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

项目摘要/摘要 大多数开发或临床试验中的降低亨廷顿蛋白的治疗方法对突变的等位基因没有选择性，但 野生型亨廷顿蛋白的表达也较低。这个项目的目的是填补我们知识中的重大空白。 关于野生型亨廷顿蛋白在灵长类动物大脑中长期降低的影响 与亨廷顿病(HD)和野生型亨廷顿蛋白功能相关的区域。来自鼠标的数据 HD的模型表明，降低皮质纹状体系统中皮质神经元中的亨廷顿蛋白可能 是最佳治疗效果所必需的。1-3尽管先前的研究已经证实，降低野生型 灵长类纹状体中45%的猎杀丁素在六个月内耐受性良好，4这项研究和其他研究5还没有 灵长类皮质纹状体神经元中野生型亨廷顿蛋白降低，其轴突组成皮质纹状体束。 亨廷顿蛋白参与脑源性神经营养因子的产生和转运。 纹状体神经元依赖的皮质神经元。8尚不清楚野生型是否减少 灵长类大脑皮质纹状体神经元/束中的亨廷顿蛋白将对动物的生存和功能产生不利影响 纹状体神经元。目标1将评估野生型Huntingtin在皮质纹状体降低的长期耐受性 通过定量测量这种降低对运动活动、脑源性神经营养因子水平的影响，在9个月内追踪 和神经递质系统。作为研究工具，该项目使用了一种已知的可降低狩猎素水平的shRNA 在灵长类4和一种已知的AAV血清型中，已知的AAV可从纹状体逆行转换皮质神经元 9丘脑的治疗已被认为是增强病媒分布的一种手段。 大脑。然而，据报道，小鼠部分脑缺血9个月后，丘脑出现病理性钙化， 野生型亨廷顿的条件敲除。10目标2将确定野生型亨廷顿蛋白在 灵长类丘脑会导致丘脑钙蓄积增加，无论是偶然发生在 纹状体输注载体或直接以丘脑为靶点的载体输注。在拟议的研究中，30- 将两(32)只成年恒河猴平均分为四个试验组(N=8只/组)和 随机分配接受MRI引导下注射编码亨廷顿蛋白降低型shRNA的AAV6或 控制shRNA，直接进入纹状体(AIM 1)或丘脑(AIM 2)。猴子将被评估为 定期进行行为和神经学检查。大脑皮质、纹状体和丘脑中的谷氨酸信号 将在接种AAV后9个月进行评估。尸检分析将包括对 纹状体组织中多巴胺代谢和脑源性神经营养因子水平以及丘脑和神经病理学中的钙 对脑冠状切片进行评估，包括皮质、纹状体和丘脑。通过此方法获得的数据 非人灵长类动物的项目将提供从人类临床上不容易获得的信息 亨廷顿降蛋白治疗的试验。这一信息将与任何当前和未来的治疗方法相关 HD使用非等位基因特异性的方法来降低亨廷顿蛋白，而不考虑毒剂或递送途径。

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.