Splice isoform-specific RNAi as therapy for Spinocerebellar Ataxia type 6

描述（由申请人提供）：脊髓小脑性共济失调6型（SCA6）是一种显性遗传性共济失调，由聚谷氨酰胺（polyQ）编码CAG重复扩增在Cav2.1钙通道基因CACNA1a的3'端附近引起。小脑浦肯野神经元的选择性丧失是SCA6的病理标志，这是一种缓慢进展且使人衰弱的疾病，目前尚无治疗方法。这里提出的临床前研究将测试一种新的剪接异型特异性RNA干扰（SIS-RNAi）治疗方法，使用一种基因准确的SCA6小鼠模型。在Cav2.1转录物的3'端选择性剪接产生的mRNA同种异构体编码两类Cav2.1变异构体，一种缺乏，一种含有靠近蛋白质c末端的polyQ结构域。在SCA6中，最近的研究表明编码突变（扩展的）含有polyq的Cav2.1变体的剪接异构体水平异常增加。含有有毒多q的Cav2.1变异水平似乎只在小脑浦肯野神经元中增加，这可能解释了它们在SCA6中的选择性变性。总的假设是，我们的研究小组开发的一种新的SIS-RNAi方法将选择性地抑制浦肯野神经元中含有polyq的Cav2.1变体的表达，从而证明对SCA6具有显着的治疗益处。目的1将建立重组腺相关（rAAV）传递的SIS-RNAi分子在SCA6敲入小鼠模型的浦肯野神经元中选择性抑制人类多q编码Cav2.1剪接异构体表达的能力。在内源性小鼠Cav2.1启动子的控制下，SCA6敲入小鼠表达扩增的polyq编码Cav2.1转录本。重要的是，这些小鼠忠实地再现了在人类SCA6中观察到的运动和组织病理特征。Aim 2将研究长期表达rAAV-SIS-RNAi对SCA6敲入小鼠进行性神经功能障碍的分子和行为影响。拟议研究的主要影响是，它们有望提供临床前数据，支持SIS-RNAi作为人类SCA6治疗的发展。此外，他们将强调SIS-RNAi作为其他遗传性疾病治疗策略的潜力，并为我们理解Cav2.1通道的功能和调控提供有用的新试剂。

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type-6 (SCA6) is a dominantly inherited ataxia caused by a polyglutamine (polyQ)- encoding CAG repeat expansion near the 3'-end of the Cav2.1 calcium channel gene, CACNA1a. Selective loss of cerebellar Purkinje neurons is the pathological hallmark of SCA6, a slowly progressive and debilitating disease for which there is no treatment. The preclinical studies proposed here will test a novel splice-isoform- specific RNA interference (SIS-RNAi) therapy approach for SCA6, using a genetically accurate mouse model of the disease. Alternative splicing at the 3'-end of the Cav2.1 transcript produces mRNA isoforms that encode two classes of Cav2.1 variants, one lacking and one containing the polyQ domain near the c-terminal end of the protein. In SCA6, recent studies have shown an aberrant increase in the levels of splice isoforms encoding for mutant (expanded) polyQ-containing Cav2.1 variants. Levels of toxic polyQ-containing Cav2.1 variants appear to increase only in cerebellar Purkinje neurons, potentially explaining their selective degeneration in SCA6. The overall hypothesis is that a novel SIS-RNAi approach developed by our research group will selectively suppress expression of polyQ-containing Cav2.1 variants in Purkinje neurons and thus will prove to be of significant therapeutic benefit in SCA6. Aim 1 will establish the capacity of recombinant adeno-associated (rAAV)-delivered SIS-RNAi molecules to selectively suppress the expression of human, polyQ-encoding Cav2.1 splice-isoforms in Purkinje neurons of an SCA6 knock-in mouse model. SCA6 knock-in mice express expanded polyQ-encoding Cav2.1 transcripts under the control of the endogenous mouse Cav2.1 promoter. Importantly, these mice faithfully recapitulate motor and histo-pathological features observed in human SCA6. Aim 2 will examine the molecular and behavioral effects of long-term rAAV-SIS-RNAi expression on the progressive neurological dysfunction observed in SCA6 knockin mice. The primary impact of the proposed studies is that they are expected to provide preclinical data supporting the development of SIS-RNAi as therapy for humans with SCA6. Moreover, they will highlight the potential of SIS-RNAi as a therapeutic strategy in other hereditary diseases and forge novel reagents useful to our understanding of Cav2.1 channel function and regulation. PUBLIC HEALTH RELEVANCE: The proposed research will test splice-isoform-specific RNA interference (SIS-RNAi) as a therapeutic strategy for Spinocerebellar Ataxia Type 6, a currently untreatable, severely debilitating neurodegenerative disorder. These studies will assess the activity, specificity and safety profiles of SIS-RNAi in vivo using a genetically accurate mouse model of the disease. Results are anticipated to support continued pre-clinical development of SIS-RNAi as a therapy for humans with SCA6.