PERIPHERAL BLOOD STEM CELL MEDIATED GENE TRANSFER

这一小额赠款(R03)提案将寻求确定使用 外周血干细胞作为基因表达的替代递送系统 到中枢神经系统，并解决了在 这是1VIH PA，“衰老中的干细胞、组织修复和细胞替换。” 神经保护策略旨在预防神经病理和 神经退行性疾病和衰老的行为后遗症可能是强有力的 因为它们可以在疾病来袭之前被介绍给患者 一连串不可逆转的终极事件。神经营养因子的细胞递送 使用转基因细胞是一种特别有吸引力的策略，它可能 提供已耗尽的重要营养因子的持续表达。那里 使用PBSC的理由有几个令人信服的优势。PBSC，输入 与骨髓来源的干细胞不同，它们很容易动员起来，而且 利用现有的技术可以很容易地通过静脉采集，避免了痛苦的， 与骨髓抽吸相关的更具侵入性的程序。临床上 应用基因转移策略，利用成熟的、未转化的神经细胞， 例如，在自体移植研究中的星形胶质细胞或神经干细胞患有 获取脑源性细胞不可避免的神经外科手术。 相比之下，PBSC可以在无需手术干预的情况下获得，并且代表着一种 潜在的可再生细胞来源，用于基因治疗的重复周期。这 研究将评估G-CSF动员的采集的CD34+浓缩的能力， 人外周血干细胞将在体外进行受控增殖和 向髓系干细胞系a(非淋巴干细胞系)和 最终走向单核/小胶质前体细胞谱系。培养的外周血干细胞- 来源的单核细胞/小胶质前体细胞将通过重组 表达选择(Neo)和标记(EGFP)基因和原型的逆转录病毒 编码睫状神经营养因子(CNTF)的神经营养因子基因。 然后，转导细胞将作为基因表达的替代传递系统 大脑和脊髓。受试者的最佳接生程序 将外周血干细胞移植到裸鼠脑内，导致持续 转基因的生存能力和构成表达将被定义。中枢神经系统PBSC 静脉输注、鞘内注射、脑室注射植入 注射和经脑内注射将被检查。美国人的命运 在裸鼠大脑中移植的、转导的干细胞将具有 关于转基因的细胞迁移和体内表达的模式。 外周血干细胞输送系统的成功演示 CNS将在设计影响组织的基因疗法中得到应用 衰老过程中的修复和细胞更替。

This small grant (R03) proposal will seek to establish the feasibility of using peripheral blood stem cell (PBSC) as surrogate delivery systems of gene expression to the central nervous -system and addresses Research Objective No. 5 outlined in this 1VIH PA, " Stem Cells, Tissue Repair and Cell Replacement in Aging." Neuroprotective strategies aimed at preventing the neuropathological and behavioral sequelae of neurodenerative disease and aging might be powerful therapeutically since they could be introduced to patients before the onslaught of a cascade of irreversible, terminal events. Cellular delivery of neurotrophic factors using genetically modified cells is a particularly attractive strategy that could provide sustained expression of a vital trophic factor that has been depleted. There are several compelling advantages for the rationale of using PBSC. PBSC, in contradistinction to bone marrow-derived stem cells, are easily mobilized and readily-harvested intravenously with existing technology and avoids the painful, more invasive procedure associated with bone marrow aspiration. Clinically applied gene transfer strategies that employ mature, non-transformed neural cells, e.g. astrocytes or neural stem cells in autologous transplantation studies suffer from the unavoidable requisite neurosurgical procedure to obtain the brain-derived cells. In contrast, PBSC can be acquired free of surgical intervention and represent a potentially renewable source of cells for repeated cycles of gene therapy. This study will evaluate the capacity of G-CSF-mobilized harvested CD34+-enriched, human peripheral blood stem cells to undergo controlled in vitro proliferation and expansion toward myeloid stem cell lines a (non-lymphoid stem cell lineage) and ultimately toward monocyte/microglial precursor cell lineage. The cultured PBSC- derived monocyte/microglial precursor cells will be transduced by recombinant retroviruses to express selection (Neo) and marker (EGFP) genes and a protype neurotrophic factor gene that encodes ciliary neurotrophic factor (CNTF).The transduced cells will then serve as surrogate delivery systems of gene expression to the brain and spinal cord. The optimal procedure for delivery o transduced human peripheral blood stem cells to the brains of nude rats that results in sustained viability and constitutive expression of the transgenes will be defined. CNS PBSC implantation via intravenous infusion, intrathecal injection, intraventricular injection and by intracerebral injection will be examined. The fate of the transplanted, - transduced stem cells in the nude rat brain will be characterized with regard to the pattern of cellular migration and in vivo expression of the transgenes. The successful demonstration of a peripheral blood stem cell delivery system to the CNS will have road application in the design of gene therapies to effect tissue repair and cell replacement in aging.