Next generation gene silencing strategies for Huntington's disease

描述（由申请人提供）：亨廷顿病（HD）是由疾病蛋白中类似的毒性功能获得突变引起的几种主要神经退行性疾病之一：多聚谷氨酰胺（polyQ）编码区的扩增。目前，尚无针对HD的治疗方法。RNA干扰（RNAi）通过靶向和降解编码mRNA来降低疾病基因表达已成为一种主要方法。我们的初步工作表明，载体介导的RNAi可以减少亨廷顿（Htt）的表达和改善疾病的小鼠模型的疾病表型。我们使用最小脱靶沉默作为载体设计的主要目标，利用生物信息学和微阵列来鉴定抑制性RNA的转录结果。这些载体，尽管最小化了序列外沉默，但仍然减少了两个HTT等位基因的表达。最近对HD人群的分析表明，有4-5个主要的单核苷酸多态性（SNP），占HD患者的近90%。该数据使我们有机会测试具有最小化脱靶的等位基因定向沉默的载体在体内是否安全有效。为此我们 开发了新的转基因小鼠，突变的转基因工程包含这些相关的SNP。我们建议现在测试是否针对疾病相关多态性的人工miRNA可以优先沉默体内突变的htt等位基因。最后，我们使用HD中转录失调的数据来鉴定、构建和初步测试用于抑制性RNA表达控制的内源性调节启动子，以进一步提高其安全性。我们现在将测试这些启动子在体外和体内以疾病响应方式驱动RNAi的能力。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is one of several dominant neurodegenerative diseases caused by a similar toxic gain of function mutation in the disease protein: expansion of a polyglutamine (polyQ)-encoding tract. Currently, no therapy exists for HD. RNA interference (RNAi) has emerged as a leading method to reduce disease gene expression by targeting and degrading the encoding mRNA. Our preliminary work demonstrates that vector-mediated RNAi can reduce huntington (Htt) expression and improve disease phenotypes in mouse models of disease. We used minimal off-target silencing as a primary objective in vector design, taking advantage of bioinformatics and microarrays to identify transcriptional consequences of the inhibitory RNAs. These vectors, though minimized for off-sequence silencing, still reduce expression of both HTT alleles. Recent analysis of the HD population suggests that there are 4-5 predominant single nucleotide polymorphisms (SNPs) representing almost 90% of HD patients. This data gives us the opportunity to test if vectors for allele-directed silencing with minimized off-targeting are safe and effective in vivo. For this, we developed novel transgenic mice, with the mutant transgenes engineered to contain these relevant SNPs. We propose to now test if artificial miRNAs targeting disease-linked polymorphisms can preferentially silence mutant htt alleles in vivo. Finally, we have used data on transcriptional dysregulation in HD to identify, build and preliminarily test endogenously regulated promoters for expression control of inhibitory RNAs to further improve their safety profile. We will now test the capabilities of these promoters to drive RNAi in a disease responsive manner in vitro and in vivo.