Targeting a novel silencer to correct SMN2 splicing in Spinal Muscular Atrophy

靶向新型消音器来纠正脊髓性肌萎缩症中的 SMN2 剪接

• 批准号: 7086017
• 负责人: RAVINDRA N SINGH
• 金额: $ 18.27万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7086017
• 关键词: atrophy; genes; model; proteins

DESCRIPTION (provided by applicant): The most frequent cause of spinal muscular atrophy (SMA) is the loss of Survival Motor Neuron 1 (SMN1) gene, which produces SMN protein. A nearly identical copy of the gene, SMN2, that produces nonfunctional SMN protein due to skipping of exon 7, fails to compensate for the loss of SMN1. My laboratory investigates regulation of SMN exon 7 splicing with the goal of identification of therapeutic targets to promote exon 7 inclusion during pre-mRNA splicing of SMN2. Using a state of the art method of iterative selection, we have recently shown that skipping of exon 7 in SMN2 is directly linked to the weak 5' splice site (5' ss) of exon 7. Upon extending our investigation, we recently discovered a novel Intronic Splicing Silencer (ISS-N1) that facilitates skipping of exon 7 by sequestering the 5' ss. Supporting the inhibitory nature of ISS-N1, mutations and deletions within ISS-N1 promoted exon 7 inclusion in SMN2 mRNA. Further confirming the inhibitory nature of ISS-N1, the antisense oligo (ASO) that blocked ISS-N1 fully restored exon 7 inclusion in both, our minigene system and SMA patient fibroblasts (from the endogenous SMN2). As a consequence, ASO- treated patient cells showed increased expression of SMN protein from SMN2. 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 ISS-N1 offers a unique target-site for ASO-mediated therapy of SMA. Antisense technology has emerged as a powerful tool to treat many human diseases. This grant proposal is aimed at designing highly efficient ASOs against ISS-N1. We will examine the effect of ASOs in SMA patient cells as well as in mice models of SMA. The findings from this study will establish the efficacy of ASO-based therapy of SMA. 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 use antisense oligos that correct aberrant splicing of SMN2 by targeting a novel intronic silencer that we discovered recently. To explore the therapeutic potential of antisense oligos, we will extend our study to the mice models of SMA.

描述（由申请人提供）：脊髓性肌萎缩症 (SMA) 最常见的原因是运动神经元存活 1 (SMN1) 基因的缺失，该基因产生 SMN 蛋白。几乎相同的基因 SMN2 拷贝由于外显子 7 的跳过而产生无功能的 SMN 蛋白，但无法补偿 SMN1 的损失。我的实验室研究 SMN 外显子 7 剪接的调节，目的是识别治疗靶点，以促进 SMN2 前 mRNA 剪接期间外显子 7 的包含。使用最先进的迭代选择方法，我们最近发现 SMN2 中外显子 7 的跳跃与外显子 7 的弱 5' 剪接位点 (5' ss) 直接相关。在扩展我们的研究后，我们最近发现了一个新颖的内含子剪接沉默器 (ISS-N1)，通过隔离 5' ss 促进外显子 7 的跳跃。 ISS-N1 内的突变和缺失促进了 SMN2 mRNA 中外显子 7 的包含，这支持了 ISS-N1 的抑制性质。进一步证实了 ISS-N1 的抑制性质，阻断 ISS-N1 的反义寡核苷酸 (ASO) 完全恢复了我们的小基因系统和 SMA 患者成纤维细胞（来自内源性 SMN2）中的外显子 7 包含。结果，ASO 处理的患者细胞显示 SMN2 的 SMN 蛋白表达增加。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 是一种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 ISS-N1 为 ASO 介导的 SMA 治疗提供了独特的靶位点。反义技术已成为治疗许多人类疾病的有力工具。该拨款提案旨在针对 ISS-N1 设计高效的 ASO。我们将研究 ASO 对 SMA 患者细胞以及 SMA 小鼠模型的影响。这项研究的结果将确定基于 ASO 的 SMA 疗法的疗效。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，我们将使用反义寡核苷酸，通过针对我们最近发现的新型内含子沉默子来纠正 SMN2 的异常剪接。为了探索反义寡核苷酸的治疗潜力，我们将把我们的研究扩展到 SMA 小鼠模型。