Non-alledle specific RNAi as a potential therapy for Huntington's disease

非等位基因特异性 RNAi 作为亨廷顿病的潜在疗法

基本信息

批准号：
7489861
负责人：
Jodi L. McBride
金额：
$ 4.96万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-11 至 2009-08-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7489861
关键词：
Address Adenine Affect Alleles Antibodies Atrophic Behavioral Binding Biochemical Brain CAG repeat Cells Cessation of life Chromosomes, Human, Pair 4 Cognitive Corpus striatum structure Cytosine Data Disease Dissection Distress Evaluation Exons Gene Expression Gene Mutation Genes Genetic Polymorphism Glial Fibrillary Acidic Protein Glutamine Guanine Harvest Histologic Huntington Disease ITGAM gene In Vitro Inclusion Bodies Individual Inherited Injection of therapeutic agent Interferons Knock-in Mouse Lasers Life Link Longitudinal Studies Messenger RNA Microglia Modeling Molecular Motor Mus Neurodegenerative Disorders Neurons Northern Blotting Nucleotides Pathway interactions Patients Polymerase Chain Reaction Process Proteins RNA RNA Interference RNA Interference Pathway RNA-Induced Silencing Complex Research Proposals Series Testing Tissue Stains Tissues Toxic effect Transcript Transcriptional Activation Trinucleotide Repeat Expansion Up-Regulation Ventricular Viral Week Weight Western Blotting Work astrogliosis base design gain of function gait examination gene repression human Huntingtin protein improved in vivo motor disorder mouse model mutant neuropathology prevent research study response small hairpin RNA tool vector

项目摘要

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal dominantly-inherited, neurodegenerative disorder caused by an expanded trinucleotide repeat (CAG) at the IT15 locus on chromosome 4. These repeats cause degeneration of striatal and cortical neurons in the brain resulting in a devastating cognitive, psychiatric, and motor disorder. RNA interference (RNAi) has emerged as a tool to decrease diseased gene expression. RNAi therapy involves the delivery of short hairpin RNAs (shRNAs) to affected cells which bind to and decrease the expression of target mRNA. It is a therapy extremely well-suited for diseases such as HD, which arise exclusively from a single, inherited gene mutation. To date, allele-specific silencing of mutant huntingtin (htt) remains unsuccessful because no RNAi-targetable polymorphism representing a large fraction of HD patients has been identified. Therefore, partial reduction of both the normal and the diseased allele should be tested as a therapy for HD. Preliminary studies in our lab demonstrate that short-term (4 months post-injection) adeno-associated viral delivery of three different shRNAs to the striata of CAG 140 mice results in an equal 50% partial reduction of htt. While all three hairpins were efficacious in partially reducing htt, two of the three hairpins induced striatal toxicity while the third did not. Toxicity was evidenced histologically as striatal atrophy and ventricular enlargement seen on Nissl and DARPP-32 stained tissue along with a robust increase in astrogliosis and a microglial response. The aims of this proposal are two- fold: 1) To address the question of why two of the three hairpins caused striatal toxicity even though they were designed using the same set of rules and were injected into the mouse striata at the same volume and titer. I hypothesize that, based on preliminary in vitro data, inappropriate strand biasing (loading both the sense and antisense strands of the hairpin) also occurs, in vivo, following viral delivery of the two toxic hairpins (sh2.4 and sh30.1), while only the correct antisense strand is loaded following viral delivery of the non-toxic hairpin (sh8.2); 2) To perform a long-term study using the non-toxic hairpin assessing the efficacy of partially reducing the expression of both htt alleles in the CAG 140 knock-in mouse as an extension of our preliminary data. Thorough behavioral, histological, molecular, and biochemical analysis will reveal whether a partial reduction of both alleles remains efficacious 10 months post-injection. Together, these two studies will address the molecular mechanisms behind differential cellular responses to viral injection of hairpins into the striatum and whether our non-toxic candidate hairpin is efficacious in the long-term, both of which are critical in developing RNAi as a therapy for HD, and more broadly, assessing RNAi in the mammalian brain.

描述（由申请人提供）：亨廷顿病 (HD) 是一种常染色体显性遗传的神经退行性疾病，由 4 号染色体 IT15 基因座上的三核苷酸重复序列 (CAG) 引起。这些重复序列会导致大脑中纹状体和皮质神经元的变性导致毁灭性的认知、精神和运动障碍。 RNA 干扰 (RNAi) 已成为减少患病基因表达的工具。 RNAi 疗法涉及将短发夹 RNA (shRNA) 递送至受影响的细胞，与目标 mRNA 结合并降低其表达。这种疗法非常适合 HD 等疾病，这些疾病完全由单一遗传基因突变引起。迄今为止，突变亨廷顿蛋白 (htt) 的等位基因特异性沉默仍然不成功，因为尚未鉴定出代表大部分 HD 患者的 RNAi 靶向多态性。因此，应该测试正常和患病等位基因的部分减少作为 HD 的治疗方法。我们实验室的初步研究表明，短期（注射后 4 个月）腺相关病毒将三种不同的 shRNA 递送至 CAG 140 小鼠的纹状体，可导致 htt 部分减少 50%。虽然所有三个发夹都能有效部分减少 htt，但三个发夹中的两个会诱导纹状体毒性，而第三个则不会。组织学上证明了毒性，尼氏和 DARPP-32 染色组织中可见纹状体萎缩和脑室扩大，同时星形胶质细胞增生和小胶质细胞反应显着增加。该提案的目的有两个：1）解决为什么三个发夹中的两个会引起纹状体毒性的问题，尽管它们是使用相同的规则集设计的，并且以相同的体积和滴度注射到小鼠纹状体中。我假设，根据初步的体外数据，在体内病毒递送两个有毒发夹（sh2.4和sh30.1）后，也会发生不适当的链偏向（加载发夹的有义链和反义链），而在病毒递送无毒发夹（sh8.2）后仅加载正确的反义链； 2) 使用无毒发夹进行长期研究，评估部分降低 CAG 140 敲入小鼠中两个 htt 等位基因表达的功效，作为我们初步数据的延伸。彻底的行为、组织学、分子和生化分析将揭示两个等位基因的部分减少在注射后 10 个月是否仍然有效。这两项研究将共同探讨病毒将发夹注射到纹状体时细胞反应差异背后的分子机制，以及我们的无毒候选发夹是否长期有效，这两个问题对于开发 RNAi 作为一种治疗方法至关重要。 HD，更广泛地说，评估哺乳动物大脑中的 RNAi。