Dopamine Neuron Protection by Macrophage GDNF Delivery

描述(申请人提供)：帕金森氏病(PD)在全球50岁以上人群中的患病率为1%-2%。帕金森病是由黑质致密部(SN)中产生多巴胺的细胞死亡引起的，SN是大脑的一个小区域。控制运动需要多巴胺，低水平的多巴胺会导致帕金森氏症的典型症状-震颤、自主运动缓慢、肌肉僵硬或僵硬、步态不稳、失去平衡、说话含糊和对他人的依赖增加。现有的治疗方法并不令人满意。基因治疗是有希望的，但DNA的局部传递和基因表达水平是具有挑战性的问题。巨噬细胞从骨髓被招募到身体的大多数组织，包括中枢神经系统，因此使它们成为一个有吸引力的基因传递选择。在小鼠模型(PPCA-/-)中，通过表达人保护性蛋白/组织蛋白酶A(PPCA)转基因的骨髓来源的巨噬细胞，纠正了Galactosialidsis(GS)。然而，中枢神经系统的校正是不完整的，部分原因是研究中使用的CSF-1R启动子的弱点。我们已经开发了一系列超级巨噬细胞启动子(SMP)，它们在体外的强度是CSF-1R启动子的100倍。在帕金森病患者和模型动物中，胶质细胞系衍生神经营养因子(GDNF)的局部应用已被发现是有益的。我们推测，使用我们的超级巨噬细胞启动子可以实现高效的中枢神经系统传递GDNF，这将为帕金森病动物模型提供多巴胺神经元保护。我们的具体目的是通过将体外转导的HSC与SMP驱动的表达GDNF基因的慢病毒载体在巨噬细胞/大胶质细胞中进行同种异基因移植，改善1-甲基-4-苯基-1，2，3，6-四氢吡啶(MPTP)帕金森病小鼠模型的神经变性。骨髓干细胞将在体外转导表达慢病毒的GDNF，并移植到致死性照射的受体小鼠体内。骨髓移植五周后，接受移植的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，并将检查脑组织对多巴胺的摄取和酪氨酸羟化酶(TH)的表达。将对多巴胺能神经元进行计数，并通过TUNEL染色和免疫组织化学方法检测活化的caspase-3来评估细胞凋亡。这项研究将作为开发潜在用于神经退行性疾病患者的载体的基础。

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.