IMMORTALIZED NEURAL PRECURSORS FOR GENE THERAPY & REPAIR

与其他器官一样，CNS功能障碍的2个研究领域 （特别是由于遗传性代谢或神经遗传性疾病） 最近融合：神经祖细胞生物学和CNS通过 移植通过逆转录病毒介导的基因转移，我们以前 产生永生化的、克隆的、多能的鼠神经祖细胞 线对它们的体外分化潜能的检测表明， 在个体祖先的水平上具有巨大的可塑性。后 移植，这些永生化的祖细胞嫁接在非- 致瘤性，细胞结构和功能上适当的方式， 在体内重现其多能性并通过逆转录病毒表达- 在脑实质内以稳健的方式转导基因， 长时间的因为血脑屏障限制了 到外周供应的酶的进入（直接或通过 基因工程体细胞），因为骨髓 移植需要对发育中的CNS有害的辐射， 中枢神经系统表现的遗传性疾病的外周治疗已经 失望将基因产物直接递送到CNS将避免 这样的问题。（为这种目的移植初级胎儿组织， 许多生物和伦理问题）。我们的数据表明 移植永生化的神经祖细胞 表达缺失的基因产物，或者通过基因工程来表达缺失的基因产物， 持续治疗中枢神经系统表现的策略 疾病，和/或作为CNS的组成部分进行修复 细胞结构这一建议试图建立一个范例， 神经前体细胞移植治疗中枢神经系统损伤 目的：（1）证实一个给定的克隆祖细胞 线可以移植（技术上容易）并正常参与 沿着神经轴和在多个神经节处的多个结构的发展 从胚胎到成年的阶段，祖细胞可以 分化成多种细胞类型，以响应占主导地位的空间 和时间线索，并表达其逆转录病毒转导的报告基因。 (This将极大地拓宽这种不朽的应用 前体）。(2)确保持续有效的植入和安全性 受体动物通过识别指导移植的变量， 确定原位移植细胞的性质和命运， 表达转基因。移植的细胞可以在体内追踪和表征; 它们可以在体外回收和重新检查;它们的效率 可以量化植入与基因表达;它们对宿主的影响 大脑可以评估。(Such特征化在 考虑人类的类似策略）。(3)将外源性 基因或因子，通过将表达的前体移植到 中枢神经系统的神经内脏疾病的原型小鼠模型，其中 这个基因的缺陷已经被确定了。(4)将祖细胞移植到 细胞类型特异性神经变性的损伤或突变小鼠模型 以确定它们是否会整合到CNS中并呈现表型 缺陷细胞类型的细胞。试验数据证明了每一种方法的可行性。 瞄准

As in other organs, 2 areas of inquiry into CNS dysfunction (particularly that due to inherited metabolic or neurogenetic disease) have recently converged: neural progenitor cell biology & CNS via transplantation. Through retrovirus-mediated gene transfer, we previously generated immortalized, clonal, multipotent murine neural progenitor lines. Examination of their differentiation potential in vitro suggested enormous plasticity at the level of the individual progenitor. Upon transplantation, these immortalized progenitors engrafted in a non- tumorigenic, cytoarchitecturally & functionally appropriate manner, recapitulating their multipotency in vivo & expressing retrovirally- transduced genes in a robust fashion within brain parenchyma for prolonged periods. Because the blood-brain barrier imposes restrictions to entry of enzyme supplied peripherally (either directly or through genetically-engineered somatic cells) & because bone marrow transplantation entails irradiation which is inimical to developing CNS, peripheral treatment of CNS manifestations of genetic disease has been disappointing. Delivery of gene products directLy to CNS would circumvent such problems. (Grafting primary fetal tissue for such purposes poses numerous biologic & ethical concerns). Our data suggest the feasibility of transplanting immortalized neural progenitors constituitively expressing missing gene products, or genetically engineered to do so, as a strategy for sustained therapy of CNS manifestations of neurovisceral disease, &/or to effect repair as integral members of CNS cytoarchitecture. This proposal attempts to establish a paradigm of neural precursor transplantation as a therapy for CNS insult through 4 aims: (1) Confirm preliminary findings that a given clonal progenitor line can engraft (with technical ease) & participate normally in the development of multiple structures along the neuraxis & at multiple stages spanning from embryo to adult, that the progenitors can differentiate into multiple cell types in response to prevailing spatial & temporal cues, & express their retrovirally-transduced reporter gene. (This would broaden enormously the applications of such an immortalized precursor.) (2) Insure consistently efficient engraftment & safety of recipient animals by identifying the variables which direct engraftment, & determining the properties & fate of cells in situ that do engraft & express transgenes. Grafted cells can be traced & characterized in vivo; they can be retrieved & re-examined in vitro; their efficiency of engraftment vs gene expression can be quantified; their impact on host brain can be assessed. (Such characterization is crucial prior to consideration of similar strategies in humans.) (3) Transfer an exogenous gene or factor, via transplantation of an expressing precursor, into the CNS of a prototypical mouse model of neurovisceral disease in which a defect in that gene has been defined. (4) Transplant progenitors into a lesioned or mutant mouse model of a cell type-specific neurodegeneration to determine if they will integrate into the CNS & assume the phenotype of the deficient cell-type. Pilot data attest to the feasibility of each aim.