rAAV-mediated knockdown for Huntington's disease

描述（由申请人提供）：亨廷顿舞蹈病（HD）是一种常染色体显性遗传的进行性神经系统疾病。神经病理症状似乎是由新纹状体神经元和/或皮质纹状体束的进行性功能障碍和死亡引起的。HD基因于1993年首次被描述，该基因缺陷被确定为编码一种名为亨廷顿蛋白的基因外显子1上CAG重复序列超过阈值水平的扩展。随后，当三核苷酸扩增达到每种疾病的典型阈值时，在其他八个基因中发现了类似的重复扩增，这些基因已知会导致不同的神经系统疾病。这些不同的疾病在概念上也有联系，因为像HD一样，症状是由中枢神经系统中特定神经元的功能障碍引起的。此外，在所有CAG扩增突变诱导的疾病中，突变蛋白似乎诱导异常核蛋白聚集，称为神经元核内包涵体。在亨廷顿舞蹈症中，最好的数据似乎表明，在亨廷顿舞蹈症突变形式中，功能的毒性增加和一些正常功能的丧失都会导致神经病理。因此，我们的建议旨在利用重组腺相关病毒载体传递基因编码结构来敲除纹状体神经元中的亨廷顿蛋白。纹状体亨廷顿蛋白的长期敲低将验证亨廷顿蛋白表达降低应限制转导神经元核内包涵体的形成，从而减缓HD小鼠模型疾病进展的假设。我们将使用核糖酶和小干扰rna （sirna）来敲除纹状体神经元中的亨廷顿蛋白。此外，我们提出了两种方法来检验正常亨廷顿蛋白表达是有益的假设。首先，我们将比较敲除亨廷顿蛋白对敲除HD小鼠和转基因HD小鼠的影响。由于转基因HD小鼠仍然表达正常数量的小鼠亨廷顿蛋白，我们的假设可以预测，与只表达突变亨廷顿蛋白的敲入HD小鼠相比，转基因小鼠的亨廷顿蛋白敲除将更有益。其次，我们将同时传递敲除纹状体亨廷顿蛋白的载体和表达亨廷顿蛋白的载体，该载体被设计成对敲入动物的核酶或干扰RNA具有抗性，作为我们假设的更临床相关的测试。我们将使用纹状体神经元核包涵体数量的减少和已知转录变化的抑制作为我们对有益效果的初步筛选，然后对运动行为进行详细的功能分析，作为该策略成功的度量。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal dominantly inherited progressive neurological disorder. The neuropathological symptoms appear to be caused by progressive dysfunction and death of neostriatal neurons and/or the cortico-striatal tract. The HD gene was first described in 1993 and the gene defect was identified as an expansion of a series of CAG repeats above a threshold level in exon 1 of a gene encoding a protein called huntingtin. Subsequently, similar repeat expansions have been identified in eight other genes that are known to cause different neurological disorders when the trinucleotide expansions reach thresholds typical for each disease. These different disorders are also conceptually linked because, like HD, symptoms are caused by the dysfunction of specific neurons in the CNS. Moreover, in all CAG expansion mutation-induced disorders, the mutant protein seems to induce abnormal nuclear protein aggregations termed neuronal intranuclear inclusions. In HD, the best data seem to indicate that both a toxic gain of function and a loss of some normal function in the mutant form of huntingtin lead to neuropathology. Thus, our proposal aims to use recombinant adeno-associated viral vectors to deliver genes encoding constructs to knock-down huntingtin in striatal neurons. Long-term knock-down of striatal huntingtin will test the hypothesis that reduced huntingtin expression should limit the formation of neuronal Intranuclear inclusions in transduced neurons and thereby slow disease progression in mouse models of HD. We will use both RNA-cleaving enzymes called ribozymes and small interfering RNAs (siRNAs) to knock-down huntingtin in striatal neurons. Moreover, we propose two ways to test the hypothesis that normal huntingtin expression is beneficial. First, we will compare the effects of huntingtin knock-down in both knock-in HD mice and transgenic HD mice. Since transgenic HD mice still express the normal amount of mouse huntingtin, our hypothesis would predict that huntingtin knock-down will be more beneficial in the transgenic mice compared to the knock-in HD mice that have only mutant huntingtin expression. Second, we will simultaneously deliver the vectors that knock-down striatal huntingtin with a vector that expresses a version of huntingtin that is engineered to be resistant to either the ribozymes or the interfering RNA in the knock-in animals as a more clinically relevant test of our hypothesis. We will use reduction in the amount of striatal neuronal nuclear inclusions and inhibition of known transcriptional changes as our initial screen for beneficial effects followed by detailed functional analysis of motor behavior as metric of success of this strategy.