Macrophage Gene Therapy of Neurodegenerative Diseases

描述（由申请人提供）：神经退行性疾病影响大量患者。现有的治疗方法并不令人满意。基因治疗有希望，但DNA的局部递送和基因表达水平具有挑战性。巨噬细胞从骨髓募集到身体的大多数组织，包括CNS，从而使它们成为基因递送的有吸引力的选择。在小鼠模型（PPCA-/-）中，通过表达人保护蛋白/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酸沉积症（GS）。然而，CNS中的校正是不完全的，部分原因是研究中使用的CSF-1 R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），其在体外比CSF-1 R启动子强100倍。在高度流行的帕金森病（PD）模型中，已发现局部递送胶质细胞系源性神经营养因子（GDNF）是有益的。我们假设，GDNF的高效CNS递送可以通过使用我们的超级巨噬细胞启动子来实现，并且这将极大地改善PD动物模型中的病理变化和神经缺陷。到 为了探索这一假说，我们的具体目标是：1）通过移植用慢病毒载体离体转导的骨髓干细胞和使用增强型绿色荧光蛋白（EGFP）作为报告基因的转基因小鼠来表征这些超级巨噬细胞启动子。具有最大强度和巨噬细胞组织特异性的启动子将用于后续目标。2)通过将用表达GDNF基因的慢病毒载体离体转导的HSC同基因移植到由SMP驱动的巨噬细胞/大胶质细胞中来改善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变性。骨髓干细胞将用表达GDNF的慢病毒离体转导，并移植到致死性照射的受体小鼠中。骨髓移植后四周，受体小鼠将皮下注射MPTP。在MPTP给药后的选定时间点，将进行PET扫描和行为测试，并检查脑组织的多巴胺摄取和酪氨酸羟化酶（TH）的表达。在黑质丘脑部（SN）中，将对多巴胺能神经元进行计数，并通过TUNEL染色和活性半胱天冬酶-3免疫组织化学评估细胞凋亡。3)以与目标2相同的方式改善神经变性，但GDNF的表达将由四环素可调控的基因表达系统控制。为了评估巨噬细胞/超级启动子介导的GDNF递送和表达对PD MPTP模型中退化的黑质纹状体神经元的影响，我们将在MPTP给药前和给药后不同时间（1、4、8、14、28天）通过四环素停药启动GDNF表达。同样，在MPTP处理后的特定时间点和GDNF表达的起始，GDNF对多巴胺能神经保护、恢复和功能增强的作用将如具体目标2中所述进行检查。这些研究将作为基础，开发潜在的用于神经退行性疾病患者的载体。

DESCRIPTION (provided by applicant): Neurodegenerative diseases affect a large population of patients. Existing therapies are not satisfactory. Gene therapy holds promise, but focal delivery of DNA and the level of gene expression are challenging. 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 l00-fold stronger in vitro than the CSF-1R promoter. In models of the highly prevalent Parkinson's disease (PD), 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 greatly ameliorate the pathologic changes and neurological defects in animal models of PD. To explore this hypothesis, our specific aims are: 1) To characterize these super macrophage promoters by transplantation of bone marrow stem cells transduced ex vivo with lentiviral vectors and in transgenic mice using EGFP (enhanced green fluorescent protein) as a reporter. Promoters with the greatest strength and tissue-specificity for macrophages will be used in the subsequent aims. 2) 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. Four weeks after bone marrow transplantation, the recipient mice will be injected subcutaneously with MPTP. At selected time points post MPTP administration, PET scan and behavioral testing will be performed, and brain tissue will be examined for dopamine uptake and expression of tyrosine hydroxylase (TH). In the substantia nigra pars compacta (SN), dopaminergic neurons will be counted and cell apoptosis will be assessed by TUNEL staining and immunohistochemistry for active easpase-3. 3) To ameliorate neurodegeneration in the same way as in Aim 2, but GDNF expression will be controlled by a tetracycline-regulatable gene expression system. To evaluate the effects of macrophage/ super promoter-mediated delivery and expression of GDNF on degenerating nigrostriatal neurons in the MPTP model of PD, we will initiate GDNF expression by tetracycline withdrawal before and at various times (1, 4, 8, 14, 28 days) after MPTP administration. Again, at specific time points post MPTP treatment and initiation of GDNF expression, GDNF effects on dopaminergic neuroprotection, restoration, and functional enhancement will be examined as in Specific Aim 2. These studies will serve as a basis for developing vectors for potential use in patients with neurodegenerative diseases.