Novel Re-engineered L DOPA probiotic therapy for Parkinsons Disease

摘要 帕金森病（PD）表现出的复杂和延长的病程首先始于非帕金森病。 运动障碍，然后缓慢发展为轻度至中度运动障碍，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引起的多巴胺缺乏症 神经元损伤最终表现为强直，运动迟缓， 震颤和姿势不稳该提案解决了当前反-- 用多巴胺前体左旋多巴替代多巴胺的帕金森病治疗。目前，口服片剂 L-DOPA/卡比多巴3-4次/天的剂量仍然是最有效和耐受性良好的治疗， 显著改善PD早期患者的运动症状和生活质量。但由于 由于L-DOPA向大脑的非连续、脉冲式递送， 有害的副作用，包括左旋多巴诱导的运动障碍（LID）以及其他运动并发症， 大多数患者。达到持续的症状缓解，而不会出现严重的左旋多巴相关运动 并发症，包括运动障碍，我们建议全身性递送基因工程，染色体- 整合和可调节的L-DOPA生产益生菌将避免血浆L-DOPA水平的波动 并向大脑提供更稳定的左旋多巴输送， 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 以及这种新型的基于微生物组的平台用于持续递送左旋多巴以缓解 运动症状而不引起严重的运动障碍。工作的科学前提是关键支撑 初步数据表明：1）基因重组，染色体整合，可调控， 产L-多巴的E.大肠杆菌Nissle 1917益生菌菌株（EcNrhaL-DOPA）在体外和体外均有效地产生L-DOPA， 和2）口服给予EcNL-DOPA容易定殖小鼠 肠，达到稳态血浆L-DOPA水平，其对应于PD中的临床有效血浆水平 患者，并增加L-DOPA和多巴胺在大脑中的水平。为了进一步扩大我们的小说初步 结果，我们将追求以下具体目标：R61阶段（i）确定口服的剂量反应效应 施用EcNrhaL-DOPA对肠道定殖的影响以及其在两种动物中的药代动力学和适应性特征。 C57 BL和MitoPark小鼠; R33阶段（ii）确定EcNrhaL-DOPA在MitoPark和MitoPark小鼠中的治疗功效。 6-OHDA-损伤的PD小鼠模型，和（iii）确定是否持续递送微生物L-DOPA 在两种LID小鼠模型中预防LID。我们的新型治疗管道策略涉及慢性递送 益生菌L-DOPA有望改变PD的多巴胺能治疗方式。

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.