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（SCA 6）是一种显性遗传性共济失调，由编码多聚谷氨酰胺（polyQ）的CAG重复序列在Cav2.1钙通道基因CACNA 1a的3 '端附近扩增引起。小脑浦肯野神经元的选择性丧失是SCA 6的病理标志，SCA 6是一种缓慢进行性和使人衰弱的疾病，目前尚无治疗方法。本文提出的临床前研究将使用遗传学上准确的SCA 6小鼠模型测试一种新的剪接异构体特异性RNA干扰（SIS-RNAi）治疗方法。Cav2.1转录物3 '端的选择性剪接产生编码两类Cav2.1变体的mRNA同种型，一类在蛋白质的C末端附近缺乏polyQ结构域，另一类含有polyQ结构域。在SCA 6中，最近的研究已经显示编码突变的（扩增的）含polyQ的Cav2.1变体的剪接同种型的水平异常增加。含有毒性polyQ的Cav2.1变体的水平似乎仅在小脑浦肯野神经元中增加，这可能解释了它们在SCA 6中的选择性变性。总体假设是，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，因此将证明在SCA 6中具有显著的治疗益处。目的1将建立重组腺相关（rAAV）递送的SIS-RNAi分子选择性抑制SCA 6敲入小鼠模型的浦肯野神经元中编码人polyQ的Cav2.1剪接异构体的表达的能力。SCA 6敲入小鼠在内源性小鼠Cav2.1启动子的控制下表达扩增的编码polyQ的Cav2.1转录物。重要的是，这些小鼠忠实地再现了在人SCA 6中观察到的运动和组织病理学特征。目的2将检查长期rAAV-SIS-RNAi表达对SCA 6敲入小鼠中观察到的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，预计它们将提供临床前数据，支持SIS-RNAi作为SCA 6人类疗法的开发。此外，他们将突出SIS-RNAi作为其他遗传性疾病治疗策略的潜力，并打造有助于我们理解Cav2.1通道功能和调节的新型试剂。 公共卫生相关性：拟议的研究将测试剪接异构体特异性RNA干扰（SIS-RNAi）作为脊髓小脑共济失调6型的治疗策略，这是一种目前无法治疗的严重衰弱的神经退行性疾病。这些研究将使用遗传上准确的疾病小鼠模型评估SIS-RNAi的活性，特异性和体内安全性。预计结果将支持SIS-RNAi作为SCA 6人类治疗的持续临床前开发。

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.