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

 描述（由申请人提供）：亨廷顿病（HD）是一种常染色体显性神经退行性疾病，由编码亨廷顿蛋白（HTT）（HD基因的蛋白产物）内一段多聚谷氨酰胺（polyQ）的CAG三联重复序列扩增引起。HD突变会导致突变HTT出现有害的功能获得和潜在的功能丧失，从而影响多种细胞途径。基因沉默是HD的一种有前途的治疗策略，它可以规避寻找靶向受突变HTT影响的所有细胞通路的治疗方法的挑战。为了确定减少突变HTT表达以实现最大治疗益处的最佳时间，并评估如果不能实现突变HTT等位基因的选择性靶向的后果，我们提出使用新的HD敲入小鼠模型（HdhLacO-140 Q和HdhLacO-20 Qhu小鼠），其中Lac操纵基因已插入小鼠HD基因座（Hdh）。在将这些小鼠与普遍表达Lac阻遏物（β-actin-LacIR-tg）的转基因小鼠品系杂交后，我们可以通过在其饮用水中施用或去除异丙基-β-D-1-硫代半乳糖苷（IPTG）来全面地去阻遏或阻遏小鼠突变亨廷顿蛋白（Htt）表达，或者在不同年龄时同时去阻遏或阻遏突变和正常Htt表达。 在目标1中，我们将描述 通过表征2- 24月龄时的行为、神经病理学和htt表达水平，抑制HdhLacO-140 Q/+、α-肌动蛋白-LacIR tg、HdhLacO-140 Q/LacO-20 Qhu、α-肌动蛋白-LacIR tg和对照小鼠在断奶、3-、6-和9-月龄时的突变型Htt或突变型和野生型Htt表达。此外，为了检查老年小鼠中去阻遏突变体htt表达的效果（模拟老年患者中基因治疗的中止），将IPTG施用至12月龄的HdhLacO-140 Q/+; β-肌动蛋白-LacIR tg小鼠。将在12至24月龄时表征其表型，并与对照组进行比较。 在目标2中，为了鉴定用于评估基因沉默疗法的功效的潜在生物标志物，我们提出通过RNA-seq表征在3月龄、6月龄和9月龄时突变型htt或突变型和野生型htt表达的抑制后在皮质和纹状体中发生的近端基因表达变化。候选基因的验证将首先用脑组织和培养的原代神经元进行。然后使用从突变htt阻遏前后的小鼠获得的血液样品进一步检查验证的基因。 总之，这些分析的结果应该有助于设计未来的基因沉默疗法，HD，并为我们的理解HD的发病机制。

 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.