Abstract The development of a neuroprotective therapy for Parkinson's disease (PD) would be a major therapeutic advance, particularly for our veterans. Glial cell line-derived neurotrophic factor (GDNF) has been shown to be the most potent protective trophic molecule to dopamine (DA) neurons affected in PD. GDNF requires CNS delivery as it does not cross the blood-brain barrier (BBB). Unfortunately, clinical trials of GDNF therapy in PD patients have given mixed results. It is challenging to deliver therapeutic levels of GDNF to all degenerating nigrostriatal neurons using traditional surgical approaches due to the large target area in the human brain and the poor brain tissue penetration of this molecule. We have developed a novel approach capable of resolving these problems: hematopoietic stem cell (HSC) transplantation (HSCT)-based macrophage-mediated GDNF delivery. This unique approach takes advantage of macrophages’ natural property of homing to sites of neuronal degeneration, capitalizes on our powerful macrophage-specific synthetic promoter (MSP), and implements recent advances in HSC gene therapy. Our previous work using conventional HSCT reproducibly demonstrated the effectiveness of our approach in various (acute and chronic, and neurotoxin induced and genetic) mouse models of PD. However, conventional HSCT requires toxic preconditioning such as irradiation and thus may not be suitable for our veterans with PD. In the last funding cycle, we introduced our newly developed novel non-cytotoxic HSCT method. Using this novel system, we again demonstrated that genetically engineered HSC-derived macrophages infiltrate the brain parenchyma and accumulate at diseased sites to provide sustained focal GDNF delivery that leads to dramatically reduced degeneration of DA neurons in the substantia nigra of PD mice. Critically, this protection ultimately results in amelioration of motor and non-motor dysfunction and is achievable with little apparent adverse effects. In this grant application, we propose to study the dose-effect relationship of this novel HSCT-based approach to identify key correlates of protection and the optimum therapeutic dose. The efficacy of this novel intervention will be validated in an additional model of PD. Finally, single-cell RNAseq sequencing will be applied to delineate the molecular and cellular mechanisms underlying this novel neuroprotective therapy. The proposed study will provide another important step in the development of our innovative disease-modifying treatment for PD.