IMMORTALIZED NEURAL PRECURSORS FOR GENE THERAPY & REPAIR

用于基因治疗的永生化神经前体

• 批准号: 2771949
• 负责人: EVAN Y SNYDER
• 金额: $ 25.59万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2771949
• 关键词: Gaucher's disease; cell differentiation; cell line; central nervous system; clone cells; disease /disorder model; gene expression; gene therapy; histology; laboratory mouse; mucopolysaccharidosis; nervous system transplantation; neural degeneration; neurogenesis; neurogenetics; tissue /cell culture

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.

与其他器官一样，CNS功能障碍的2个调查区域 （特别是由于遗传的代谢或神经遗传疾病） 最近融合了：神经祖细胞生物学和CNS 移植。通过逆转录病毒介导的基因转移，我们先前 产生永生的，克隆的，多能鼠神经祖细胞 线。表明其在体外的分化潜力的检查 在单个祖细胞水平上的巨大可塑性。之上 移植，这些永生的祖细胞植入了非 - 肿瘤症，细胞结构和功能合适的方式， 概括其在体内的多能力，并逆转录病毒 在脑实质内以强大的方式转导基因 长时间。因为血脑屏障施加限制 向外围提供的酶进入（直接或直接通过 遗传工程的体细胞）和因为骨髓 移植需要辐照，这与发展中枢神经系统无关 CNS遗传疾病表现的外围治疗一直是 令人失望。将基因产品直接传递到中枢神经系统 这样的问题。 （用于此目的的原代胎儿组织构成 许多生物学和道德问题）。我们的数据表明可行性 未永生的神经祖细胞 表达丢失的基因产品或经过基因工程来这样做，例如 CNS持续治疗神经守望液的策略 作为中枢神经系统不可或缺的成员，疾病和/或进行维修 细胞结构。该提议试图建立 神经前体移植作为中枢神经系统损伤的治疗 目的：（1）确认给定克隆祖细胞的初步发现 线可以（技术轻松）植入并正常参加 开发沿着神经和多种结构的多个结构 从胚胎到成人的阶段，祖细胞可以 响应流行空间而分化为多种细胞类型 和时间提示，并表达其逆转录病毒转导的记者基因。 （这将大大扩大这种永生的应用 （2）确保持续有效的植入和安全 接受动物通过识别直接植入的变量， 并确定植入型细胞的特性和命运 快递转基因。接枝细胞可以在体内追踪和表征； 它们可以在体外检索和重新检查；他们的效率 植入与基因表达可以进行量化；他们对主机的影响 可以评估大脑。 （这种表征在之前至关重要 考虑人类类似策略。）（3）转移外源性 通过将表达前体移植到基因或因子中 神经疾病的原型小鼠模型的中枢神经系统，其中 该基因的缺陷已被定义。 （4）移植祖细胞 细胞类型特异性神经变性的病变或突变小鼠模型 确定它们是否会集成到中枢神经系统中并假定表型 缺陷的细胞类型。飞行员数据证明了每个的可行性 目的。