Long-term effects of wildtype huntingtin lowering in the primate corticostriatal tract and thalamus

项目总结/摘要 大多数在开发或临床试验中的降低亨廷顿蛋白的治疗对突变等位基因没有选择性， 也降低野生型亨廷顿蛋白表达。这个项目的目的是填补我们知识的关键空白 关于野生型亨廷顿蛋白在灵长类动物大脑中降低的影响， 与亨廷顿病（HD）和野生型亨廷顿蛋白功能相关的区域。小鼠数据 HD模型表明，降低皮质纹状体系统的皮质神经元中的亨廷顿蛋白可能 1 -3尽管先前的研究已经确定，降低野生型 在灵长类纹状体中，亨廷顿蛋白的45%在六个月内耐受良好，4这项研究和其他研究5没有 灵长类皮质纹状体神经元（其轴突包括皮质纹状体束）中野生型亨廷顿蛋白降低。 亨廷顿蛋白参与脑源性神经营养因子（BDNF）的产生和转运速率。 皮质神经元的纹状体，其中纹状体神经元依赖。8目前还不清楚是否减少野生型 在灵长类大脑的皮质纹状体神经元/束中的亨廷顿蛋白将不利地影响 纹状体神经元AIM 1将评估皮质纹状体中野生型亨廷顿蛋白降低的长期耐受性。 通过定量测量这种降低对自发活动、BDNF水平的影响， 和神经递质系统。作为研究工具，该项目使用了一种已知可以降低亨廷顿蛋白的shRNA 在灵长类动物中，4和已知的AAV血清型可以逆行地从纹状体神经元中分离皮质神经元。 9丘脑的治疗已被提出作为一种手段，加强载体分布， 大脑16.然而，在部分， 条件性敲除野生型亨廷顿蛋白。10 AIM 2将确定是否减少野生型亨廷顿蛋白， 灵长类动物丘脑会导致丘脑钙积累增加，无论是偶然发生， 载体的纹状体输注或直接通过靶向丘脑的载体输注。在研究中，30- 将两只（32）成年恒河猴平均分为4个实验组（N= 8只动物/组）， 随机分配接受MRI引导的编码亨廷顿蛋白降低shRNA或 对照shRNA，直接进入纹状体（AIM 1）或丘脑（AIM 2）。将通过以下方式评估猴 定期进行行为和神经检查。大脑皮层、纹状体和丘脑中的谷氨酸信号 将在AAV施用后9个月进行评估。尸检分析将包括评估 纹状体组织中的多巴胺周转和BDNF水平以及丘脑和神经病理学中的钙 冠状脑切片的评价，包括皮质、纹状体和丘脑。由此获得的数据 在非人类灵长类动物中的项目将提供无法从人类临床获得的信息。 亨廷顿蛋白降低治疗的试验。这些信息将与任何当前和未来的治疗有关， 使用非等位基因特异性手段降低亨廷顿蛋白的HD，无论药物或递送途径如何。

PROJECT SUMMARY / ABSTRACT Most huntingtin-lowering treatments in development or clinical trials are not selective for the mutant allele, but also lower wildtype huntingtin expression. The purpose of this project is to fill critical gaps in our knowledge about the effects of wildtype huntingtin lowering in the primate brain over an extended period of time in brain regions relevant to Huntington’s disease (HD) and to the functions of wildtype huntingtin. Data from mouse models of HD indicate that lowering huntingtin in cortical neurons that are part of the corticostriatal system may be necessary for optimal therapeutic efficacy.1-3 Although prior research has established that lowering wildtype huntingtin by 45% in the primate striatum is well-tolerated for six months,4 this and other research5 has not lowered wildtype huntingtin in primate corticostriatal neurons whose axons comprise the corticostriatal tract. Huntingtin is involved in the rate of production6 and transport7 of BDNF (brain-derived neurotrophic factor) from cortical neurons to the striatum, on which striatal neurons depend.8 It is unknown whether reduction of wildtype huntingtin in corticostriatal neurons/tract of the primate brain will adversely affect the survival and functioning of striatal neurons. AIM 1 will assess the long-term tolerability of wildtype huntingtin lowering in the corticostriatal tract over 9 months by quantitatively measuring the effects of this lowering on locomotor activity, BDNF levels and neurotransmitter systems. As research tools, the project uses an shRNA already known to lower huntingtin in the primate4 and a serotype of AAV already known to retrogradely transduce cortical neurons from a striatal point of infusion.9 Treatment of the thalamus has been proposed as a means of enhancing vector distribution in the brain16. However, pathologic calcification of the thalamus has been reported in mice 9 months after partial, conditional knockout of wildtype huntingtin.10 AIM 2 will determine if reduction of wildtype huntingtin in the primate thalamus will result in an increased thalamic calcium accumulation, whether occurring incidentally from striatal infusion of vectors or directly by vector infusion targeting the thalamus. In the proposed studies, thirty- two (32) adult rhesus monkeys will be equally divided into four experimental groups (N= 8 animals/group) and randomly assigned to receive MRI-guided injections of AAV6 encoding a huntingtin-lowering shRNA or a control shRNA, directly into either the striatum (AIM 1) or thalamus (AIM 2). Monkeys will be assessed by periodic behavioral and neurological exams. In vivo glutamate signaling in the cortex, striatum and thalamus will be assessed 9 months after AAV administration. Post-mortem analysis will include assessments of dopamine turnover and BDNF levels in striatal tissue as well as calcium in the thalamus and neuropathological evaluations of coronal brain sections, including cortex, striatum and thalamus. The data obtained by this project in the nonhuman primate will provide information that cannot be readily obtained from human clinical trials of huntingtin-lowering treatments. This information will be pertinent to any current and future therapies for HD that use non-allele-specific means to lower huntingtin irrespective of the agent or route of delivery.