Takeshima Yasuhiro, Yagi Mariko, Matsuo Masafumi
Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.
Methods Mol Biol. 2012;867:131-41. doi: 10.1007/978-1-61779-767-5_9.
A molecular therapy for Duchenne muscular dystrophy (DMD) that converts dystrophin mRNA from out-of-frame to in-frame transcripts by inducing exon skipping with antisense oligonucleotides (AOs) is now approaching clinical application. To exploit the broad therapeutic applicability of exon skipping therapy, it is necessary to identify AOs that are able to induce efficient and specific exon skipping. To optimize AOs, we have established an in vitro splicing system using cultured DMD myocytes. Here, we describe the process of identifying the best AO.Cultured DMD myocytes are established from a biopsy sample and the target exon is chosen. A series of AOs are designed to cover the whole target exon sequence. As AOs, we use 15-20-mer chimeric oligonucleotides consisting of 2'-O-methyl RNA and modified nucleic acid (2'-O, 4'-C-ethylene-bridged nucleic acid). Each AO is transfected individually into cultured DMD myocytes, and the resulting mRNA is analyzed by reverse transcription-PCR. The ability of each AO to induce exon skipping is examined by comparing the amount of cDNA with and without exon skipping. If necessary, having roughly localized the target region, another set of AOs are designed and the exon skipping abilities of the new AOs are examined. Finally, one AO is determined as the best for the molecular therapy.Our simple and reliable methods using an in vitro splicing system have enabled us to identify optimized AOs against many exons of the DMD gene.
一种用于杜氏肌营养不良症（DMD）的分子疗法正接近临床应用阶段，该疗法通过反义寡核苷酸（AO）诱导外显子跳跃，将肌营养不良蛋白的信使核糖核酸（mRNA）从框外转录本转变为框内转录本。为了拓展外显子跳跃疗法的广泛治疗适用性，有必要鉴定出能够高效且特异性诱导外显子跳跃的AO。为了优化AO，我们利用培养的DMD肌细胞建立了一种体外剪接系统。在此，我们描述鉴定最佳AO的过程。从活检样本中培养出DMD肌细胞，并选择目标外显子。设计一系列AO以覆盖整个目标外显子序列。作为AO，我们使用由2'-O-甲基核糖核酸和修饰核酸（2'-O，4'-C-乙烯桥连核酸）组成的15 - 20个核苷酸的嵌合寡核苷酸。将每个AO单独转染到培养的DMD肌细胞中，然后通过逆转录-聚合酶链反应（RT-PCR）分析产生的mRNA。通过比较有或没有外显子跳跃的互补脱氧核糖核酸（cDNA）量来检测每个AO诱导外显子跳跃的能力。如有必要，在大致定位目标区域后，设计另一组AO并检测新AO的外显子跳跃能力。最后，确定一个AO作为分子疗法的最佳选择。我们使用体外剪接系统的简单可靠方法，使我们能够针对DMD基因的许多外显子鉴定出优化的AO。
