Macrophage Gene Therapy of Neurodegenerative Diseases

神经退行性疾病的巨噬细胞基因治疗

• 批准号: 6919823
• 负责人: SENLIN LI
• 金额: $ 27.01万
• 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: 2004-07-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6919823
• 关键词: Parkinson' s disease; age difference; bone marrow transplantation; central nervous system; cysteine endopeptidases; disease /disorder model; dopamine; enzyme activity; gene delivery system; gene expression; gene therapy; genetically modified animals; green fluorescent proteins; hematopoietic stem cells; immunocytochemistry; laboratory mouse; macrophage; methylphenyltetrahydropyridine; neural degeneration; neurotrophic factors; nonhuman therapy evaluation; substantia nigra; tetracyclines; transfection; tyrosine 3 monooxygenase

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.

描述（由申请人提供）：神经退行性疾病会影响大量患者。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具有挑战性。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），它们的体外强度高于CSF-1R启动子。在高度普遍的帕金森氏病（PD）的模型中，发现局部细胞系衍生的神经营养因子（GDNF）的局部递送有益。我们假设使用超级巨噬细胞启动子可以实现高效的GDNF的中枢神经系统传递，这将大大减轻PD动物模型中的病理变化和神经缺陷。到 探讨这一假设，我们的具体目的是：1）通过使用EGFP（增强的绿色荧光蛋白）作为报告剂，用慢病毒载体移植了骨髓干细胞和在转基因小鼠中转导的这些超巨噬细胞启动子。随后的目标将使用具有最大强度和组织特异性的启动子。 2）通过在帕金森氏病的MPTP（1-甲基-4-苯基1,2,3,6-四氢吡啶）小鼠模型中通过HSC转导的HSC转导的帕金森氏病小鼠模型，该模型通过表达GDNF基因的借离基因/macroglogages in Macroglogages in Macroglagages intervivo tendivotors tendive。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。骨髓移植后四个星期，将从MPTP皮下注入受体小鼠。在MPTP给药后选定的时间点，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化酶（TH）的表达。在黑质Nigra Pars Cmpacta（SN）中，将计算多巴胺能神经元，并通过TUNEL染色和免疫组织化学评估细胞凋亡，以进行活性EAPPase-3。 3）以与AIM 2相同的方式改善神经变性，但是GDNF表达将由四环素调节基因表达系统控制。为了评估巨噬细胞/超级启动子介导的GDNF的递送和表达对PD的MPTP模型中的染色性纹状体神经元的反应，我们将在MPTP给药后在不同时间和在不同时间之前和在不同时间启动Tetracycline撤回GDNF表达（1、4、8、14、28天）。同样，在MPTP治疗后的特定时间点和GDNF表达的开始，GDNF对多巴胺能神经保护，恢复和功能增强的影响将被检查与特定目标2一样。这些研究将作为开发媒介的基础，以在神经减退性疾病患者中使用潜在使用。