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

脊髓性肌萎缩症(SMA)最常见的原因是存活运动神经元1(SMN1)的缺失 基因，它产生SMN蛋白。该基因的一个几乎相同的拷贝，SMN2，产生了不起作用的 SMN蛋白由于跳过外显子7，不能补偿SMN1的丢失。我的实验室调查 调控SMN外显子7的剪接，以确定促进外显子7的治疗靶点 结论SMN2mRNA前剪接过程中。使用最先进的迭代选择方法，我们有 最近的研究表明，SMN2中外显子7的跳跃与外显子7的5‘端弱剪接位点(5’ss)直接相关。 随着研究的深入，我们最近发现了一种新型的内含子剪接消音器(ISS-N1)，它可以 通过隔离5‘s来帮助跳过外显子7。支持ISS-N1基因突变的抑制性 ISS-N1中的缺失促进了SMN2mRNA中第7外显子的包含。进一步证实了抑制作用 ISS-N1的性质，阻断ISS-N1完全恢复外显子7的反义寡核苷酸(ASO)，我们的 迷你基因系统和SMA患者成纤维细胞(来自内源性SMN2)。因此，ASO- 经处理的患者细胞显示SMN蛋白的表达增加。值得注意的是，ASO- 即使在较低的ASO剂量下也观察到了介导的刺激效应，表明ISS-N1是一种高度的 可接近的反义靶点。反义效应是ISS-N1病毒的两个或两个以上突变所特有的 ISS-N1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治疗提供了一个独特的靶点。反义技术已经 成为治疗许多人类疾病的有力工具。这项拨款提案旨在设计高度 针对ISS-N1的高效ASO。我们将研究ASOS对SMA患者细胞和小鼠的影响 SMA的模型。这项研究的结果将确定基于ASO的SMA治疗的有效性。 脊髓性肌萎缩症(SMA)最常见的原因是SMN1基因缺失，伴随着 SMN2基因因剪接异常而不能补偿。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消音器来纠正SMN2的异常剪接。至 为了探索反义寡核苷酸的治疗潜力，我们将把研究扩展到SMA的小鼠模型。

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.