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

保护性修饰剂 NCALD 开发脊髓性肌萎缩症有效组合疗法的力量

• 批准号: 398410809
• 负责人: Professorin Dr. Brunhilde Wirth
• 金额: --
• 依托单位: Institut für Humangenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/398410809?language=en
• 关键词: power; protective; modifier; NCALD; develop

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.

脊髓性肌萎缩症（SMA）是一种常见的神经肌肉疾病，约60%的患者在儿童早期死亡。SMN1基因突变导致脊髓α -运动神经元（MNs）功能丧失，主要影响神经肌肉连接（NMJs）的发育和成熟。突触传递受损导致肌无力和近端随意肌萎缩。该疾病的严重程度主要受拷贝基因SMN2的影响，SMN2在90%的转录本中异常剪接，缺乏外显子7，很少受其他遗传修饰因子的影响。SMN2被认为是SMA治疗的主要靶点。首个smn - aso （SPINRAZA）最近已获得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SMN2拷贝的患者，SPINRAZA诱导的SMN蛋白可能仍不足以终生抵消MN功能障碍。基于所谓的SMA不一致家族，在这些家族中，smn1缺失的个体仍然没有症状，我们确定了两种人类SMA保护修饰剂：plastin 3 （PLS3）和神经钙素δ (NCALD)。PLS3是一种f -肌动蛋白结合和捆绑蛋白，通过过表达来保护SMA，而NCALD是一种神经元钙传感器蛋白，通过抑制来保护SMA。我们假设这些修饰剂将向我们展示如何开发不依赖于SMA的治疗方法的新途径，并将有助于更好地理解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，证明这两种修饰剂对各种转基因或morpholino诱导的物种（包括蠕虫、斑马鱼和小鼠）都有保护作用。最重要的是，我们发现低剂量的SMN-ASOs加上PLS3（来自转基因）的过表达或NCALD（作为杂合敲除等位基因）的减少可以挽救SMA病理并显着延长生存期，证明了联合治疗的力量。此外，这两种修饰剂的发现指向了SMA的主要病理细胞紊乱，即内吞作用受损。在这里，我们的目标是在小鼠身上开发一种组合疗法，为未来的人类疗法奠定基础，并了解信号传导和细胞保护网络。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。本研究将重点研究三个方面：1)对重度SMA小鼠进行Ncald-ASOs联合低剂量SPINRAZA的症状前和症状前注射试验，并进行详细的功能和组织学分析。2)开发人类特异性NCALD ASOs，并测试ipscs衍生的MN、NMJs和来自对照和SMA个体的脑类器官的有效性。3)揭示NCALD的分子和蛋白网络，更好地理解SMA的保护作用。