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.

Narrative: Dopamine replacement therapy with the dopamine precursor L-DOPA remains the gold standard treatment for Parkinson's disease (PD), as it dramatically improves motor performance and the quality of life of patients in the early stages of the disease. However, due to L-DOPA’s pulsatile delivery following oral tablet administration, chronic L-DOPA treatment is often associated in most patients with deleterious side effects known as L-DOPA-induced dyskinesia (LID), and therefore, a more practical, patient-friendly, non-invasive therapy for stabilizing a patient’s plasma L-DOPA level is urgently needed. The main goals of this proposal are to develop a novel, tightly regulated and tunable microbiome-based platform for the non-pulsatile delivery of L- DOPA produced by genetically engineered probiotics and to evaluate its efficacy to alleviate symptoms and prevent the development of LID in preclinical animal models of PD.