Development of Therapy for Spinal Muscular Atrophy Based on the Spliring Modulation Technology

1.（背景）超过90%的脊髓性肌萎缩症（SMA）患者为SMN 1缺失纯合子。然而，SMN 2在这些患者中保留。SMN 2是与SMN 1几乎相同的基因，并且编码与SMN 1相同的蛋白。SMN 2的主要产物是缺少外显子7的转录物，产生截短的SMN蛋白。这就是为什么SMN 2不能补偿大多数SMA患者的SMNI损失的原因。SMN 1和SMN 2的外显子7中的单个核苷酸变化，即SMN 1中的6C和SMN 2中的6 T，使得它们之间的剪接差异。目前认为SMN 2外显子7剪接的纠正是SMA的一种治疗策略。2。（合成的外显子特异性剪接激活剂）基于Cartegni，et.例如，我们合成了一种肽核酸，SMN-PNA（ESSENCE），它可以激活SMN 2外显子7的剪接。SMN-PNA具有SMN 2外显子7结合结构域和丝氨酸-精氨酸重复结构域。我们的研究使用来自SMA患者的成纤维细胞系，没有显示SMN-PNA完全纠正剪接模式，而使用SMN 2 EX 6 -7质粒的体外剪接试验显示了对剪接模式的一些影响.丙戊酸（Valproic acid，VPA）是一种抗癫痫药物，广泛应用于临床。最近，有报道称VPA可增加SMN 2的表达并改变其剪接模式。我们测试了VPA是否可以增加SMA患者成纤维细胞中SMN 2基因的表达。通过定量PCR方法评价VPA（浓度为0.5-1000 μM）对成纤维细胞中SMN 2总转录水平和外显子7剪接模式的影响。在本研究中，VPA未显著改变SMN 2的总转录水平和剪接模式，表明存在VPA治疗无应答者。

1. (Background) More than 90% of patients with spinal muscular atrophy (SMA) are homozygous for SMN1 deletion. However, SMN2 is retained in such patients. SMN2 is an almost identical gene to SMN1 and codes the same protein as SMN1 does. The main product of SMN2 is a transcript lacking exon7, producing truncated SMN protein. That is the reason why SMN2 cannot compensate the loss of SMNI in most SMA patients. A single nucleotide change in exon 7, 6C in SMN1 and 6T in SMN2, makes the splicing difference between them. Correction of the exon 7 splicing of SMN2 is now considered as a treatment strategy for SMA.2. (Synthetic exon-specific splicing activator) Based on the report of Cartegni, et. al., we synthesized a peptide nucleic acid, SMN-PNA (ESSENCE), which may activate splicing of SMN2 exon 7. SMN-PNA had SMN2 exon 7 binding domain and serine-arginine repeat domain. Our study using fibroblast cell line from an SMA patient did not show at all that SMN-PNA corrected the splicing pattern, while in-vitro splicing assay with SMN2EX6-7 plasmid showed some effects on the splicing pattern.3. (Splicing modulating drug) Valproic acid (VPA) is widely used as an antiepileptic drug. Recently, it has been reported that VPA may increase SMN2 expression and alter its splicing pattern. We tested whether VPA can increase SMN2 gene expression in the fibroblasts from our SMA patients. The effect of VPA (concentration of 0.5-1000 μM) on total transcription level and exon 7-splicing pattern of SMN2 in the fibroblasts was evaluated by quantitative PCR method. VPA did not alter significantly the total transcription level and splicing pattern of SMN2 in this study, suggesting that there are non-responders to VPA treatment.