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

模拟减少亨廷顿蛋白和 Hdh 选择性剪接对小鼠的影响

• 批准号: 8911911
• 负责人: Scott Zeitlin
• 金额: $ 34.56万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8911911
• 关键词: Actins; Adverse effects; Affect; Age; Age-Months; Alleles; Alternative Splicing; Antisense Oligonucleotides; Behavior; Behavioral; Biogenesis; Biological Markers; Blood; Blood specimen; Candidate Disease Gene; Cholesterol Homeostasis; Chronic; Clinical; Corpus striatum structure; Degradation Pathway; Disease; Disease Progression; Event; Fingers; Future; Gene Expression; Gene Expression Profile; Gene Silencing; Genes; Hereditary Disease; Huntington Disease; Intervention; Knock-in Mouse; Laboratories; Lac Repressors; Lactose; Membrane Protein Traffic; Methods; Microtubules; Mitochondria; Modeling; Monitor; Mouse Strains; Mus; Mutant Strains Mice; Mutation; Neurodegenerative Disorders; Neurons; Pathogenesis; Pathway interactions; Phenotype; RNA Interference; Repression; Stretching; Therapeutic; Therapeutic Intervention; Time; Tissues; Transgenic Mice; Trinucleotide Repeats; Validation; Viral Vector; Weaning; Withdrawal; aged; analog; base; brain tissue; cell motility; cilium biogenesis; design; drinking water; gain of function; gene therapy; human Huntingtin protein; loss of function; mouse model; mutant; neurogenesis; neuropathology; novel; nuclease; older patient; polyglutamine; pre-clinical; preclinical study; protein degradation; public health relevance; success; synaptic function; tissue culture; transcriptome sequencing; treatment strategy

 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.

 描述（由适用提供）：亨廷顿疾病（HD）是一种常染色体显性神经退行性疾病，是由CAG三重症重复膨胀编码Huntingtin（HTT）（HTT）（HTT）（HTT）（HTT）的蛋白质蛋白质产物中的一系列聚谷氨酰胺（PolyQ）引起的。 HD突变赋予了影响多种细胞途径的突变体HTT的有害功能和潜在的功能丧失。基因溶解是HD的有前途的理论策略，它可以规定寻找针对所有受突变HTT影响的细胞途径的治疗的挑战。为了确定减少突变体HTT表达以实现最大治疗益处的最佳时间，并评估如果无法实现突变体HTT等位基因的选择性靶向，我们建议使用新型的HD敲击鼠标模型（HDHLACO-140Q和HDHLACO-20QHU-20QHU鼠标），该模型已成为lectors（laC）的鼠标（HUD）。将这些小鼠与普遍表达LAC阻遏物（ß-Actin-Lacir-tg）的转基因小鼠交叉后，我们可以在不同年龄或戒断含水液在不同年龄的情况下全球抑制或反映突变体和正常表达的突变体和正常表达的小鼠突变体表达（HTT）表达（HTT）。在AIM 1中，我们将表征 在HDHLACO-140Q/+中，在断奶，3，6-和9个月的断奶，3，6-和9个月时，抑制突变体HTT或突变体和野生型HTT表达； ß-Actin-lacir TG，HDHLACO-140Q/LACO-20QHU; β-肌动蛋白 - lacir tg和通过表征2至24个月时代的HTT表达水平来控制小鼠。此外，为了检查抑制突变体HTT表达在老年患者中衰老的小鼠（建模）中断的效果），IPTG将用于12个月大的HDHLACO-140Q/+； ß-Actin-Lacir TG小鼠。它们的表型将在12至24个月的年龄和对照组中进行表征。在AIM 2中，为了确定潜在的生物标志物来评估基因沉默疗法的效率，我们建议通过RNA-Seq来表征proxy基因表达的变化在突变体HTT表达或突变体和野生型HTT表达后，在3--，6-，6-，6-，6-，6-，6-，6-，9-1个月的年龄表达表达后出现的代理基因表达变化。候选基因的验证将首先用脑组织和培养的原发性神经元进行。然后，将使用从小鼠突变htt表达之前和之后从小鼠获得的血液样本进一步检查经过验证的基因。总之，这些分析的结果应为HD的未来基因沉默疗法的设计以及我们对HD发病机理的理解做出贡献。