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：
8721561
负责人：
RAVINDRA N SINGH
金额：
$ 32.81万
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 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8721561
关键词：
Acylation Affect Agreement Amino Acid Sequence Antisense Oligonucleotides Antisense Technology Binding Cells Collaborations Complex Comprehension Deposition Development Devices Elements Exons Genes Genetic Hereditary Disease Human Human Genetics Hydroxyl Radical In Vitro Infant Mortality Introns Iowa Lead Licensing Link Mediating Methods Modeling Molecular Motor Neurons Mutation Nucleic Acids Oligonucleotides Outcome Pathogenesis Patients Peptide Sequence Determination Pharmaceutical Preparations Pilot Projects Point Mutation Positioning Attribute Primer Extension Proteins RNA RNA Processing RNA Splicing RNA-Protein Interaction Randomized Regulation Regulatory Element Reporting Role SMN protein (spinal muscular atrophy)SMN1 gene SMN2 gene Sample Size Severities Site Slice Small Interfering RNA Spinal Muscular Atrophy Structure Therapeutic Transact Transcript Treatment Efficacy Universities Validation Welander Distal Myopathy base cell type crosslink design in vivo mRNA Precursor mouse model new therapeutic target novel phosphorodiamidate morpholino oligomer protein complex public health relevance stem survival motor neuron gene

项目摘要

DESCRIPTION (provided by applicant): Humans have two nearly identical copies of Survival Motor Neuron (SMN) gene, SMN1 and SMN2. Low SMN levels due to deletion and/or mutation of SMN1 lead to spinal muscular atrophy (SMA), a major genetic cause of infant mortality. SMN2 fails to compensate for the loss of SMN1 due to a C to T mutation at the 6th position (C6U in transcript) in exon 7. C6U weakens the 32-splice site and triggers SMN2 exon 7 skipping, resulting in synthesis of a truncated protein (SMN¿7), which is unstable. It is known that strategies aimed at correction of SMN2 exon 7 splicing hold the promise for a cure. This proposal emanates from our recent discovery of a unique RNA structure formed by a long-distance interaction (LDI) as a regulator of SMN2 exon 7 splicing (Singh et al., Nucleic Acids Res., 2013, doi:10.1093/nar/gkt609). We call this structure internal stem through LDI-1 (ISTL1). Employing the SHAPE (Selective 22-Hydroxyl Acylation analyzed by Primer Extension) method, we confirmed the formation and functional significance of ISTL1. We showed that an antisense oligonucleotide (ASO)-mediated sequestration of the 32 strand of ISTL1 fully corrects SMN2 exon 7 splicing and restores high levels of SMN and Gemin2, an SMN-interacting protein, in SMA patient cells. Our results also revealed that the 32 strand of ISTL1 is located within a large inhibitory region that we termed intronic splicing silencer N2 (ISS-N2). To continue with our lead, here we propose to characterize additional (novel) intronic cis-elements and their cognate transacting factors in regulation of SMN2 exon 7 splicing. A part of the proposal is aimed at validating the therapeutic efficacy of ISS-N2- targeting ASOs. In Aim 1, we will use overlapping deletions, ASO-based strategies and SHAPE analyses to determine the significance of novel cis-elements within SMN2 intron 7. We will validate our findings in different cell types including motor neuron-like NSC34 cells. We will examine the effect of a larger structural context on the accessibility of the splice sites of SMN2 exon 7. We will determine whether SMN2-specific mutations lead to a structural difference between SMN1 and SMN2 pre-mRNAs, particularly at the splice sites of SMN exon 7. In addition, we will evaluate whether critical cis-elements within intron 6 affect the structural context of intron 7 and potentially lead to the remodeling of the 52 slice site of exon 7. In Aim 2, we will employ over-expression and siRNA-based strategies to identify splicing factors that assist ISTL1 formation and/or use ISTL1 as a site for self-recruitment. We will use a biotinylated oligonucleotide as a trapping device to capture novel RNA-protein complexes that are deposited on SMN2 intron 7 and are critical for the inhibitory effect of LDI. We have previously shown that TIA1 stimulates SMN2 exon 7 splicing by binding to intron 7. We also demonstrated critical role of the Q-rich domain of TIA1 in regulation of SMN2 exon 7 splicing. Recently, a point mutation within Q-rich domain of TIA1 has been shown to cause Welander distal myopathy as well as promote SMN2 exon 7 skipping. Based on these findings and our preliminary results, we will examine the role of another Q-rich domain containing splicing regulator, SFPQ, in SMN2 exon 7 splicing. We will employ UV-crosslinking, footprinting, in vitro binding and SHAPE-based approaches to characterize RNA-protein interactions. In Aim 3, we will perform in vivo studies in a mild as well as in a severe mouse model of SMA to determine the therapeutic efficacy of an ISS-N2 targeting lead phosphorodiamidate morpholino oligomer (PMO). We will design our experimental plan based on several successful in vivo studies reported recently and will employ rigorous criteria of sample-size estimation, randomization and blinding. A successful outcome will lead to the development of a novel ASO-based therapy for SMA.

