of an antisense sequence optimized for exon skipping in duchenne patients reveals no off target effect |
| topic | exon skipping off-target RNA sequencing Duchenne U7snRNA |
| url | http://www.sciencedirect.com/science/article/pii/S2162253117303074 |
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