Biomarkers and additive therapies to enhance symptomatic treatment of Spinal Muscular Atrophy

项目摘要/摘要 脊髓性肌萎缩症(SMA)是一种神经退行性疾病，会导致运动神经元丧失，导致 瘫痪，在最严重的情况下会导致死亡。SMA的发病率为每10,000名活产儿中就有1名，使 这种疾病是导致婴儿死亡的主要原因之一。SMA是由低水平的生存运动引起的 神经元(SMN)蛋白。最近的实验表明，SMA小鼠的表型显著挽救了 使用scAAV9交付SMN。同样，使用反义寡核苷酸(ASO)纠正SMN2剪接 可扩大存活率，挽救电生理缺陷。这些充满希望的临床前研究已经导致了 治疗SMA的几项临床试验。至关重要的是，为SMA建立生物标记物可以 帮助预测治疗反应和衡量治疗效果。一组被称为SMA的蛋白质标记- SMA患者MAP与功能呈正相关。然而，目前还不清楚这些标记是否能量化 治疗反应。这些血浆标志物中的一个子集在SMA小鼠中异常，并在 用反义寡核苷酸(ASO)对SMA小鼠进行症状前治疗以增加SMN。的能力 这些用于量化ASO治疗后的治疗反应以增加SMN的标记物将在 在不同疾病阶段对SMA小鼠进行治疗。这些发现将通过测试SMA进一步调查- 基因治疗1期临床试验中接受SMA治疗的I型婴儿血液样本的MAP面板 在我们的中心。这些样本将与未经治疗的SMA 1型婴儿的样本进行比较，匹配的 年龄和SMN2拷贝数，来自NeuroNEXT临床试验，以允许确定 对治疗有反应。虽然早期使用SMN疗法非常有效，但在晚期使用效果较差。 疾病的病程。在目标2中，结合SMN改善肌肉功能的组合疗法 治疗方法将在SMA小鼠身上进行测试。导致钙升高的肌钙蛋白C基因突变 将使用腺相关病毒(AAV)载体传递敏感性，并测试对肌肉的影响 即使运动神经元输入减少，也能产生收缩力量。自我互补的AAV卵泡抑素将用于 增加肌肉质量，以确定结合使用时是否存在增加肌肉大小的相加效应 SMN.在这些联合治疗实验中，小鼠将被给予ASO，以增加不同时间的SMN 疾病分期以模拟临床试验情况。我们将监测肌肉力量和电生理 测量这些小鼠的运动单位功能。最后，目标3将调查更高水平的SMN是否可以 促进运动神经元修复和改善症状治疗后小鼠的治疗反应。最后一点 将使用CRISPR/Cas9系统对内含子6和7进行调查，以找到控制 外显子7的剪接。新位点的发现将扩大可用于ASOS和 有可能使用AAV载体在SMN2中进行意想不到的更改。

Project Summary/Abstract Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes loss of motor neurons, results in paralysis, and in the most severe forms leads to death. The incidence of SMA is 1 in 10,000 live births, making this disease one of the leading causes of infant mortality. SMA is caused by low levels of the survival motor neuron (SMN) protein. Recent experiments have shown a remarkable rescue of phenotype in SMA mice upon delivery of SMN using scAAV9. Likewise, correction of SMN2 splicing using antisense oligonucleotides (ASO) can expand survival and rescue electrophysiology defects. Such promising pre-clinical studies have led to several clinical trials for the treatment of SMA. It is crucial that biomarkers are established for SMA that can help predict treatment response and to measure therapeutic effect. A panel of protein markers known as SMA- MAP shows correlation with function in SMA patients. Yet, it is unknown if these markers can quantify therapeutic response. A subset of these plasma markers are both abnormal in SMA mice and normalize in SMA mice treated presymptomatically with anti-sense oligonucleotides (ASO) to increase SMN. The ability of these markers to quantify treatment response following treatment with ASO to increase SMN will be tested in SMA mice treated at different disease stages. These findings will be further investigated by testing the SMA- MAP panel in blood samples from treated SMA type I infants enrolled in the phase 1 gene therapy clinical trial at our center. These samples will be compared with samples from untreated SMA type 1 infants, matched for age and SMN2 copy number, from the NeuroNext clinical trial to allow determination of the markers that respond to treatment. While SMN therapies are very effective when given early, they are less effective late in the course of disease. In aim 2, combinatorial therapies to improve muscle function in combination with SMN therapies, will be tested in SMA mice. Mutations in the troponin C gene that result in increased calcium sensitivity will be delivered using adeno associated virus (AAV) vectors and tested for an effect on muscle contraction force even with reduced motor neuron input. Self-complementary AAV follistatin will be used to increase muscle mass to determine if there is an additive effect of increased muscle size when combined with SMN. In these combinatorial therapy experiments, mice will be treated with ASOs to increase SMN at different disease stages to mimic the clinical trial situation. We will monitor muscle force and electrophysiological measures of motor unit function in these mice. Finally, aim 3 will investigate whether higher levels of SMN can enhance motor neuron repair and improve therapeutic response in post symptomatically treated mice. Lastly intron 6 and 7 will be investigated using the CRISPR/Cas9 system to find new regulatory sites that control splicing of exon 7. Identification of new sites will expand therapeutic targets that can be used with ASOs and open the possibility of using AAV vector to make a permeant change in SMN2.