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

基于Spliring调制技术的脊髓性肌萎缩症治疗方法研究进展

基本信息

批准号：
18591151
负责人：
NISHIO Hisahide
金额：
$ 2.43万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591151/
关键词：
spinal muscular atrophy the SMN1 gene the SMN2 gene thetic exon-specific splicing activator valproic acid ヒストン脱アセチル化酵素阻害剤転写効率スプライシング効率エクソン7 スプライシングペプチド核酸遺伝子変異 Tudorドメイン

项目摘要

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.

1. （背景）超过90%的脊髓性肌萎缩（SMA）患者是SMN1缺失的纯合子。然而，SMN2在这类患者中保留。SMN2与SMN1几乎是一模一样的基因，并编码与SMN1相同的蛋白质。SMN2的主要产物是缺乏外显子7的转录物，产生截断的SMN蛋白。这就是为什么SMN2在大多数SMA患者中不能补偿SMNI的损失。SMN1的外显子7,6c和SMN2的外显子6T上的一个核苷酸变化导致了它们之间剪接的差异。纠正SMN2的外显子7剪接现在被认为是sma的一种治疗策略。（合成外显子特异性剪接激活剂）根据Cartegni等人的报道，我们合成了一种可能激活SMN2外显子7剪接的肽核酸SMN-PNA （ESSENCE）。SMN-PNA具有SMN2外显子7结合域和丝氨酸-精氨酸重复结构域。我们使用来自SMA患者的成纤维细胞系进行的研究完全没有显示SMN-PNA纠正剪接模式，而SMN2EX6-7质粒的体外剪接实验显示对剪接模式有一定的影响。丙戊酸（Valproic acid， VPA）是一种广泛应用的抗癫痫药物。最近有报道称VPA可能增加SMN2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SMN2基因的表达。采用定量PCR方法观察VPA （0.5 ~ 1000 μM）对成纤维细胞SMN2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SMN2的总转录水平和剪接模式，提示存在对VPA治疗无反应的患者。