Characterization of a complex regulatory element of Spinal Muscular Atrophy genes

描述（由申请人提供）：人类有两个拷贝的运动神经元生存（SMN）基因，SMN1和SMN2。SMN1的缺失导致脊髓性肌萎缩症（SMA），这是一种婴儿和儿童的衰弱性疾病。SMN2无法弥补SMN1由于外显子7的跳变而导致的截断蛋白的合成而造成的损失，这是不稳定的。大多数SMA患者中SMN2的存在提供了一个难得的机会，通过纠正外显子7的异常剪接来恢复SMN水平。使用一种新的体内选择方法，我们之前报道了SMN2中一个弱5'剪接位点（51 s）是外显子7包含的限制因素。基于这一观察，我们最近发现了一个独特的内含子抑制元件对7号外显子的弱5' s起作用。我们称之为内含子剪接沉默者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的发病机制中起重要作用。最重要的是，我们发现针对ISS-N1的反义寡核苷酸（简称ASOs）可以纠正包括SMA患者细胞在内的所有细胞类型中SMN2的异常剪接。因此，抗ISS-N1的ASO完全恢复了患者细胞中的SMN水平。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是一个高度可及的反义靶标。反义效应对ISS-N1非常特异性，因为ISS-N1中的两个或多个突变完全消除了aso介导的刺激作用。基于这些结果，我们相信我们已经发现了aso介导的SMA治疗的理想靶点。在本次拨款申请中，我们将(1)详细描述ISS-N1及其RNA结构和相互作用因素；(2)开发针对ISS-N1的高效ASOs，纠正患者细胞中SMN2剪接；最后(3)进行体内研究，利用小鼠SMA模型验证抗ISS-N1的ASOs作为可能的候选药物。脊髓性肌萎缩症（SMA）最常见的原因是SMN1基因的缺失，同时SMN2基因由于剪接异常而无法补偿。在这里，我们将描述一个在SMA发病机制中起关键作用的新内含子元件。此外，我们将使用该元件作为反义介导的SMA异常剪接纠正的靶标。

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.