A Novel Monkey Model for Parkinson's Drug Discovery

描述（由申请人提供）：帕金森病（PD）是一种神经退行性疾病，其特征在于黑质（SN）多巴胺神经元（DAN）的死亡、纹状体内的多巴胺缺乏和临床运动障碍。大多数PD是散发性/特发性的，可能由基因-环境相互作用引起。在家族性形式中，LRRK 2中的突变是最常见的，导致超过10%的常染色体显性遗传家族性PD和3.6%的散发病例。最常见的突变G2019 S发生在这个2527个氨基酸的蛋白质的激酶结构域内。GT3结构域内的突变（例如，R1441 C）也与人类疾病有关。我们已经表明，病毒载体递送至小鼠黑质纹状体途径的人LRRK 2-G2019 S，而不是人野生型LRRK 2，导致SN中显著的多巴胺能神经元损失。鉴于需要开发可能在药物开发中更具预测性的其他无毒模型，我们计划将小鼠观察扩展到非人灵长类动物（NHP）。在此，我们研究了HSV扩增子介导的G2019 S或R1441 C在NHP纹状体中的表达是否会建立一个有用的新型PD模型用于药物发现。我们的方法包括：1）HSV扩增子LRRK 2突变体病毒载体的产生和表征。将野生型LRRK 2（LRRK 2 WT）、G2019 S（LRRK 2G 2019 S）、LRRK 2激酶死亡（LRRK 2KD）和R1441 C（Roc/GTK结构域; LRRK 2 R1441 C）亚克隆到共表达增强型绿色荧光蛋白（eGFP; HSVPrPuc/CMVegfp）的HSV扩增子载体中。然后将其包装到无辅助病毒的HSV扩增子载体中，滴定并进一步检测表达。2和3）用于药物发现的新型PD模型的建立、验证和表征。将每个扩增子载体单向地对流增强递送到恒河猴的纹状体中，随后纵向进行18氟甲基酪氨酸PET成像和临床评定量表（CRS），停用和停用左旋多巴和阿朴吗啡。在安乐死时，将收获大脑用于神经病理学、神经化学和基因表达的组织学分析。4)LRRK 2激酶抑制剂（KI）的翻译评价。KI化合物库的筛选产生了一种分子GW 5074，作为对LRRK 2-G2019 S具有特异性的候选物。已经制备了一种改进的新化合物，并将进一步评价。将进行ADME以确定活性剂量、给药方案和代谢产物。使用DAN数作为读数，将确认所选剂量在HSV扩增子转导的小鼠模型中具有神经保护作用。将对药效团进行SAR以得到优化的分子。针对LRRK 2G 2019 S毒性的化合物神经保护的证据将使NHP测试成为可能。将使用PET成像和临床评级评价化合物。将对受试者实施安乐死，并收获大脑进行神经病理学、神经化学和基因表达。将收获其他器官用于未来的病理学分析。将采集血液用于未来的药物和代谢物分析。这项翻译工作旨在使IND成为可能。 公共卫生相关性：帕金森病（PD）是第二常见的神经退行性疾病，其特征在于黑质多巴胺神经元（DAN）的死亡。目前非常需要新型无毒的PD模型，这些模型可能在药物开发中更具预测性。我们建议通过过度表达各种形式的最常见的突变PD基因，将我们以前的小鼠观察扩展到非人灵长类动物模型。该模型还将用于测试新型治疗性小分子LRRK 2抑制剂。

DESCRIPTION (Provided by Applicant): Parkinson's disease (PD) is a neurodegenerative disorder characterized by death of substantia nigra (SN) dopamine neurons (DANs), dopamine deficiency within the striatum and a clinical movement disorder. Most PD is sporadic/idiopathic that may arise from gene-environmental interactions. Among familial forms, mutations in the LRRK2 are most common contributing to over 10% of autosomal dominant familial PD and 3.6% of sporadic cases. The most frequent mutation, G2019S, occurs within the kinase domain of this 2527 amino acid protein. Mutations within the GTPase domain (e.g., R1441C) have also been associated with disease in the human population. We have shown that viral vector delivery to the mouse nigrostriatal pathway of human LRRK2-G2019S, but not human wild-type LRRK2, results in marked dopaminergic neuron loss in the SN. Given the need to develop additional non-toxicant models that may possibly be more predictive in drug development, we plan to extend the murine observations to the non-human primate (NHP). Herein, we examine whether HSV amplicon-directed expression of G2019S or R1441C in the striatum of NHPs will establish a useful and novel PD model for drug discovery. Our approach involves: 1) Production and characterization of HSV amplicon LRRK2 mutant viral vectors. Wild-type LRRK2 (LRRK2WT), G2019S (LRRK2G2019S), LRRK2 kinase dead (LRRK2KD) and R1441C (Roc/GTPase domain; LRRK2R1441C) will be subcloned into HSV amplicon vectors that co-express enhanced green fluorescent protein (eGFP; HSVPrPuc/CMVegfp). These will then be packaged into helper virus-free HSV amplicon vectors, titred and further tested for expression. 2 and 3) Establishment, Validation and Characterization of a novel PD model for drug discovery. Convection-enhanced delivery of each amplicon vector unilaterally into the striatum of rhesus macaque monkeys, which will be followed longitudinally by 18fluoro-methyl tyrosine PET imaging and a Clinical Rating Scale (CRS) off and on levodopa and apomorphine. At euthanasia brains will be harvested for histological analysis of neuropathology, neurochemistry and gene expression. 4) Translational evaluation of a LRRK2 Kinase Inhibitor (KI). Screening of a library of KI compounds has yielded one molecule, GW5074, as a candidate with specificity against LRRK2-G2019S. An improved novel compound has been prepared and will be further evaluated. ADME will be undertaken to establish an active dose, schedule of administration and metabolites. The selected dose will be confirmed as neuroprotective in the HSV amplicon transduced mouse model using DAN number as a readout. SAR will be undertaken on the pharmacophore to derive an optimized molecule. Evidence of compound neuroprotection against LRRK2G2019S toxicity will enable NHP testing. The compound will be evaluated using PET imaging and clinical rating. Subjects will be euthanized and brains harvested for neuropathology, neurochemistry and gene expression. Other organs will be harvested for future pathological analyses. Blood will be collected for future drug and metabolite analyses. This translational effort is intended to be IND-enabling. Public Health Relevance: Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by death of substantia nigra dopamine neurons (DANs). There is a great need for novel non-toxicant models of PD that may possibly be more predictive in drug development. We propose to extend our previous murine observations to non-human primate models by overexpressing various forms of the most commonly mutated PD gene. This model will also be used to test novel therapeutic small molecule LRRK2 inhibitors.