Ferlini Alessandra, Rimessi Paola
Section of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy.
Methods Mol Biol. 2012;867:189-99. doi: 10.1007/978-1-61779-767-5_12.
Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach for subsets of Duchenne muscular dystrophy (DMD) patients to ameliorate the severe DMD phenotype. Several groups have successfully induced exon skipping by AONs to reframe the mRNA in various patients carrying deletions, and phase I/II clinical trials are ongoing. The approach is based on targeting specific splicing motifs, both exonic and located on the exon borders, thus interfering with the spliceosome assembly by steric hindrance. Evaluation of the effectiveness of treatment with AONs in cells, animal models, and humans requires a sensitive, specific, and highly reproducible method. We have developed a real-time PCR-based protocol that uses the probe-based approach to recognize specific sequences internal to the target exon (exon-specific real-time assay). The methods for this protocol are described in this chapter.
反义寡核苷酸（AON）介导的外显子跳跃是一种针对杜氏肌营养不良症（DMD）患者亚群的治疗方法，旨在改善严重的DMD表型。多个研究小组已成功通过AON诱导外显子跳跃，从而在各种携带缺失的患者中重新构建mRNA，并且I/II期临床试验正在进行中。该方法基于靶向特定的剪接基序，这些基序既存在于外显子内部，也位于外显子边界，从而通过空间位阻干扰剪接体组装。评估AON在细胞、动物模型和人类中的治疗效果需要一种灵敏、特异且高度可重复的方法。我们开发了一种基于实时PCR的方案，该方案使用基于探针的方法来识别目标外显子内部的特定序列（外显子特异性实时检测）。本章将介绍该方案的方法。
