权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Reduction of the polyQ repeats in the P/Q type Calcium Channel Splice Specific Variant causing Spinocerebellar Ataxia Type 6 using CRISPR-Cas9 System.

使用 CRISPR-Cas9 系统减少导致脊髓小脑共济失调 6 型的 P/Q 型钙通道剪接特异性变体中的 polyQ 重复。

基本信息

批准号：
310649331
负责人：
Professorin Dr. Melanie D. Mark
金额：
--
依托单位：
Arbeitsgruppe Verhaltensneurobiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2022-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/310649331?language=en
关键词：
Reduction polyQ repeats type Calcium

项目摘要

Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant, debilitating neurological disorder caused by CAG expansions of 20-33 repeats in the C terminus (CT) of the P/Q type calcium channel gene. CAG encodes the amino acid glutamine, leading to poly-glutamine repeats in the calcium channel protein. In humans, an alternative splicing event occurs at C-terminal end of P/Q type calcium channel gene resulting in two isoforms, one devoid or one containing the CT CAG expansion (1, 2). Both isoform transcripts are equally abundant in adult cerebellar Purkinje cells (PC). However in individuals suffering from SCA6, the diseased isoform transcript containing the CAG expansion is increased (2) compared to control individuals. Moreover, the CT of the alpha subunit of the P/Q type calcium channel (Cava12.1) undergoes proteolytic degradation leading to a more stable diseased CT peptide fragment which specifically accumulate in nuclear and cytosolic PC protein aggregates in adult SCA6 patients (2-4). These diseased CT fragments may potentially be acting as a dominant negative mutant to interfere with the normal interactions between P/Q type calcium channels and its substrates (binding partners) which eventually leads to the SCA6 disease state. I have recently demonstrated in mice that PC specific expression of only the CT polyQ27 fragment (containing a stretch of 27 glutamines as found in SCA6 patients) is sufficient to cause SCA6 like symptoms such as PC degeneration, ataxia and motor learning deficits where the CT fragment lacking the polyQ domain did not (5). Since there are no therapeutic treatments available for these individuals, I propose to develop a potentially, new therapeutic method where I specifically reduce the CAG repeats in the CACNA1A gene using the CRISPR-Cas9 (Cas9 nucleases from microbial clustered regularly interspaced short palindromic repeat) system. I will first test the targeting efficiency of the method in a neuroblastoma cell line. Then I will introduce a viral (adeno-associated virus) form of these CRISPR targeting vectors into the cerebellum of our transgenic SCA6 mouse model expressing the diseased polyQ27 expansion and examine the extent and efficiency of eliminating the production of the diseased CT fragment and the SCA6-like symptoms.

脊髓小脑性共济失调6型（SCA 6）是一种常染色体显性遗传的神经系统疾病，由P/Q型钙通道基因C端（CT）20-33个重复的CAG扩增引起。CAG编码氨基酸谷氨酰胺，导致钙通道蛋白中的多聚谷氨酰胺重复。在人类中，在P/Q型钙通道基因的C末端发生选择性剪接事件，导致两种亚型，一种缺乏或一种含有CT CAG扩增（1，2）。这两种异构体转录同样丰富的成年小脑浦肯野细胞（PC）。然而，在患有SCA 6的个体中，与对照个体相比，含有CAG扩增的患病同种型转录物增加（2）。此外，P/Q型钙通道的α亚基的CT（Cava12.1）经历蛋白水解降解，导致更稳定的患病CT肽片段，其特异性地在成人SCA 6患者的细胞核和细胞溶质PC蛋白聚集体中积累（2-4）。这些患病的CT片段可能潜在地充当显性负突变体以干扰P/Q型钙通道与其底物（结合伴侣）之间的正常相互作用，这最终导致SCA 6疾病状态。我最近在小鼠中证明，仅CT polyQ 27片段（如在SCA 6患者中发现的，含有一段27个谷氨酰胺）的PC特异性表达足以引起SCA 6样症状，如PC变性、共济失调和运动学习缺陷，而缺乏polyQ结构域的CT片段则不会（5）。由于这些人没有可用的治疗方法，我建议开发一种潜在的新治疗方法，使用CRISPR-Cas9（来自微生物成簇的规则间隔短回文重复序列的Cas9核酸酶）系统特异性减少CACNA 1A基因中的CAG重复序列。我将首先在神经母细胞瘤细胞系中测试该方法的靶向效率。然后，我将这些CRISPR靶向载体的病毒（腺相关病毒）形式引入我们表达患病polyQ 27扩增的转基因SCA 6小鼠模型的小脑中，并检查消除患病CT片段和SCA 6样症状的程度和效率。