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

点击翻译按钮获取中文摘要

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.