Macrophage Gene Therapy of Neurodegenerative Diseases

描述(申请人提供)：神经退行性疾病影响了大量的患者。现有的治疗方法并不令人满意。基因治疗是有希望的，但DNA的集中传递和基因表达的水平是具有挑战性的。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。我们已经开发出一系列超巨噬细胞启动子(SMP)，它们在体外的强度是CSF-1R启动子的100倍。在高度流行的帕金森病(PD)模型中，胶质细胞系衍生神经营养因子(GDNF)的局部注射被发现是有益的。我们推测，使用我们的超巨噬细胞启动子可以实现高效的中枢神经系统传递GDNF，这将极大地改善帕金森病动物模型的病理变化和神经功能缺陷。至 探索这一假说，我们的具体目的是：1)通过慢病毒载体体外转导的骨髓干细胞移植和以增强型绿色荧光蛋白(EGFP)为报告基因的转基因小鼠，对这些超巨噬细胞启动子进行表征。对巨噬细胞具有最大强度和组织特异性的启动子将在随后的目标中使用。2)将体外转导的小鼠骨髓间充质干细胞与表达胶质细胞源性神经营养因子基因的慢载体联合移植到巨噬细胞/大胶质细胞中，以改善(1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine)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。骨髓移植四周后，受体小鼠皮下注射MPTP。在MPTP注射后的选定时间点，将进行PET扫描和行为测试，并将检查脑组织的多巴胺摄取和酪氨酸羟基酶(TH)的表达。对黑质致密部(SN)内的多巴胺能神经元进行计数，并用原位末端标记法和免疫组织化学方法检测细胞的凋亡情况。3)以与目标2相同的方式改善神经退行性变，但GDNF的表达将由四环素可调控的基因表达系统控制。为了评价巨噬细胞/超启动子介导的GDNF转导和表达对PD模型黑质纹状体神经元变性的影响，我们在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的表达。同样，在MPTP治疗和启动GDNF表达后的特定时间点，GDNF在多巴胺能神经保护、恢复和功能增强方面的作用将在特定目标2中被检测。这些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): 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.