A Novel Monkey Model for Parkinson's Drug Discovery

描述(申请人提供)：帕金森病(PD)是一种神经退行性疾病，以黑质(SN)多巴胺神经元(DAN)死亡、纹状体内多巴胺缺乏和临床运动障碍为特征。大多数帕金森病是散发性/特发性的，可能是基因-环境相互作用引起的。在家族性形式中，LRRK2基因突变是最常见的，导致10%以上的常染色体显性遗传性家族性帕金森病和3.6%的散发性病例。最常见的突变是G2019S，发生在这个2527个氨基酸的蛋白的激活域内。GTPase结构域(例如，R1441C)中的突变也与人类群体中的疾病有关。我们已经证明，将病毒载体运送到人类LRRK2-G2019S的小鼠黑质纹状体通路中，而不是人类野生型LRRK2，会导致黑质中显著的多巴胺能神经元丢失。考虑到需要开发更多可能在药物开发中更具预测性的无毒模型，我们计划将小鼠的观察扩展到非人类灵长类动物(NHP)。在这里，我们研究了HSV扩增导向的G2019S或R1441C在NHP纹状体内的表达是否将为药物开发建立一个有用的新的PD模型。我们的方法包括：1)HSV扩增子LRRK2突变型病毒载体的制备和鉴定。野生型LRRK2(LRRK2WT)、G2019S(LRRK2G2019S)、LRRK2激酶DEAD(LRRK2KD)和R1441C(Roc/GTPase结构域；LRRK2R1441C)将被亚克隆到共表达增强型绿色荧光蛋白(EGFP；HSVPrPuc/CMVegfp)的HSV扩增载体中。然后将这些载体包装成无辅助病毒的HSV扩增子载体，命名并进一步测试表达。2和3)新的药物发现PD模型的建立、验证和表征。对流增强的每个扩增载体单侧进入恒河猴纹状体，随后将进行18F-甲基酪氨酸PET成像和临床评定量表(CRS)关闭和使用左旋多巴和阿朴吗啡。在安乐死时，将采集大脑进行神经病理、神经化学和基因表达的组织学分析。4)LRRK2激酶抑制剂(KI)的翻译评价。对KI化合物文库的筛选产生了一个分子，GW5074，作为对LRRK2-G2019S具有特异性的候选分子。一种改进的新型化合物已经制备出来，并将进行进一步的评估。将进行ADME以确定有效剂量、给药时间表和代谢物。在以丹数为读数的单纯疱疹病毒扩增基因转导的小鼠模型中，所选剂量将被确认为神经保护。将在药效团上进行SAR，以获得优化的分子。针对LRRK2G2019S毒性的化合物神经保护证据将使NHP测试成为可能。该化合物将使用PET成像和临床评级进行评估。受试者将被实施安乐死，并采集大脑进行神经病理、神经化学和基因表达。其他器官将被采集用于未来的病理分析。血液将被采集用于未来的药物和代谢物分析。这项翻译工作的目的是支持IND。 公共卫生相关性：帕金森病(PD)是第二种最常见的神经退行性疾病，其特征是黑质多巴胺神经元(DAN)死亡。迫切需要新的无毒的帕金森病模型，这种模型在药物开发中可能更具预测性。我们建议通过过度表达各种形式的最常见的突变PD基因，将我们之前的小鼠观察扩展到非人类灵长类动物模型。该模型还将用于测试新型治疗性小分子LRRK2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.