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

脊髓小脑型共济失调6型(SCA6)是一种常染色体显性遗传性衰弱神经系统疾病，由P/Q型钙通道基因C末端20-33个重复序列的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，P/Q型钙通道基因的C端发生选择性剪接事件，导致两种异构体，一种缺失或一种含有CT CAG扩展(1，2)。这两种异构体转录本在成人小脑浦肯野细胞(PC)中同样丰富。然而，在患有SCA6的个体中，与对照组相比，含有CAG扩展的患病异构体转录本增加(2)。此外，P/Q型钙通道α亚单位(Cava12.1)的CT经历了蛋白降解，导致了更稳定的病态CT多肽片段，该片段特异性地积聚在成年SCA6患者的细胞核和胞浆PC蛋白聚集体中(2-4)。这些患病的CT片段可能作为显性负性突变体，干扰P/Q型钙通道与其底物(结合伙伴)之间的正常相互作用，最终导致SCA6疾病状态。我最近在小鼠身上证明，只有CT PolyQ27片段(如在SCA6患者中发现的那样，包含27种谷氨酸)的PC特异性表达足以导致类似SCA6的症状，如PC变性、共济失调和运动学习障碍，而CT片段缺乏PolyQ结构域则不能(5)。由于目前还没有针对这些个体的治疗方法，我建议开发一种潜在的新的治疗方法，其中我使用CRISPR-Cas9(来自微生物簇的规则间隔的短回文重复序列的Cas9核酸酶)系统特异性地减少CACNA1A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，我将把这些CRISPR靶向载体的病毒(腺相关病毒)形式引入我们的转基因SCA6小鼠模型的小脑，表达疾病的PolyQ27扩展，并检查消除疾病CT片段和SCA6样症状产生的程度和效率。

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.