Goyenvalle Aurélie
MRC Functional Genomics Unit, Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford, UK.
Methods Mol Biol. 2012;867:259-71. doi: 10.1007/978-1-61779-767-5_17.
Antisense-mediated splicing modulation of premessenger RNA represents a novel therapeutic strategy for several types of pathologies such as genetic disorders, cancers, and infectious diseases. Antisense oligonucleotides designed to bind to specific mRNA molecules have been actively developed for more than 20 years as a form of molecular medicine to modulate splicing patterns or inhibit protein translation. More recently, small nuclear RNA such as U7 or U1 small nuclear RNA have been used to carry antisense sequences, offering the advantage of long-term effect when delivered to cells using viral vectors. We have previously demonstrated the therapeutic potential of U7snRNA targeting dystrophin mRNA as a treatment for Duchenne muscular dystrophy. In particular, we showed that bifunctional U7 snRNAs harboring silencer motifs induce complete skipping of exon 51, and thus restore dystrophin expression in DMD patients cells to near wild-type levels. These new constructs are very promising for the optimization of therapeutic exon skipping for DMD, but also offer powerful and versatile tools to modulate pre-mRNA splicing in a wide range of applications. Here, we outline the design of these U7snRNA constructs to achieve efficient exon-skipping and describe methods to evaluate the efficacy of such U7snRNA constructs in vitro using the dystrophin gene as an example.
反义介导的前体信使核糖核酸剪接调控代表了一种针对多种类型疾病（如遗传疾病、癌症和传染病）的新型治疗策略。作为一种分子医学形式，旨在与特定信使核糖核酸分子结合的反义寡核苷酸已被积极研发超过20年，用于调控剪接模式或抑制蛋白质翻译。最近，诸如U7或U1小核核糖核酸等小核核糖核酸已被用于携带反义序列，当使用病毒载体递送至细胞时具有长期效应的优势。我们之前已证明靶向抗肌萎缩蛋白信使核糖核酸的U7小核核糖核酸作为杜氏肌营养不良症治疗方法的治疗潜力。具体而言，我们表明携带沉默子基序的双功能U7小核核糖核酸可诱导外显子51完全跳跃，从而将杜氏肌营养不良症患者细胞中的抗肌萎缩蛋白表达恢复至接近野生型水平。这些新构建体对于优化杜氏肌营养不良症的治疗性外显子跳跃非常有前景，而且还为在广泛应用中调控前体信使核糖核酸剪接提供了强大且通用的工具。在此，我们概述这些U7小核核糖核酸构建体的设计以实现高效外显子跳跃，并描述以抗肌萎缩蛋白基因为例在体外评估此类U7小核核糖核酸构建体功效的方法。
