Signaling Mechanisms in Neuronal Degeneration

描述（由申请方提供）：亨廷顿病（HD）是最常见的常染色体显性遗传三核苷酸重复神经退行性疾病。亨廷顿蛋白（htt）基因编码一种350 kD的蛋白质;致病突变导致氨基末端多聚谷氨酰胺重复序列扩增，超过36个连续谷氨酰胺。我们广泛的研究目标是了解HD发病机制的分子基础，以靶向治疗。由于亨廷顿氏病是一种遗传性疾病，我们预期突变等位基因将与野生型至少有一个单核苷酸多态性（SNP）不同。我们的核心思想是RNA沉默可以用于选择性地减少突变htt的产生，从而减缓或阻断HD疾病中的神经元功能障碍和死亡。我们提出五个目标。目的1将鉴定htt mRNA中存在的所有SNP，以鉴定可以设计SNP选择性siRMAs的那些SNP 9。目的2将开发SNP选择性siRNA，然后研究它们在细胞模型中的功效：在HeLa细胞、用人htt转染的X-57永生化神经元细胞以及通过慢病毒转导表达人htt的原代纹状体和皮质神经元中的荧光素酶报告基因测定。将测量htt RNAi对HD发病机制的分子相关性（htt片段积累、htt聚集和自噬）的影响。目的3将检查针对非SNP选择性的htt mRNA的siRNA的有效性。将研究htt siRNA的脱靶效应。目标4将寻求一种替代战略来阻止。htt mRNA，通过创建一个分子系链，招募miRNA靶向rriRNA。目的5将在亨廷顿病小鼠模型中通过递送缀合的htt siRNA和慢病毒htt shRNA来获得最有活性的SNP选择性siRNA。将测量HD分子相关性、神经生理学和行为。初步和已发表的研究支持每个目标，包括开发htt SNP选择性超功能siRNA，将siRNA递送至原代神经元，以及分子和整个动物终点。这些目标预计将提供HD治疗和深入了解标志其发病机制的分子相关性。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is the most prevalent autosomal dominant, trinucleotide repeat neurodegenerative disease. The huntingtin (htt) gene encodes a protein of 350 kD; the disease-causing mutation causes an expansion of an amino-terminal polyglutamine repeat of more than 36 successive glutamines. Our broad research goal is to understand the molecular basis of HD pathogenesis, to target therapy. Because Huntington's disease is an inherited disease, we expect that the mutant allele will differ from wild-type by at least a single nucleotide polymorphism (SNP). Our core idea is that RNA silencing could be used) to selectively reduce mutant htt production and thereby slow or block neuronal dysfunction and death in HD disease. We propose 5 Aims. Aim 1 will identify all the SNPs present in the htt mRNA, to identify those SNP9 to which SNP-selective siRMAs can be designed. Aim 2 will develop SNP-selective siRNAs and then study their efficacy in cell models: luciferase reporter assays in HeLa cells, X-57 immortalized neuronal cells transfected with human htt, and primary striatal and cortical neurons expressing human htt through lentiviral transduction. Effects of htt RNAi on molecular correlates of HD pathogenesis (htt fragment accumulation, htt aggregation and autophagy) will be measured. Aim 3 will examine the effectiveness of siRNAs directed against htt mRNA that are not SNPselective. Off-target effects of htt siRNAs will be studied. Aim 4 will pursue an alternative strategy to block. htt mRNA, by creating a molecular tether that recruits miRNA to target rriRNAs. Aim 5 will iesi the most active, SNP-selective siRNA in mouse models of Huntington's disease, through delivery of conjugated htt siRNAs and lentivirus htt shRNA. HD molecular correlates, neurophysiology and behavior will be measured. Preliminary and published studies support each of the aims, including development of htt SNP-selective hyper functional siRNAs, delivery of siRNAs to primary neurons, and molecular and whole animal endpoints. These aims are expected to provide HD therapy and insights into molecular correlates that mark its pathogenesis.