Dopamine Neuron Protection by Macrophage GDNF Delivery

巨噬细胞 GDNF 传递对多巴胺神经元的保护

• 批准号: 6936457
• 负责人: SENLIN LI
• 金额: $ 5.99万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-15 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6936457
• 关键词: Parkinson' s disease; behavior test; cytoprotection; dopamine; gene therapy; glia; hematopoietic tissue transplantation; immunocytochemistry; intracellular transport; laboratory mouse; macrophage; methylphenyltetrahydropyridine; nervous system disorder therapy; neural degeneration; neurons; neuroprotectants; neurotrophic factors; oxygenases; stem cell transplantation; terminal nick end labeling; tissue /cell culture; transfection /expression vector; ultracentrifugation; western blottings

DESCRIPTION (provided by applicant): Parkinson's disease (PD) has a prevalence of 1-2% worldwide in people over the age of 50. PD results from the death of dopamine-producing cells in the substantia nigra pars compacta (SN), a small region of the brain. Dopamine is required to control movement and low levels of dopamine result in the typical symptoms of Parkinson's - tremor, slowness of voluntary movement, muscle stiffness or rigidity, shuffling gait, loss of balance, slurred speech and increasing dependence on others. Existing therapies are not satisfactory. Gene therapy holds promise, but focal delivery of DNA and the level of gene expression are challenging problems. Macrophages are recruited from bone marrow to most tissues of the body including the CNS, thus making them an attractive option for gene delivery. Galactosialidosis (GS) has been corrected by bone marrow-derived macrophages expressing human protective protein/cathepsin A (PPCA) transgene in a mouse model (PPCA-/-). However, correction in the CNS was incomplete due in part to weakness of the CSF-1R promoter used in the study. We have developed a series of super macrophage promoters (SMP) that are up to 100-fold stronger in vitro than the CSF-1R promoter. In PD patients and model animals, local delivery of glial cell line-derived neurotrophic factor (GDNF) has been found beneficial. We hypothesize that highly effective CNS delivery of GDNF can be achieved with the use of our super macrophage promoters and this will provide dopamine neuron protection in animal models of PD. Our specific aim is to ameliorate neurodegeneration in the MPTP (1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease by syngeneic transplantation of HSC transduced ex vivo with lentivectors expressing GDNF gene in macrophages/macroglia driven by the SMP. Bone marrow stem cells will be transduced ex vivo with GDNF expressing lentivirus and transplanted into lethally irradiated recipient mice. Five weeks after bone marrow transplantation, the recipient mice will be subject to either acute or chronic MPTP treatment. At selected time points post MPTP, behavioral testing will be performed, and brain tissue will be examined for dopamine uptake and expression of tyrosine hydroxylase (TH). Dopaminergic neurons will be counted and cell apoptosis will be assessed by TUNEL staining and immunohistochemistry for active caspase-3. The study will serve as a basis for developing vectors for potential use in patients with neurodegenerative diseases.

描述（由申请人提供）：帕金森氏病（PD）在50岁以上的人中，全球患病率为1-2％。PD是由于大脑小区域的Nigra Pars Compacta（SN）的多巴胺产生细胞的死亡而导致的。需要多巴胺来控制运动和低水平的多巴胺会导致帕金森氏症的典型症状 - 震颤，自愿运动的缓慢，肌肉僵硬或僵化，步态降解，平衡丧失，言语流失和对其他人的依赖。现有疗法并不令人满意。基因疗法有望，但是DNA的焦点递送和基因表达水平是具有挑战性的问题。巨噬细胞从骨髓募集到人体的大多数组织，包括中枢神经系统，从而使其成为基因递送的有吸引力的选择。在小鼠模型（PPCA - / - ）中，表达人保护蛋白/组织蛋白酶A（PPCA）转基因的骨髓来源的巨噬细胞（PPCA）校正了半乳糖菌病（GS）。但是，CNS的校正是由于研究中使用的CSF-1R启动子的弱点而不完整的。我们已经开发了一系列超级巨噬细胞启动子（SMP），它们在体外比CSF-1R启动子高100倍。在PD患者和模型动物中，已发现局部分娩神经胶质细胞系的神经营养因子（GDNF）有益。我们假设使用超级巨噬细胞启动子可以实现高效的GDNF中枢神经系统递送，这将在PD动物模型中提供多巴胺神经元保护。 Our specific aim is to ameliorate neurodegeneration in the MPTP (1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease by syngeneic transplantation of HSC transduced ex vivo with lentivectors expressing GDNF gene in macrophages/macroglia driven by the SMP.骨髓干细胞将用GDNF表达慢病毒，并将其移植到致命的辐照受者小鼠中。骨髓移植后五周，受体小鼠将接受急性或慢性MPTP治疗。在MPTP后选定的时间点，将进行行为测试，并将检查脑组织的多巴胺摄取和酪氨酸羟化酶（TH）的表达。将计算多巴胺能神经元，并通过主动caspase-3进行TUNEL染色和免疫组织化学评估细胞凋亡。这项研究将作为开发媒介在神经退行性疾病患者中潜在使用的基础。