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的局部传递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。在高度流行的帕金森病（PD）模型中，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，这将极大地改善PD动物模型的病理改变和神经缺陷。来

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.