The power of protective modifier NCALD to develop an efficient combinatorial therapy for spinal muscular atrophy

脊髓性肌萎缩症（SMA）是一种常见的神经肌肉疾病，导致约60%的患者在儿童早期死亡。SMN 1基因突变导致脊髓中α运动神经元（MN）的功能丧失，主要影响神经肌肉接头（NMJ）的发育和成熟。受损的突触传递导致近端随意肌的肌无力和萎缩。疾病的严重程度主要受一个拷贝基因SMN 2的影响，该基因在90%的转录本中被异常剪接，缺乏外显子7，很少受其他遗传修饰剂的影响。SMN 2被认为是SMA治疗的主要靶点。第一批SMN-ASO（SPINRAZA）最近获得了FDA和EMA的批准。虽然SPINRAZA显示出令人鼓舞的结果，但对于大多数仅携带两个SMN 2拷贝的患者，SPINRAZA诱导的SMN蛋白可能仍然不足以终身抵消MN功能障碍。基于所谓的SMA不一致家族，其中SMN 1缺失的个体保持无症状，我们鉴定了两种人SMA保护性修饰物：plastin 3（PLS 3）和neurocalcin delta（NCALD）。PLS 3是一种F-肌动蛋白结合和捆绑蛋白，其通过过表达起SMA保护作用，而NCALD是一种神经元钙传感器蛋白，并通过抑制起保护作用。我们假设，这些修饰剂将为我们展示如何开发SMN非依赖性SMA疗法的新途径，并将有助于更好地了解SMA的细胞机制。在过去的资助期内，我们进行了广泛的功能研究，提供了强有力的证据表明，这两种修饰剂对各种转基因或吗啉诱导的物种（包括蠕虫、斑马鱼和小鼠）具有保护作用。最重要的是，我们表明低剂量SMN-ASO加上PLS 3的过表达（来自转基因）或减少的NCALD（作为杂合敲除等位基因）挽救了SMA病理学并显著延长了存活，证明了组合疗法的功效。此外，这两种修饰剂的发现指出我们对SMA的主要病理细胞干扰，这是一个受损的内吞作用。在这里，我们的目标是开发一种组合疗法在小鼠和奠定基础，为未来的治疗在人类和了解信号和细胞网络的保护。由于特定基因的激活比下调更困难，我们将通过使用特定的ASO来集中研究NCALD。将研究三个重要领域：1）在症状前和症状后注射的重度SMA小鼠中测试小鼠Ncald-ASO与低剂量SPINRAZA，随后进行详细的功能和组织学分析。2)开发人特异性NCALD ASO并测试来自对照和SMA个体的iPSC衍生的MN、NMJ和脑类器官中的有效性。3)揭示NCALD的分子和蛋白质网络，以更好地了解SMA保护。

Spinal muscular atrophy (SMA) is a common neuromuscular disorder leading to early childhood lethality in about 60% of patients. Mutations in the SMN1 gene cause functional loss of the alpha-motor neurons (MNs) in the spinal cord mainly affecting development and maturation of neuromuscular junctions (NMJs). Impaired synaptic transmission causes muscle weakness and atrophy of proximal voluntary muscles. The disease severity is mainly influenced by a copy gene, SMN2, which is aberrantly spliced lacking exon 7 in 90% of transcripts and, rarely, by additional genetic modifiers. SMN2 is considered as the main target for SMA therapy. The first SMN-ASOs (SPINRAZA) have recently been FDA and EMA approved. While SPINRAZA shows encouraging results, for the majority of patients, who carry only two SMN2 copies, the SMN protein induced by SPINRAZA may still be insufficient to counteract MN dysfunction lifelong. Based on so called SMA discordant families, in whom SMN1-deleted individuals remain asymptomatic, we identidied two human SMA protective modifiers: plastin 3 (PLS3) and neurocalcin delta (NCALD). PLS3 is an F-actin binding and bundling protein, which acts SMA protective by overexpression, whereas NCALD is a neuronal calcium sensor protein and acts protective by supression. We hypothesized that these modifiers will show us new avenues how to develop SMN-independent SMA therapies and will help to better understand the cellular mechanism underlying SMA. In the past funding period we did extensive functional studies, providing strong evidence that both modifiers are acting protective across various genetically-modified or morpholino-induced species including worm, zebrafish and mice. Most important, we showed that low dose SMN-ASOs plus overexpression of PLS3 (from a transgene) or reduced NCALD (as heterozygous knockout allele) rescues SMA pathology and significantly prolongs survival, proving the power of combinatorial therapy. Moreover, the discovery of both modifiers pointed us towards the main pathocellular disturbance in SMA, which is an impaired endocytosis.Here we aim to develop a combinatorial therapy in mice and set the ground for future therapies in humans and to understand the signalling and cellular network of protection. Since activation of a specific gene is more difficult than downregulation, we will concentrate on NCALD, by using specific ASOs. Three important areas wil be studied: 1) Test of murine Ncald-ASOs together with low-dose of SPINRAZA in severe SMA mice injected presymptomatically and symptomatically followed by detailed functional and histological analysis. 2) Development of human-specific NCALD ASOs and test for effiicacy in iPSCs-derived MN, NMJs and brain organoids from control and SMA individuals. 3) Unveil the molecular and protein network of NCALD to better understand the SMA protection.