Novel Re-engineered L DOPA probiotic therapy for Parkinsons Disease

摘要 帕金森氏病(PD)表现出的复杂而漫长的病程首先始于非 运动障碍，然后慢慢发展为轻度到中度的运动障碍，最终造成严重的 运动障碍和认知能力下降。黑质纹状体多巴胺能致多巴胺缺乏症 神经元损伤最终表现为主要的锥体外系运动症状，如僵直，运动迟缓， 震颤和姿势不稳。这项提案解决了当前反政府武装面临的最大挑战之一 用多巴胺前体L多巴替代多巴胺治疗帕金森病。目前，口服片 每天服用L多巴/卡比多巴3-4次仍然是最有效和耐受性良好的治疗方法， 显著改善帕金森病早期患者的运动症状和生活质量。然而，由于 由于其非连续、脉冲式的脑部给药，L-多巴长期给药引起 有害的副作用，包括L-多巴诱导的运动障碍(LID)和其他运动并发症，在 大多数患者。在没有严重L-多巴相关运动的情况下实现持续的症状缓解 并发症，包括运动障碍，我们建议系统性地传递基因工程的染色体- 整合和可调节的L-多巴产生益生菌将避免血浆L-多巴水平的波动 并将L-多巴更稳定地输送到大脑，在那里它可以转化为连续的供应 黑质纹状体通路中的多巴胺。因此，我们的目标是系统地评估治疗的可行性。 基于微生物组的L多巴持续给药平台的临床疗效 运动症状不会引起严重的运动障碍。这项工作的科学前提是有关键支撑的 初步数据表明：1)基因重组、染色体整合和可调控 L-多巴产生菌Nissle1917菌株(EcNrhaL-DOPA)在体外和体外均高效生产L-多巴 2)口服EcNL-DOPA很容易在小鼠体内定植 Gut，达到与PD临床有效血浆水平相对应的稳态血浆L-多巴水平 并增加脑内L多巴胺和多巴胺的水平。为了进一步扩展我们的小说初步 结果，我们将追求以下具体目标：R61阶段(I)测定口服的剂量-反应效应 ECNrhaL-DOPA对两种动物肠道定植及其药代动力学和适应性的影响 C57BL和MitoPark小鼠；R33期(II)测定EcNrhaL-DOPA对MitoPark和MitoPark小鼠的治疗效果 6-羟基多巴胺损毁的小鼠帕金森病模型，以及(Iii)确定微生物L-多巴的持续释放 在两种盖子的鼠标模型中防止盖子。我们新的治疗流水线策略包括慢性给药 益生菌L多巴有望改变多巴胺能治疗帕金森病的方式。

Abstract The complex and prolonged disease course exhibited by Parkinson’s disease (PD) first starts with non- motor disturbances and then slowly progresses to mild-to-moderate motor deficits, ultimately inflicting severe movement impairment and cognitive decline. Dopamine deficiency resulting from nigrostriatal dopaminergic neuronal damage ultimately manifests as the cardinal extrapyramidal motor symptoms of rigidity, bradykinesia, tremors, and postural instability. This proposal addresses one of the greatest challenges facing the current anti- Parkinsonian therapy of dopamine replacement with the dopamine precursor L-DOPA. Currently, oral tablet dosing of L-DOPA/carbidopa 3-4 times/day remains the most effective and well-tolerated treatment, one that significantly improves the motor symptoms and quality of life of patients in the early stages of PD. However, due to its non-continuous, pulsatile delivery of L-DOPA to the brain, long-term L-DOPA administration causes deleterious side effects, including L-DOPA-induced dyskinesia (LID) among other motor complications, in the majority of patients. To achieve sustained symptomatic relief without severe L-DOPA-associated motor complications, including dyskinesia, we propose that systemic delivery of genetically engineered, chromosome- integrated, and regulatable L-DOPA-producing probiotic bacteria will avoid fluctuations in plasma L-DOPA levels and provide a more consistent delivery of L-DOPA to the brain where it can be converted to a continuous supply of dopamine in the nigrostriatal pathway. Thus, we aim to systematically evaluate the treatment feasibility and efficacy of this novel microbiome-based platform for the continuous delivery of L-DOPA in relieving motor symptoms without inducing severe dyskinesia. The scientific premise of the work is supported by key preliminary data demonstrating that: 1) the genetically reengineered, chromosome-integrated, and regulatable L-DOPA-producing E. coli Nissle 1917 probiotic strain (EcNrhaL-DOPA) efficiently produce L-DOPA both in vitro and in vivo than the older plasmid-based system, and 2) oral administration of EcNL-DOPA readily colonizes the mouse gut, achieves a steady-state plasma L-DOPA level that corresponds to the clinically effective plasma level in PD patients, and increases L-DOPA and dopamine levels in the brain. To further expand our novel preliminary results, we will pursue the following specific aims: R61 phase (i) determine the dose-response effect of orally administered EcNrhaL-DOPA on gut colonization as well as its pharmacokinetic and adaptation profiles in both C57BL and MitoPark mice; R33 phase (ii) determine the therapeutic efficacy of EcNrhaL-DOPA in the MitoPark and 6-OHDA-lesioned mouse models of PD, and (iii) determine whether sustained delivery of microbial L-DOPA prevents LID in two mouse models of LID. Our novel therapeutic pipeline strategy involving chronic delivery of probiotic L-DOPA is expected to transform the dopaminergic therapeutic modalities for PD.