Modeling the effects of reducing huntingtin and Hdh alternative splicing in mice

 描述(申请人提供)：亨廷顿病(HD)是一种常染色体显性遗传性神经退行性疾病，由编码一段聚谷氨酰胺(PolyQ)的CAG三联体重复序列在亨廷顿蛋白(HD基因的蛋白质产物)内扩张而引起。HD突变给突变的HTT带来了有害的功能获得和潜在的功能丧失，影响了多种细胞通路。基因沉默是一种很有前景的HD治疗策略，它可以绕过寻找针对受突变HTT影响的所有细胞通路的治疗挑战。为了确定减少突变hTt表达以获得最大治疗效益的最佳时间，并评估如果不能实现对突变htt等位基因的选择性靶向的后果，我们建议使用新的HD敲入小鼠模型(HdhLacO-140Q和HdhLacO-20Qhu小鼠)，在该模型中已将Lac操作符插入到小鼠HD基因座(HDH)中。将这些小鼠与一株普遍表达Lac抑制子的转基因小鼠(？-Actin-Lacir-Tg)杂交后，我们可以通过在它们的饮用水中添加或停用异丙基-？D-1-硫代半乳糖苷(IPTG)来全局地抑制或抑制小鼠突变的Huntingtin(Htt)的表达，或在不同年龄段同时抑制突变和正常的Htt的表达。在目标1中，我们将描述 在断奶、3个月、6个月和9个月龄的HdhLacO-140Q/+中抑制突变型Htt或或同时抑制突变型和野生型Htt的表达；在2-24个月龄的HdhLacO-140Q/LACO-20Qhu；？-Actin-Lacir TG中，通过表征小鼠的行为、神经病理和HTT表达水平来抑制它们的Htt表达。此外，为了检验在老年小鼠中解除抑制突变HTT表达的效果(在老年患者中建立停止基因治疗的模型)，将对12个月大的HdhLacO-140Q/+；？-actin-Lacir TG小鼠实施IPTG。他们的表型将在12至24个月龄时进行表征，并与对照组进行比较。在目标2中，为了确定评估基因沉默治疗效果的潜在生物标志物，我们建议用RNA-SEQ来表征在3、6和9个月龄时，在突变型HTT或突变型和野生型HTT同时表达后，皮质和纹状体中发生的近端基因表达的变化。候选基因的验证将首先在脑组织和培养的原代神经元中进行。然后，将使用在突变HTT抑制之前和之后从小鼠身上获得的血液样本来进一步检查经过验证的基因。总之，这些分析的结果应该有助于设计未来的HD基因沉默疗法，并有助于我们对HD发病机制的理解。

 DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that is caused by the expansion of a CAG triplet repeat encoding a stretch of polyglutamine (polyQ) within Huntingtin (HTT), the protein product of the HD gene. The HD mutation confers a deleterious gain-of-function and potential loss-of- function on mutant HTT that affects a variety of cellular pathways. Gene-silencing is a promising therapeutic strategy for HD which can circumvent the challenge of finding treatments targeting all the cellular pathways that are affected by mutant HTT. To determine the optimal time for reducing mutant HTT expression for achieving maximal therapeutic benefit, and to evaluate the consequences if selective targeting of the mutant HTT allele cannot be achieved, we propose using novel HD knock-in mouse models (HdhLacO-140Q and HdhLacO- 20Qhu mice) in which Lac operators have been inserted into the mouse HD locus (Hdh). After crossing these mice with a strain of transgenic mice ubiquitously expressing the Lac repressor (ß-actin-LacIR-tg), we can globally de-repress or repress mouse mutant huntingtin (Htt) expression, or both mutant and normal Htt expression at different ages by administering or withdrawing Isopropyl-ß-D-1-thiogalactopyranoside (IPTG) in their drinking water. In Aim 1, we will characterize the effect of repressing either mutant Htt or or both mutant and wild-type Htt expression at weaning, 3-, 6-, and 9-months of age in HdhLacO-140Q/+; ß-actin-LacIR tg, HdhLacO-140Q/LacO-20Qhu; ß-actin-LacIR tg, and control mice by characterizing their behavior, neuropathology, and htt expression levels at 2- to 24- months of age. In addition, to examine the effect of de-repressing mutant htt expression in an aged mouse (modeling discontinuation of a gene therapy in an older patient), IPTG will be administered to 12-month old HdhLacO-140Q/+; ß-actin-LacIR tg mice. Their phenotypes will be characterized at 12- to 24-months of age and compared to controls. In Aim 2, in order to identify potential biomarkers for evaluating the efficacy of a gene-silencing therapy, we propose to characterize by RNA-seq the proximal gene expression changes that occur in the cortex and striatum following repression of mutant htt or both mutant and wild-type htt expression at 3-, 6-, and 9- months of age. Validation of candidate genes will be performed first with brain tissue and cultured primary neurons. Validated genes will then be further examined using blood samples obtained from mice prior to and following mutant htt repression. Together, the results of these analyses should contribute to the design of future gene-silencing therapies for HD, and to our understanding of HD pathogenesis.