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

描述（由申请人提供）：脊髓性肌萎缩症（SMA）最常见的原因是产生SMN蛋白的运动神经元生存基因1（SMN 1）缺失。由于外显子7的跳跃，产生无功能SMN蛋白的基因SMN 2的几乎相同的拷贝不能补偿SMN 1的损失。我的实验室研究了SMN外显子7剪接的调控，目的是确定治疗靶点，以促进SMN 2前体mRNA剪接期间的外显子7包含。使用最先进的迭代选择方法，我们最近发现SMN 2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，我们最近发现了一种新的内含子剪接沉默子（ISS-N1），它通过隔离5' ss来促进外显子7的跳跃。支持ISS-N1的抑制性质，ISS-N1内的突变和缺失促进SMN 2 mRNA中的外显子7包含。进一步证实了ISS-N1的抑制性质，阻断ISS-N1的反义寡核苷酸（阿索）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SMN 2）中的外显子7包含。因此，阿索处理的患者细胞显示来自SMN 2的SMN蛋白表达增加。值得注意的是，即使在低阿索剂量下也观察到阿索介导的刺激作用，表明ISS-N1是高度可及的反义靶标。反义效应对ISS-N1非常特异，因为ISS-N1内的两个或更多个突变完全消除了ASO介导的刺激效应。基于这些结果，我们认为ISS-N1为ASO介导的SMA治疗提供了独特的靶点。反义技术已成为治疗多种人类疾病的有力工具。该拨款提案旨在设计针对ISS-N1的高效ASO。我们将检查ASO在SMA患者细胞以及SMA小鼠模型中的作用。本研究的结果将确定基于ASO的SMA治疗的疗效。脊髓性肌萎缩症（SMA）最常见的原因是SMN 1基因的丢失，伴随着SMN 2基因由于异常剪接而无法补偿。在这里，我们将使用反义寡核苷酸，通过靶向我们最近发现的一种新的内含子沉默子来纠正SMN 2的异常剪接。为了探索反义寡核苷酸的治疗潜力，我们将我们的研究扩展到SMA小鼠模型。

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.