Characterization of a complex regulatory element of Spinal Muscular Atrophy genes

描述（由申请方提供）：人类有两个运动神经元存活（SMN）基因拷贝，SMN 1和SMN 2。SMN 1缺失导致脊髓性肌萎缩症（SMA），这是一种使婴儿和儿童衰弱的疾病。SMN 2无法补偿由于外显子7跳跃导致的SMN 1损失，导致合成不稳定的截短蛋白。大多数SMA患者中存在SMN 2，这为通过纠正外显子7的异常剪接恢复SMN水平提供了难得的机会。使用一种新的体内选择方法，我们之前报道过弱5'剪接位点（51 ss）是SMN 2中包含第7号外显子的限制因素。基于这一观察，我们最近发现，一个独特的内含子抑制元件有助于对外显子7的弱5' ss。我们称这种元件为Intronic Splicing Silencer N1（缩写为ISS-N1）。有趣的是，ISS-N1似乎是人类独有的。根据我们的初步结果，ISS-N1在SMA的发病机制中起着重要作用。最重要的是，我们发现靶向ISS-N1的反义寡核苷酸（简称ASO）可纠正所有检测细胞类型（包括SMA患者细胞）中SMN 2的异常剪接。因此，针对ISS-N1的阿索完全恢复了患者细胞中的SMN水平。值得注意的是，即使在低阿索剂量下也观察到ASO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，我们认为我们已经发现了一个理想的靶点为ASO介导的治疗SMA。在这项资助提案中，我们将（1）详细表征ISS-N1，其RNA结构和相互作用因子;（2）开发针对ISS-N1的有效ASO，以纠正患者细胞中的SMN 2剪接;最后（3）进行体内研究，使用SMA小鼠模型验证针对ISS-N1的ASO作为可能的候选药物。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，我们将描述一种新的内含子元件，在SMA的发病机制中起着至关重要的作用。此外，我们将使用该元件作为SMA中异常剪接的反义介导校正的靶点。

DESCRIPTION (provided by applicant): Humans have two copies of Survival of Motor Neuron (SMN) gene, SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), a debilitating disease of infants and children. SMN2 fails to compensate for the loss of SMN1 due to skipping of exon 7 resulting in the synthesis of a truncated protein, which is unstable. Presence of SMN2 in most SMA patients provides a rare opportunity to restore SMN levels by correcting the aberrant splicing of exon 7. Using a novel method of in vivo selection, we previously reported that a weak 5' splice site (51 ss) serves as the limiting factor for exon 7 inclusion in SMN2. Based on this observation, we have recently discovered that a unique intronic inhibitory element contributes towards the weak 5' ss of exon 7. We call this element Intronic Splicing Silencer N1 (abbreviated as ISS-N1). Interestingly, ISS-N1 appears to be unique to humans. Based on our preliminary results, ISS-N1 plays an important role in pathogenesis of SMA. Most importantly, we discovered that Antisense Oligonucleotides (abbreviated as ASOs) that targeted ISS-N1 corrected aberrant splicing of SMN2 in all cell types tested including SMA patient cells. Consequently, ASO against ISS-N1 fully restored SMN levels in patient cells. 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 we have discovered an ideal target-site for the ASO-mediated therapy of SMA. In this grant proposal, we will (1) characterize ISS-N1, its RNA structure and interacting factors in details; (2) develop efficient ASOs against ISS-N1 to correct SMN2 splicing in patient cells; and finally (3) conduct in vivo studies, using mice models of SMA to validate ASOs against ISS-N1 as the possible drug candidates. 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 characterize a novel intronic element that plays a critical role in pathogenesis of SMA. In addition, we will use this element as a target for the antisense-mediated correction of aberrant splicing in SMA.