Dopamine Neuron Protection by Macrophage GDNF Delivery

描述（由申请人提供）：帕金森病（PD）在全球50岁以上人群中的患病率为1-2%。PD是由大脑的一个小区域黑质致密部（SN）中产生多巴胺的细胞死亡引起的。多巴胺是控制运动所必需的，低水平的多巴胺会导致帕金森的典型症状——震颤、自主运动缓慢、肌肉僵硬或僵硬、步履蹒跚、失去平衡、言语不清以及对他人的依赖性增加。现有的治疗方法并不令人满意。基因治疗有希望，但DNA的局部传递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身体的大多数组织，包括中枢神经系统，因此使它们成为基因传递的一个有吸引力的选择。在小鼠模型（PPCA-/-）中，通过表达人保护性蛋白/组织蛋白酶A （PPCA）转基因的骨髓源性巨噬细胞纠正了半乳糖唾液中毒（GS）。然而，由于研究中使用的CSF-1R启动子的缺陷，中枢神经系统的校正是不完整的。我们已经开发了一系列超级巨噬细胞启动子（SMP），在体外比CSF-1R启动子强100倍。在PD患者和模型动物中，局部递送胶质细胞系来源的神经营养因子（GDNF）已被发现是有益的。我们假设使用我们的超级巨噬细胞启动子可以实现GDNF在中枢神经系统的高效递送，这将在PD动物模型中提供多巴胺神经元保护。我们的具体目标是通过在SMP驱动的巨噬细胞/大胶质细胞中，用表达GDNF基因的慢载体在体外转导的HSC同基因移植，改善MPTP（1-甲基-4-苯基- 1,2,3,6-四氢吡啶）帕金森病小鼠模型的神经退行性变。骨髓干细胞将在体外用表达GDNF的慢病毒转导，并移植到受致死性辐照的受体小鼠中。骨髓移植5周后，受体小鼠将接受急性或慢性MPTP治疗。在MPTP后的选定时间点，将进行行为测试，并检查脑组织的多巴胺摄取和酪氨酸羟化酶（TH）的表达。对多巴胺能神经元进行计数，并通过TUNEL染色和活性caspase-3免疫组化评估细胞凋亡。该研究将为开发用于神经退行性疾病患者的潜在载体奠定基础。

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.