描述（由申请人提供）：人类有几乎两个相同的运动神经元生存基因（SMN1 和 SMN2），由于 SMN1 的缺失和/或突变导致 SMN 水平低，导致脊髓性肌萎缩症（SMA），这是一种主要的遗传病。 SMN2 无法补偿由于外显子 7 中第 6 位（转录物中的 C6U）C 至 T 突变而造成的 SMN1 损失。削弱 32 剪接位点并触发 SMN2 外显子 7 跳跃，导致合成截短的蛋白质 (SMN¿7)，该蛋白质不稳定。众所周知，旨在纠正 SMN2 外显子 7 剪接的策略有望治愈。这一提议源自我们最近发现的一种独特的 RNA 结构，该结构由长距离相互作用 (LDI) 形成，作为 SMN2 外显子 7 剪接的调节因子（Singh 等人） al., Nucleic Acids Res., 2013, doi:10.1093/nar/gkt609)，我们通过 LDI-1 (ISTL1) 方法将此结构称为内部茎。证实了 ISTL1 的形成和功能意义，我们发现反义寡核苷酸 (ASO) 介导的隔离作用。 ISTL1 的 32 链完全纠正了 SMA 患者细胞中 SMN2 外显子 7 的剪接，并恢复了高水平的 SMN 和 SMN 相互作用蛋白 Gemin2。我们的结果还表明，ISTL1 的 32 链位于一个大的区域内。我们称之为内含子剪接沉默子 N2 (ISS-N2) 的抑制区域为了继续我们的研究，我们建议表征 SMN2 外显子 7 剪接调节中的其他（新颖）内含子顺式元件及其同源反式作用因子。该提案旨在验证 ISS-N2 靶向 ASO 的治疗效果。在目标 1 中，我们将使用重叠删除、基于 ASO 的策略和方法。 SHAPE 分析以确定 SMN2 内含子 7 内新顺式元件的重要性。我们将在不同细胞类型中验证我们的发现，包括我们将检查更大的结构背景对 SMN2 外显子 7 剪接位点可及性的影响。我们将确定 SMN2 特异性突变是否导致 SMN1 和 SMN2 前 mRNA 之间的结构差异，特别是在 SMN 外显子 7 的剪接位点。此外，我们将评估是否存在关键的顺式元件内含子 6 影响内含子 7 的结构背景，并可能导致 52 的重塑外显子 7 的切片位点。在目标 2 中，我们将采用过表达和基于 siRNA 的策略来识别有助于 ISTL1 形成的剪接因子和/或使用 ISTL1 作为自我招募位点。捕获装置捕获沉积在 SMN2 内含子 7 上的新型 RNA-蛋白质复合物，对于 LDI 的抑制作用至关重要。通过与内含子 7 结合来调节外显子 7 剪接。我们还证明了 TIA1 的 Q 丰富结构域在调节 SMN2 外显子 7 剪接中的关键作用。最近，TIA1 的 Q 丰富结构域内的点突变已被证明会导致 Welander 远端肌病。以及促进 SMN2 外显子 7 跳跃基于这些发现和我们的初步结果，我们将研究另一个包含剪接调节因子的富含 Q 的结构域 SFPQ 在 SMN2 中的作用。我们将采用 UV 交联、足迹、体外结合和基于 SHAPE 的方法来表征 RNA-蛋白质相互作用。在目标 3 中，我们将在轻度和重度小鼠模型中进行体内研究。 SMA 以确定 ISS-N2 靶向磷酸二酰胺吗啉低聚物 (PMO) 的治疗效果我们将根据最近报道的几项成功的体内研究设计我们的实验计划，并将采用严格的样本量标准。估计、随机化和盲法的成功结果将导致基于 ASO 的新型 SMA 疗法的开发。