Next generation gene silencing strategies for Huntington's disease

描述（由申请人提供）：亨廷顿氏病（HD）是几种主要的神经退行性疾病之一，由疾病蛋白中类似的毒性功能突变获得引起：聚谷氨酰胺（polyQ）编码通道的扩增。目前，还没有针对HD的治疗方法。RNA干扰（RNAi）是一种通过靶向和降解编码mRNA来降低疾病基因表达的主要方法。我们的初步工作表明，载体介导的RNAi可以降低亨廷顿（Htt）的表达，改善疾病小鼠模型的疾病表型。我们将最小的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识别抑制性rna的转录后果。这些载体，虽然最小化了序列外沉默，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）代表了几乎90%的HD患者。这一数据使我们有机会测试等位基因定向沉默载体在体内是否安全有效。为此，我们

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.