El Boujnouni Najoua, Ripken Lise, Willemse Marieke, van der Sanden Bart, Neveling Kornelia, Hoischen Alexander, Brock Roland, Wansink Derick G
Department of Medical BioSciences, Radboud University Medical Center, Nijmegen 6525 GA, the Netherlands.
Department of Human Genetics, Radboud University Medical Center, Nijmegen 6525 GA, the Netherlands.
Mol Ther Methods Clin Dev. 2025 Jun 2;33(3):101502. doi: 10.1016/j.omtm.2025.101502. eCollection 2025 Sep 11.
Myotonic dystrophy type 1 (DM1) is caused by a (CTG) expansion in the gene, leading to a multisystemic manifestation and broad disease presentation. Although the DM1 phenotype and onset correlate with expansion length, understanding DM1 etiology and developing effective therapies remains challenging. Here, we investigated the contribution of repeat length on aberrant splicing and response to antisense oligonucleotides (ASOs). In primary DM1 myoblasts bearing repeat lengths of 800, 1,200, or >3,000, downregulation was achieved by blocking and gapmer ASOs, though splicing correction was inefficient. Further analyses revealed profound differences in mRNA levels. To exclude such confounding effects, we generated an isogenic myoblast panel with repeats from 0 to 2,900 triplets using a repeat-targeted CRISPR/Cas9 nickase approach. This panel revealed repeat-length dependency of aberrant splicing and nuclear MBNL1 abundance. While the blocker ASO marginally induced downregulation with longer repeats, its effect on splicing correction was evident, though decreased as repeat length increased. The gapmer ASO led to substantial downregulation and essentially normalized splicing levels throughout. Our study demonstrates that repeat length is central to therapeutic effectiveness, but this correlation may be obscured by genetic background, underscoring the need to consider genotypic heterogeneity in DM1 clinical trials.
1型强直性肌营养不良症（DM1）由该基因中的（CTG）重复序列扩增引起，导致多系统表现和广泛的疾病呈现。尽管DM1的表型和发病与扩增长度相关，但了解DM1的病因并开发有效的治疗方法仍然具有挑战性。在这里，我们研究了重复长度对异常剪接和反义寡核苷酸（ASO）反应的影响。在携带800、1200或>3000个重复长度的原发性DM1成肌细胞中，通过阻断型和间隙型ASO实现了下调，尽管剪接校正效率低下。进一步分析揭示了mRNA水平上的显著差异。为了排除这些混杂效应，我们使用重复靶向的CRISPR/Cas9切口酶方法生成了一个具有0至2900个三联体重复序列的同基因成肌细胞组。该组揭示了异常剪接和核MBNL1丰度的重复长度依赖性。虽然阻断型ASO对较长重复序列仅轻微诱导下调，但其对剪接校正的作用很明显，尽管随着重复长度增加而降低。间隙型ASO导致大量下调并基本使整个剪接水平正常化。我们的研究表明，重复长度是治疗效果的关键，但这种相关性可能会被遗传背景掩盖，强调在DM1临床试验中需要考虑基因型异质性。
