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.

近年来，非编码富尿苷的小核RNA（UsnRNA）成为一种有效的工具，可用于跳过外显子，以治疗杜氏肌营养不良症（DMD），这是一种退化性肌肉遗传性疾病。我们最近发现的重组腺相关病毒的高容量（腺相关病毒）-U7snRNA矢量恢复的阅读框DMD基因在DMD狗的肌肉。我们现在正朝着使用rAAV-U7snRNA-E53的I / II期临床试验迈进，它带有旨在与人类DMD外显子53杂交的反义序列信使。如对基因组编辑工具所观察到的，反义序列存在脱靶效应的风险，反映了与非预期转录物的部分杂交。为了表征临床反义序列，我们研究了其表达并探讨了其在人体外骨骼肌和肝脏模型中脱靶作用的发生。我们提出了一种综合的方法，结合了RNA测序和计算机滤波来分析脱靶。我们显示U7snRNA-E53诱导DMD的有效外显子跳跃在基因表达或mRNA剪接水平上都不会在人类细胞中引起转录本显着失调的转录本。总之，这些结果表明，在合格的DMD患者中使用rAAV-U7snRNA-E53载体进行外显子跳过可能是安全的。