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针对导致人类疾病的前 mRNA 剪接缺陷进行治疗的方法。脊髓性肌萎缩症 (SMA) 是一种可以通过靶向前 mRNA 剪接来治愈的疾病。 SMA 是一种小儿神经退行性疾病，目前尚无治愈或有效疗法。该疾病是由于运动神经元存活基因SMN1纯合性缺失引起的。人类还有第二个基因 SMN2，它与 SMN1 几乎相同。 SMN1 和 SMN2 均编码 SMN 蛋白。然而，大多数 SMN2 mRNA 转录物都会剪出外显子 7。这种替代的 mRNA 同工型编码一种截短的、不稳定的蛋白质。因此，SMA 是由于 SMN1 缺失导致 SMN 蛋白水平降低所致。大多数 SMA 患者中 SMN2 的存在为通过增加 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来增加 SMN2 外显子 7 剪接，从而提高 SMN 蛋白水平。该化合物作为治疗剂特别有吸引力，因为四环素家族分子具有有利的药代动力学特征，并且该化合物似乎在基因表达途径中的非常特定的步骤发挥作用。我们申请的目标是开发这种有效的 SMN2 剪接激活剂，作为治疗 SMA 和剪接缺陷引起的其他潜在疾病的治疗剂。该研究的中心假设是，针对 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 是利用源自 SMA 患者的诱导多能干细胞来确定剪接效应分子（例如四环素衍生物）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。目标 2 是确定四环素衍生物增强 SMN2 剪接的分子靶标和作用机制。目标 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 的治疗提供见解和治疗方法，还将推进治疗由剪接缺陷引起的其他疾病的治疗方法。这些研究还将对我们对诱导多能干细胞作为人类疾病模型的理解以及 SMA 患者运动神经元中 SMN 蛋白丢失的细胞缺陷的理解产生广泛的影响。

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.