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

脊髓性肌萎缩症（SMA）最常见的原因是运动神经元1（SMN 1）的丧失。 基因，产生SMN蛋白。一个几乎相同的基因拷贝，SMN 2， 由于外显子7的跳跃，SMN蛋白不能补偿SMN 1的丢失。我的实验室研究 调节SMN外显子7剪接，目的是鉴定促进外显子7的治疗靶点 在SMN 2的前体mRNA剪接过程中的融合。使用最先进的迭代选择方法，我们有 最近显示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型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。支持ISS-N1的抑制性质，突变 ISS-N1内的缺失促进SMN 2 mRNA中外显子7的包含。进一步证实了 ISS-N1的性质，阻断ISS-N1的反义寡核苷酸（阿索）完全恢复了两者中的外显子7包含，我们的研究表明， 小基因系统和SMA患者成纤维细胞（来自内源性SMN 2）。因此，阿索- 经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，阿索- 即使在低阿索剂量下也观察到介导的刺激作用，这表明ISS-N1是一种高度的 可接近的反义靶。反义效应对ISS-N1具有特异性，因为两个或多个突变 在ISS-N1内完全消除了ASO介导的刺激作用。根据这些结果，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 成为治疗许多人类疾病的有力工具。这项拨款计划旨在设计高度 针对ISS-N1的高效ASO。我们将研究ASO在SMA患者细胞以及小鼠中的作用， SMA模型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基因的缺失， 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型内含子沉默子来纠正SMN 2的异常剪接。到 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。

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 nclusion 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.