KINETICS OF MICROGLIA--IMPLICATIONS FOR GENE THERAPY

基因治疗是将遗传物质转移到哺乳动物细胞中， 治疗疾病。许多疾病如溶酶体贮积病， 涉及单核细胞谱系的细胞可能有助于基因 疗法多能干细胞是基因治疗的有吸引力的靶点 在这些疾病中。移植工程干细胞将导致 在所有血细胞中持续存在的基因， 生物体。然而，基因治疗在临床上有许多局限性。 造血干细胞使用逆转录病毒的一个限制是 用于基因转移的载体的一个重要特征是前病毒需要细胞分裂 整合到靶细胞中。因此，基因不能维持在 非复制细胞;如静止干细胞。第二限制 是转移基因的持续高水平表达。单核细胞 不静止的前体细胞应该是更理想的靶细胞 多能干细胞的基因治疗。一旦 感染后，这些细胞可以移植到受体动物体内， 它们会在肝脏或脾脏（或其他组织）中定居， 分化成表达所需基因的组织巨噬细胞。一个关键 问题是，是否早期细胞致力于单核细胞谱系， 作为组织巨噬细胞的祖细胞， 在特定组织部位的植入。 这项建议的具体目标是： 1. 确定组织巨噬细胞和小胶质细胞的动力学 骨髓移植后的移植。 为此将 使用ROSA 26移植模型，其可以检测个体 移植细胞 2.为了确定除了静止的造血干细胞以外的细胞 可以作为小鼠单核细胞和组织巨噬细胞的祖细胞 移植模型这些研究最初将与ROSA一起完成 26例移植模型。以后的研究将涉及使用 逆转录病毒载体作为基因治疗的模型。 3.将这些研究扩展到临床前大型动物模型（EAT） 在出生后和子宫内使用逆转录病毒标记的细胞 移植系统。

Gene therapy is the transfer of genetic material into mammalian cells to treat disease. Numerous diseases such as lysosomal storage diseases which involve cells of the monocytic lineage could be ammenable to gene therapy. Pluripotent stem cells are attractive targets for gene therapy in such diseases. Transplantation of engineered stem cells would result in the continuous presence of the gene in all blood cells for the life of the organism. However, there are many limitations to gene therapy in hematopoietic stem cells. One restriction to the use of retroviral vectors for gene transfer is that cell division is required for proviral integration into the target cell. Thus genes can not be maintained in nonreplicating cells; such as quiescent stem cells. A second limitation is the continued high level expression of the transferred gene. Monocytic precursors that are not quiescent should be a more desirable target cell population for gene therapy than the pluripotent stem cell. Once infected, these cells could be transplanted into recipient animals where they would take up residence in liver or spleen (or other tissues) and differentiate into tissue macrophages expressing the desired gene. A key question is whether early cells committed to the monocytic lineage can serve as progenitors of tissue macrophages and result in long term engraftment at specific tissue sites. The specific aims of this proposal are: 1. To determine the kinetics of tissue macrophage and microglial engraftment following bone marrow transplantation. This will be done using the ROSA 26 transplantation model which can detect individual transplanted cells. 2. To determine if cells other than quiescent hematopoietic stem cells can serve as progenitors of monocytes and tissue macrophages in a mouse transplantation model. These studies will initially be done with the ROSA 26 transplantation model. Later studies will involve the use of retroviral vectors as a model of gene therapy. 3. To extend these studies to a preclinical large animal model (eat) using retrovirally marked cells in both postnatal and in utero transplantation systems.