Exon skipping therapies for Duchenne muscular dystrophy that restore an open reading frame can be induced by the use of noncoding U7 small nuclear RNA (U7snRNA) modified by an antisense exon-targeting sequence delivered by an adeno-associated virus (AAV) vector. We have developed an AAV vector (AAV9.U7-ACCA) containing four U7snRNAs targeting the splice donor and acceptor sites of dystrophin exon 2, resulting in highly efficient exclusion of DMD exon 2. We assessed the specificity of splice variation induced by AAV9.U7-ACCA delivery in the Dmd exon 2 duplication (Dup2) mouse model through an unbiased RNA-seq approach. Treatment-related effects on pre-mRNA splicing were quantified using local splicing variation (LSV) analysis. Filtering the transcriptome for differences in treatment-related splicing resulted in only 16 candidate off-target LSVs. Only a single candidate off-target LSV was found in both skeletal and cardiac muscle tissue and occurred at a known variable cassette exon. In contrast, four LSVs represented significant on-target correction of Dmd exon 2 splicing and transcriptome analysis showed correction of known dystrophin-deficient gene dysregulation. We conclude that the absence of off-target splicing induced by treatment with the U7-ACCA vector supports the continued clinical development of this approach.

中文翻译：

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靶向 DMD 外显子 2 重复的 U7snRNA 载体不存在显着的脱靶剪接变异

可以通过使用由腺相关病毒 (AAV) 载体传递的反义外显子靶向序列修饰的非编码 U7 小核 RNA (U7snRNA) 诱导用于恢复开放阅读框的 Duchenne 肌营养不良症的外显子跳跃疗法。我们开发了一种 AAV 载体 (AAV9.U7-ACCA)，其中包含四个 U7snRNA，靶向抗肌萎缩蛋白外显子 2 的剪接供体和受体位点，从而高效排除DMD外显子 2。我们评估了 AAV9.U7 诱导的剪接变异的特异性- Dmd 中的ACCA交付外显子 2 复制 (Dup2) 小鼠模型通过无偏 RNA-seq 方法。使用局部剪接变异 (LSV) 分析量化对前 mRNA 剪接的治疗相关影响。过滤转录组的治疗相关剪接差异导致只有 16 个候选脱靶 LSV。在骨骼肌和心肌组织中都只发现了一个候选脱靶 LSV，并且发生在一个已知的可变盒式外显子上。相比之下，四个 LSV 代表Dmd外显子 2 剪接的显着靶向校正，转录组分析显示已知抗肌萎缩蛋白缺陷基因失调的校正。我们得出结论，U7-ACCA 载体治疗诱导的脱靶剪接不存在支持这种方法的持续临床开发。