IMMORTALIZED NEURAL PRECURSORS FOR GENE THERAPY & REPAIR

与其他器官一样，中枢神经系统功能障碍的两个调查领域 (尤其是因遗传性代谢或神经遗传疾病引起的疾病) 最近融合在一起：神经前体细胞生物学和中枢神经系统 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、克隆性、多潜能的小鼠神经前体细胞 台词。对它们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。vt.在.的基础上 移植后，这些永生化的祖细胞被植入非 致瘤，细胞结构和功能上适当的方式， 概述它们在体内的多能性并逆转录病毒表达- 在脑实质内以强健的方式转导基因 很长的时间。因为血脑屏障限制了 进入外周供应的酶(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中枢神经系统有害的辐射， 外周治疗表现为中枢神经系统遗传性疾病 令人失望。将基因产品直接运送到CNS将绕过 这样的问题。(为此目的移植原始胎儿组织的姿势 许多生物和伦理问题)。我们的数据表明这是可行的 有组织地移植永生化神经前体细胞 表达缺失的基因产物，或通过基因工程做到这一点，就像 中枢神经内脏表现的持续治疗策略 疾病，和/或作为CNS不可或缺的成员进行修复 细胞结构。这项提议试图建立一种 神经前体移植治疗中枢神经系统损伤 目的：(1)确认初步发现一个给定的克隆祖细胞 LINE可以(在技术上容易地)嫁接并正常参与 沿神经轴的多种结构的发育&在多个 从胚胎到成体的各个阶段，祖先可以 根据流行的空间情况区分为多种细胞类型 和时间线索，并表达其逆转录病毒转导的报告基因。 (这将极大地扩大这种不朽的 前身)(2)确保一贯有效的嫁接和安全 受体动物通过识别指导植入的变量， 确定进行移植的原位细胞的特性和命运(&D) 表达转基因。移植的细胞可以在体内追踪和表征； 它们可以在体外被取回和重新检查；它们的效率 嫁接对基因表达的影响可以量化；它们对宿主的影响 可以对大脑进行评估。(这样的特征在 在人类中考虑类似的策略。)(3)转移一个外源 基因或因子，通过将表达的前体移植到 神经内脏病典型小鼠模型的中枢神经系统 该基因的缺陷已被定义。(4)将祖细胞移植到 细胞类型特异性神经变性的受损或突变小鼠模型 确定它们是否会整合到CNS中，并假定其表型 有缺陷的细胞型。试点数据证明了每种方法的可行性 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.