Peretto Laura, D'angiolillo Claudia, Ferraresi Paolo, Balestra Dario, Pinotti Mirko
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, 44121, Italy.
Mol Med. 2025 Mar 27;31(1):121. doi: 10.1186/s10020-025-01176-8.
Aberrant mRNA splicing is a well-established pathogenic mechanism for human disease, but its real impact is hardly predictable and underestimated. Splicing can be therefore modulated for therapeutic purposes, and splicing-switching molecules are in clinics for some diseases. Here, conscious that over 10% of all pathogenic mutations occurs at 5'ss, we aimed at characterizing and rescuing nine 5'ss mutations in three models of defective F8 exons whose skipping would lead to factor VIII (FVIII) deficiency (Hemophilia A), the most frequent coagulation factor disorder.
HEK293T cells were transfected with F8 minigene variants, alone or with engineered U1 small nuclear RNAs (U1snRNAs), and splicing patterns analysed via RT-PCR.
All 5'ss mutations induced exon skipping, and the proportion of correct transcripts, not predictable by computational analysis, was consistent with residual FVIII levels in patients. For each exon we identified a unique engineered U1snRNAs, either compensatory or Exon Specific (ExSpeU1), able to rescue all mutations. Overall, ExSpeU1s were more effective than compensatory U1snRNAs, particularly in the defective exons 6 and 22.
Data highlight the importance of splicing assays to elucidate genotype-phenotype relationships and proved the correction efficacy of ExSpeU1s for each targeted defective F8 exon, thus expanding their translational potential for HA.
异常的mRNA剪接是一种公认的人类疾病致病机制，但其实际影响难以预测且被低估。因此，可以对剪接进行调节以用于治疗目的，并且剪接转换分子已在临床上用于某些疾病。在此，鉴于超过10%的致病突变发生在5'剪接位点（5'ss），我们旨在表征和挽救三种F8外显子缺陷模型中的九个5'ss突变，这些外显子的跳跃会导致因子VIII（FVIII）缺乏（甲型血友病），这是最常见的凝血因子疾病。
将F8小基因变体单独或与工程化的U1小核RNA（U1snRNAs）一起转染到人胚肾293T细胞（HEK293T细胞），并通过逆转录聚合酶链反应（RT-PCR）分析剪接模式。
所有5'ss突变均诱导外显子跳跃，并且计算分析无法预测的正确转录本比例与患者的残余FVIII水平一致。对于每个外显子，我们鉴定出一种独特的工程化U1snRNAs，其要么是补偿性的，要么是外显子特异性的（ExSpeU1），能够挽救所有突变。总体而言，ExSpeU1s比补偿性U1snRNAs更有效，尤其是在缺陷外显子6和22中。
数据突出了剪接分析对于阐明基因型-表型关系的重要性，并证明了ExSpeU1s对每个靶向的缺陷F8外显子的校正效果，从而扩大了它们在甲型血友病中的转化潜力。
