RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect

Molecular Therapy: Nucleic Acids

Marine Allais¹  Caroline Le Guiner²  Laurent Servais³  Claire Domenger⁴  Virginie François⁴  Marie Montus⁴  Emilie Lecomte⁴  Adrien Léger⁴  Yann Audic⁴  Thomas Voit⁵  Philippe Moullier⁶ 

[1] Corresponding author: Claire Domenger, INSERM UMR 1089, IRS 2 Nantes Biotech, Université de Nantes 22, Boulevard Bénoni Goullin, 44200 Nantes, France.;CNRS, UMR 6290 Institut Génétique et Développement de Rennes, Université de Rennes 1, 35000 Rennes, France;Généthon, 91000 Evry, France;INSERM UMR 1089, Université de Nantes, CHU de Nantes, 44200 Nantes, France;Institute I-Motion, Hôpital Armand Trousseau, 75012 Paris, France;NIHR Biomedical Research Centre, UCL Institute of Child Health/Great Ormond Street Hospital NHS Trust, WC1N 1EH London, UK;

关键词: exon skipping;    off-target;    RNA sequencing;    Duchenne;    U7snRNA;   

DOI  :  10.1016/j.omtn.2017.12.008

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown