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用于治疗导致人类疾病的前mrna剪接缺陷的方法。一种可以通过靶向前mRNA剪接来治愈的疾病是脊髓性肌萎缩症（SMA）。SMA是一种儿童神经退行性疾病，目前没有治愈或有效的治疗方法。该病是由运动神经元SMN1基因的纯合子缺失引起的。人类有第二个基因SMN2，它几乎与SMN1完全相同。SMN1和SMN2都编码SMN蛋白。然而，大多数SMN2 mRNA转录本剪切掉了外显子7。这种替代的mRNA异构体编码一个截断的、不稳定的蛋白质。因此，SMA是由SMN1缺失导致的SMN蛋白水平降低引起的。SMN2在大多数SMA患者中的存在为通过增加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。由于四环素分子家族具有良好的药代动力学特性，并且该化合物似乎在基因表达途径的一个非常特定的步骤上起作用，因此该化合物作为一种治疗药物特别有吸引力。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和由剪接缺陷引起的潜在其他疾病的治疗方法。该研究的中心假设是，针对SMA等疾病中的剪接缺陷将导致治疗性蛋白表达的增加。本研究的目的1是利用来自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方面的有效性，并表征运动神经元变性过程中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是发现新的靶点并开发SMA治疗的替代策略。所提出的实验将为SMA的治疗提供见解和治疗方法，并将推进治疗由剪接缺陷引起的其他疾病的治疗方法。这些研究也将对我们理解作为人类疾病模型的诱导多能干细胞以及SMA患者运动神经元中SMN蛋白丢失的细胞缺陷产生广泛的影响。

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.