rAAV-mediated knockdown for Huntington's disease

亨廷顿病(HD)是一种常染色体显性遗传性进行性神经系统疾病。这个 神经病理症状似乎是由新纹状体的进行性功能障碍和死亡引起的 神经元和/或皮质纹状体束。HD基因于1993年首次被描述，该基因缺陷是 被识别为编码A基因的外显子1上一系列CAG重复序列的扩展 一种叫做亨廷顿蛋白的蛋白质。随后，在其他八个基因中也发现了类似的重复扩展 当三核苷酸扩增达到阈值时，已知会导致不同的神经疾病 每种疾病的典型症状。这些不同的障碍在概念上也是有联系的，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扩展突变中- 诱导的紊乱，突变蛋白似乎诱导异常的核蛋白聚集，称为 神经元核内包涵体。在HD中，最好的数据似乎表明，有毒的功能和 亨廷顿蛋白突变形式的一些正常功能的丧失会导致神经病理。因此，我们的建议 目的是使用腺相关重组腺病毒载体来传递编码构建物的基因以击倒 纹状体神经元中的亨廷顿蛋白。纹状体亨廷顿蛋白的长期击倒将检验这一假设 亨廷顿蛋白表达降低应限制转导后神经元核内包涵体的形成 从而延缓HD小鼠模型的疾病进展。我们将使用小干扰RNA (SiRNAs)击倒纹状体神经元中的亨廷顿蛋白。此外，我们还提出了两种方法来测试 假设正常的亨廷顿蛋白表达是有益的。首先，我们将比较狩猎的效果 在敲入HD小鼠和转基因HD小鼠中都存在敲除。由于转基因HD小鼠仍然表达 正常的老鼠狩猎量，我们的假说预测狩猎素击倒会更多 与仅具有突变Huntingtin的敲入HD小鼠相比，在转基因小鼠中有益 表情。其次，尚不清楚纹状体功能障碍是否起源于细胞自主方式。 纹状体神经元或由突变的亨廷顿蛋白引起的皮质-纹状体功能障碍。我们将直接测试 这一假说是通过转换皮层区域与纹状体区域来实现的。我们将使用纹状体数量的减少 神经元核包涵体和已知转录变化的逆转作为我们最初的有益筛选 随后对运动行为进行详细的功能分析，以此作为这些策略成功与否的衡量标准。

Huntington's disease (HD) is an autosomal dominant inherited progressive neurological disorder. The neuorpathological 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 leads 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 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 predicts 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, it is unclear whether striatal dysfunction originates in a cell autonomous fashion in striatal neurons or is caused by mutant huntingtin-induced cortico-striatal dysfunction. We will directly test this hypothesis by transducing cortical areas vs. striatal areas. We will use reduction in the amount of striatal neuronal nuclear inclusions and reversal of known transcriptional changes as our initial screen for beneficial effects followed by detailed functional analysis of motor behavior as metric of success of these strategies.