Maruna Matea, Sušjan-Leite Petra, Meško Maja, Miroševič Špela, Jerala Roman
Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Graduate School of Biomedicine, University of Ljubljana, Kongresni trg 12, 1000 Ljubljana, Slovenia.
Mol Ther Nucleic Acids. 2025 Aug 12;36(3):102680. doi: 10.1016/j.omtn.2025.102680. eCollection 2025 Sep 9.
Loss-of-function mutations in the gene cause β-catenin deficiency, resulting in CTNNB1 syndrome, a rare neurodevelopmental disorder characterized by motor and cognitive impairments. Given the wide variety of mutations across and its dosage sensitivity, a mutation-independent therapeutic approach that preserves endogenous gene regulation is critically needed. This study introduces spliceosome-mediated RNA -splicing as a novel approach to restore β-catenin production. Pre--splicing RNA molecules (PTMs) targeting introns 2, 5, and 6 were designed and evaluated using a split yellow fluorescent protein reporter system. Rationally designed short antisense RNAs, which mask splicing regulatory elements, significantly enhanced PTM-mediated -splicing at both mRNA and protein levels. Additionally, introducing a self-cleaving ribozyme at the PTM's 5' end further improved -splicing efficiency, likely due to increased nuclear retention. CMV promoter-driven PTM expression yielded the highest efficiency. Importantly, successful -splicing of the endogenous transcript confirmed the physiological relevance of this strategy. This study is the first to apply and optimize spliceosome-mediated RNA -splicing (SMaRT) for mRNA correction, providing a promising, mutation-agnostic approach for treating CTNNB1 syndrome.
该基因的功能丧失突变会导致β-连环蛋白缺乏，进而引发CTNNB1综合征，这是一种罕见的神经发育障碍，其特征为运动和认知障碍。鉴于该基因存在各种各样的突变及其剂量敏感性，迫切需要一种能保留内源性基因调控的与突变无关的治疗方法。本研究引入了剪接体介导的RNA剪接作为恢复β-连环蛋白产生的新方法。使用分裂型黄色荧光蛋白报告系统设计并评估了靶向该基因第2、5和6内含子的前体剪接RNA分子（PTMs）。合理设计的短反义RNA可掩盖剪接调控元件，在mRNA和蛋白质水平上均显著增强了PTM介导的剪接。此外，在PTM的5'端引入自切割核酶进一步提高了剪接效率，这可能是由于核滞留增加所致。由巨细胞病毒（CMV）启动子驱动的PTM表达产生了最高效率。重要的是，内源性该基因转录本的成功剪接证实了这一策略的生理相关性。本研究首次应用并优化了剪接体介导的RNA剪接（SMaRT）来校正该基因的mRNA，为治疗CTNNB1综合征提供了一种有前景的、与突变无关的方法。
