Expansion of an unstable CTG repeat in the 3'UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1). CUG-expanded DMPK transcripts (CUGexp) sequester Muscleblind-like (MBNL) alternative splicing regulators in ribonuclear inclusions (foci), leading to abnormalities in RNA processing and splicing. To alleviate the burden of CUGexp, we tested therapeutic approach utilizing antisense oligonucleotides (AONs)-mediated DMPK splice-switching and degradation of mutated pre-mRNA. Experimental design involved: (i) skipping of selected constitutive exons to induce frameshifting and decay of toxic mRNAs by an RNA surveillance mechanism, and (ii) exclusion of the alternative exon 15 (e15) carrying CUGexp from DMPK mRNA. While first strategy failed to stimulate DMPK mRNA decay, exclusion of e15 enhanced DMPK nuclear export but triggered accumulation of potentially harmful spliced out pre-mRNA fragment containing CUGexp. Neutralization of this fragment with antisense gapmers complementary to intronic sequences preceding e15 failed to diminish DM1-specific spliceopathy due to AONs' chemistry-related toxicity. However, intronic gapmers alone reduced the level of DMPK mRNA and mitigated DM1-related cellular phenotypes including spliceopathy and nuclear foci. Thus, a combination of the correct chemistry and experimental approach should be carefully considered to design a safe AON-based therapeutic strategy for DM1.

中文翻译：

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AON诱导的剪接转换和DMPK前mRNA降解是1型强直性营养不良的潜在治疗方法。

DMPK基因3'UTR中不稳定的CTG重复序列的扩增会导致1型肌强直性营养不良（DM1）。CUG扩展的DMPK转录本（CUGexp）隔离了核核内含物（病灶）中的Muscleblind-like（MBNL）选择性剪接调节剂，导致RNA加工和剪接异常。为了减轻CUGexp的负担，我们测试了利用反义寡核苷酸（AON）介导的DMPK剪接转换和突变的pre-mRNA降解的治疗方法。实验设计涉及：（i）通过RNA监测机制跳过选定的组成性外显子，以诱导毒性mRNA的移码和衰减，以及（ii）从DMPK mRNA中排除携带CUGexp的替代外显子15（e15）。尽管第一个策略未能刺激DMPK mRNA的降解，排除e15会增强DMPK核输出，但会触发潜在有害的剪接的含CUGexp的mRNA片段积聚。由于AON的化学相关毒性，用与e15之前的内含子序列互补的反义空聚物对该片段进行中和未能减少DM1特异性剪接病。然而，仅内含性缺口器降低了DMPK mRNA的水平并减轻了DM1相关的细胞表型，包括剪接病和核灶。因此，应仔细考虑正确的化学方法和实验方法的组合，以设计用于DM1的基于AON的安全治疗策略。由于AON的化学相关毒性，用与e15之前的内含子序列互补的反义空聚物对该片段进行中和未能减少DM1特异性剪接病。然而，仅内含性缺口器降低了DMPK mRNA的水平并减轻了DM1相关的细胞表型，包括剪接病和核灶。因此，应仔细考虑正确的化学方法和实验方法的组合，以设计用于DM1的基于AON的安全治疗策略。由于AON的化学相关毒性，用与e15之前的内含子序列互补的反义空聚物对该片段进行中和未能减少DM1特异性剪接病。然而，仅内含性缺口器降低了DMPK mRNA的水平并减轻了DM1相关的细胞表型，包括剪接病和核灶。因此，应仔细考虑正确的化学方法和实验方法的组合，以设计用于DM1的基于AON的安全治疗策略。