Targeting SMN2 Alternative Splicing for the Treatment of Spinal Muscular Atrophy

描述（由申请人提供）：该项目的长期目标是开发治疗靶向导致人类疾病的前体mRNA剪接缺陷的方法。一种可以通过靶向前体mRNA剪接潜在治愈的疾病是脊髓性肌萎缩症（SMA）。SMA是一种儿科神经退行性疾病，目前尚无治愈或有效治疗方法。该疾病是由运动神经元存活基因SMN1的纯合缺失引起的。人类有第二个基因SMN2，它几乎与SMN1相同。SMN1和SMN2都编码SMN蛋白。然而，大多数SMN2 mRNA转录本剪接出外显子7。这种替代mRNA同种型编码截短的不稳定蛋白质。因此，SMA是由SMN1缺失引起的SMN蛋白水平降低引起的。大多数SMA患者中存在SMN2，这为通过增加SMN2外显子7剪接从而恢复SMN水平来治疗该疾病提供了独特的机会。我们最近发现了一种四环素衍生物，通过直接靶向剪接反应增加SMN2外显子7剪接来提高SMN蛋白水平。该化合物作为治疗剂特别有吸引力，这是由于四环素家族分子的有利的药代动力学特征，并且因为该化合物似乎在基因表达途径中的非常特定的步骤起作用。我们申请的目的是开发这种SMN2剪接的强效激活剂，作为治疗SMA和可能由剪接缺陷引起的其他疾病的治疗药物。该研究的中心假设是，靶向SMA等疾病中的缺陷剪接将导致蛋白质表达的治疗性增加。本研究的目的1是使用来自SMA患者的诱导多能干细胞来确定剪接效应分子（如四环素衍生物）在挽救运动神经元变性中的有效性，并表征运动神经元变性期间SMN蛋白丢失的细胞后果。目的2是确定四环素衍生物增加SMN2剪接的分子靶点和作用机制。目的3是发现SMA治疗的新靶点并开发替代策略。拟议的实验将为SMA的治疗提供见解和疗法，并将推进治疗方法，以治疗由剪接缺陷引起的其他疾病。这些研究也将对我们理解诱导多能干细胞作为人类疾病模型和SMA患者运动神经元中SMN蛋白丢失的细胞缺陷产生广泛影响。

DESCRIPTION (provided by applicant): The long term objective of this project is to develop methods to therapeutically target defects in pre-mRNA splicing that cause human disease. One disease that can potentially be cured by targeting pre- mRNA splicing is spinal muscular atrophy (SMA). SMA is a pediatric neurodegenerative disease for which there is currently no cure or effective therapy. The disease is caused by the homozygous loss of the survival of motor neuron, SMN1, gene. Humans have a second gene, SMN2, which is nearly identical to SMN1. Both SMN1 and SMN2 code for SMN protein. However, the majority of SMN2 mRNA transcripts splice out exon 7. This alternative mRNA isoform codes for a truncated, unstable protein. Thus, SMA results from the reduction of SMN protein levels caused by the loss of SMN1. The presence of SMN2 in most SMA patients provides a unique opportunity to treat the disease by increasing SMN2 exon 7 splicing and thereby restoring SMN levels. We have recently identified a tetracycline derivative that improves SMN protein levels by directly targeting the splicing reaction to increase SMN2 exon 7 splicing. This compound is particularly attractive as a therapeutic due to the favorable pharmacokinetic characteristics of the tetracycline family of molecules and because the compound appears to act at a very specific step in the gene expression pathway. The goal of our application is to develop this potent activator of SMN2 splicing as a therapeutic for the treatment of SMA and potentially other diseases caused by splicing defects. The central hypothesis of the study is that targeting defective splicing in diseases such as SMA will lead to a therapeutic increase in protein expression. Aim 1 of this study is to use induced pluripotent stem cells derived from SMA patients to determine the effectiveness of splicing effector molecules, such as the tetracycline derivatives, in rescuing motor neuron degeneration and also to characterize the cellular consequences of SMN protein loss during motor neuron degeneration. Aim 2 is to identify the molecular target and mechanism of action by which tetracycline derivatives increase SMN2 splicing. Aim 3 is to discover novel targets and develop alternative strategies for SMA therapy. The proposed experiments will provide insights and therapeutics for the treatment of SMA and will also advance therapeutic approaches to treat other diseases caused by splicing defects. These studies will also have a broad impact on our understanding of induced pluripotent stem cells as models for human disease and the cellular defects of SMN protein loss in motor neurons of SMA patients.