rAAV-mediated knockdown for Huntington's disease

描述（申请人提供）：亨廷顿病（HD）是一种常染色体显性遗传的进行性神经系统疾病。神经病理学症状似乎是由进行性功能障碍和死亡的新纹状体神经元和/或皮质-纹状体束。HD基因于1993年首次被描述，该基因缺陷被鉴定为编码亨廷顿蛋白的基因的外显子1中一系列CAG重复序列超过阈值水平的扩展。随后，在其他八个基因中发现了类似的重复扩增，当三核苷酸扩增达到每种疾病的典型阈值时，这些基因已知会引起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CNS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，突变蛋白似乎诱导称为神经元核内包涵体的异常核蛋白聚集。在HD中，最好的数据似乎表明，亨廷顿蛋白突变形式的毒性功能获得和一些正常功能的丧失都会导致神经病理学。因此，我们的建议旨在使用重组腺相关病毒载体，提供基因编码结构敲低亨廷顿蛋白在纹状体神经元。纹状体亨廷顿蛋白的长期敲低将检验以下假设：降低的亨廷顿蛋白表达应限制转导神经元中神经元核内包涵体的形成，从而减缓HD小鼠模型中的疾病进展。我们将使用核糖酶和小干扰RNA（siRNA）来敲低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测试的假设，正常的亨廷顿蛋白表达是有益的。首先，我们将比较亨廷顿蛋白敲低在敲入HD小鼠和转基因HD小鼠中的作用。由于转基因HD小鼠仍然表达正常量的小鼠亨廷顿蛋白，我们的假设将预测，亨廷顿蛋白敲低将是更有益的转基因小鼠相比，敲入HD小鼠，只有突变亨廷顿蛋白表达。第二，我们将同时提供载体，敲低纹状体亨廷顿蛋白与载体，表达一种版本的亨廷顿蛋白，被改造为耐核酶或干扰RNA在敲入动物作为一个更临床相关的测试我们的假设。我们将使用纹状体神经元核内含物的量的减少和已知的转录变化的抑制作为我们的有益效果的初始筛选，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.