PERIPHERAL BLOOD STEM CELL MEDIATED GENE TRANSFER

这项小额赠款（R 03）提案将寻求建立使用 外周血干细胞（PBSC）作为基因表达的替代递送系统 研究目标5概述， 这1VIH PA，“干细胞，组织修复和细胞替代老化。" 神经保护策略旨在预防神经病理学和 神经退行性疾病和衰老的行为后遗症可能是强大的 治疗上，因为它们可以在一种病毒的攻击之前引入患者。 一系列不可逆的终末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， 提供已经耗尽的重要营养因子的持续表达。那里 使用PBSC的理由有几个引人注目的优点。方案支助和预算支助中心， 与骨髓来源的干细胞相反， 通过现有技术容易地静脉内获得，并且避免了痛苦， 与骨髓抽吸相关的更具侵入性的手术。临床 应用基因转移策略，采用成熟的，非转化的神经细胞， 例如自体移植研究中的星形胶质细胞或神经干细胞遭受 获得脑源性细胞的不可避免的必要神经外科手术。 相比之下，PBSC可以在没有手术干预的情况下获得，并且代表了一种免疫学治疗。 潜在的可再生细胞来源，用于基因治疗的重复循环。这 本研究将评价G-CSF动员的收获的富含CD 34+的， 人外周血干细胞进行受控的体外增殖， 向骨髓干细胞系a（非淋巴干细胞谱系）扩增， 最终朝向单核细胞/小胶质前体细胞谱系。培养的PBSC- 衍生的单核细胞/小胶质细胞前体细胞将被重组转导 逆转录病毒，以表达选择（Neo）和标记（EGFP）基因和原型 睫状神经营养因子是编码睫状神经营养因子（CNTF）的神经营养因子基因。 转导的细胞将作为基因表达的替代传递系统， 大脑和脊髓转基因人的最佳递送程序 外周血干细胞移植到裸鼠的大脑中， 将定义转基因的活力和组成型表达。CNS PBSC 通过静脉输注、鞘内注射、脑室内植入 将检查注射和脑内注射。的命运 在裸大鼠脑中移植的、转导的干细胞的特征在于： 关于转基因的细胞迁移和体内表达的模式。 外周血干细胞输注系统的成功示范， CNS将在基因治疗的设计中有道路应用， 修复和细胞替换。

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.