Characterization of a complex regulatory element of Spinal Muscular Atrophy genes

脊髓性肌萎缩症基因复杂调控元件的表征

基本信息

批准号：
7496967
负责人：
RAVINDRA N SINGH
金额：
$ 32.01万
依托单位：
IOWA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-07-06 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7496967
关键词：
5&apos Splice Site Antisense Oligonucleotides Applications Grants Binding Cells Chemistry Child Complex Development Dimensions Disease Disease Progression Dose Elements Exclusion Exons Genes Global Change Grant Growth and Development function Heterogeneous Nuclear RNA Human Infant Knowledge Laboratories Length Letters Libraries Maine Mediating Messenger RNA Methods Molecular Profiling Monitor Motor Neurons Mus Muscle Mutation Nucleotides Numbers Oligonucleotides Outcome Study Pathogenesis Patients Pharmaceutical Preparations Play Positioning Attribute Proteins RNA RNA Probes RNA Splicing Range Regulatory Element Reporting Research Research Personnel Role SMN1 gene SMN2 gene Site Spinal Cord Spinal Muscular Atrophy Structure Testing Tissues Transact Transgenic Mice Work base cell type design in vivo mRNA Precursor mouse model novel novel strategies novel therapeutics portability research study size survival motor neuron gene therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Humans have two copies of Survival of Motor Neuron (SMN) gene, SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), a debilitating disease of infants and children. SMN2 fails to compensate for the loss of SMN1 due to skipping of exon 7 resulting in the synthesis of a truncated protein, which is unstable. Presence of SMN2 in most SMA patients provides a rare opportunity to restore SMN levels by correcting the aberrant splicing of exon 7. Using a novel method of in vivo selection, we previously reported that a weak 5' splice site (51 ss) serves as the limiting factor for exon 7 inclusion in SMN2. Based on this observation, we have recently discovered that a unique intronic inhibitory element contributes towards the weak 5' ss of exon 7. We call this element Intronic Splicing Silencer N1 (abbreviated as ISS-N1). Interestingly, ISS-N1 appears to be unique to humans. Based on our preliminary results, ISS-N1 plays an important role in pathogenesis of SMA. Most importantly, we discovered that Antisense Oligonucleotides (abbreviated as ASOs) that targeted ISS-N1 corrected aberrant splicing of SMN2 in all cell types tested including SMA patient cells. Consequently, ASO against ISS-N1 fully restored SMN levels in patient cells. Significantly, the ASO-mediated stimulatory effect was observed even at low ASO doses, suggesting that ISS-N1 is a highly accessible antisense target. The antisense effect was very specific to ISS-N1 as two or more mutations within ISS-N1 completely eliminated the ASO-mediated stimulatory effect. Based on these results we believe that we have discovered an ideal target-site for the ASO-mediated therapy of SMA. In this grant proposal, we will (1) characterize ISS-N1, its RNA structure and interacting factors in details; (2) develop efficient ASOs against ISS-N1 to correct SMN2 splicing in patient cells; and finally (3) conduct in vivo studies, using mice models of SMA to validate ASOs against ISS-N1 as the possible drug candidates. The most frequent cause of spinal muscular atrophy (SMA) is the loss of SMN1 gene accompanied by the inability of SMN2 gene to compensate due to aberrant splicing. Here, we will characterize a novel intronic element that plays a critical role in pathogenesis of SMA. In addition, we will use this element as a target for the antisense-mediated correction of aberrant splicing in SMA.

描述（由申请人提供）：人类有运动神经元存活（SMN）基因的两个拷贝，SMN1和SMN2。 SMN1 缺失会导致脊髓性肌萎缩症 (SMA)，这是一种使婴儿和儿童衰弱的疾病。 SMN2 无法补偿 SMN1 的损失，因为外显子 7 的跳过导致合成了不稳定的截短蛋白质。大多数 SMA 患者中 SMN2 的存在为通过纠正外显子 7 的异常剪接来恢复 SMN 水平提供了难得的机会。使用一种新的体内选择方法，我们之前报道了弱 5' 剪接位点 (51 ss) 作为SMN2 中外显子 7 包含的限制因素。基于这一观察，我们最近发现一种独特的内含子抑制元件有助于外显子7的弱5'ss。我们将此元件称为内含子剪接沉默器N1（缩写为ISS-N1）。有趣的是，ISS-N1 似乎是人类独有的。根据我们的初步结果，ISS-N1在SMA的发病机制中发挥着重要作用。最重要的是，我们发现靶向 ISS-N1 的反义寡核苷酸（缩写为 ASO）纠正了所有测试的细胞类型（包括 SMA 患者细胞）中 SMN2 的异常剪接。因此，针对 ISS-N1 的 ASO 完全恢复了患者细胞中的 SMN 水平。值得注意的是，即使在低 ASO 剂量下也能观察到 ASO 介导的刺激作用，这表明 ISS-N1 是一个易于接近的反义靶标。反义效应对 ISS-N1 非常具有特异性，因为 ISS-N1 内的两个或多个突变完全消除了 ASO 介导的刺激效应。基于这些结果，我们相信我们已经发现了 ASO 介导的 SMA 治疗的理想靶位点。在本次资助提案中，我们将 (1) 详细描述 ISS-N1、其 RNA 结构和相互作用因素； (2) 开发针对 ISS-N1 的有效 ASO，以纠正患者细胞中的 SMN2 剪接；最后 (3) 进行体内研究，使用 SMA 小鼠模型来验证 ASO 与 ISS-N1 作为可能的候选药物。脊髓性肌萎缩症 (SMA) 最常见的原因是 SMN1 基因缺失，同时 SMN2 基因因剪接异常而无法补偿。在这里，我们将描述一种在 SMA 发病机制中发挥关键作用的新型内含子元件。此外，我们将使用该元件作为反义介导的 SMA 异常剪接校正的靶标。