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.
Federoff, Howard, J. 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. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page
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