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，例如溶酶体贮积病 涉及单核细胞谱系的细胞可能适合基因 治疗。多能干细胞是基因治疗的有吸引力的靶标 在此类疾病中。工程干细胞移植将导致 该基因终生持续存在于所有血细胞中 有机体的。然而，基因治疗有很多局限性。 造血干细胞。使用逆转录病毒的一项限制 用于基因转移的载体是原病毒需要细胞分裂 整合到靶细胞中。因此基因不能维持在 非复制细胞；例如休眠干细胞。第二个限制 是转移基因的持续高水平表达。单核细胞 非静止的前体细胞应该是更理想的靶细胞 与多能干细胞相比，基因治疗的人群。曾经 被感染后，这些细胞可以被移植到受体动物体内 它们会驻留在肝脏或脾脏（或其他组织）中并且 分化为表达所需基因的组织巨噬细胞。一把钥匙 问题是定型为单核细胞谱系的早期细胞是否可以 作为组织巨噬细胞的祖细胞并导致长期 在特定的组织部位植入。 该提案的具体目标是： 1. 确定组织巨噬细胞和小胶质细胞的动力学 骨髓移植后的植入。 这将完成 使用ROSA 26移植模型，可以检测个体 移植的细胞。 2. 确定是否存在除静止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。这些研究最初将由 ROSA 完成 26移植模型。后续研究将涉及使用 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（吃） 在产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.