HUMAN GC GENE TRANSDUCTION INTO HEMATOPOIETIC CELLS

人类 GC 基因转导至造血细胞

• 批准号: 2148441
• 负责人: FRIEDRICH G SCHUENING
• 金额: $ 21.4万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2148441
• 关键词: Gaucher's disease; Macaca nemestrina; dogs; gene therapy; genetic transduction; glucosylceramidase; hematopoietic stem cells; human genetic material tag; human subject; tissue /cell culture; transfection /expression vector

Gaucher's disease is a debilitating sphingolipid storage disorder affecting approximately 30,000 patients in the United States. The goal of this project is to develop improved gene transduction methods which would allow to cure this disease by expressing functional levels of glucocerebrosidase (GC) in macrophages of Gaucher's disease patients by transducing their hematopoietic repopulating cells with a human GC gene. We want to develop more efficient in vitro transduction methods for hematopoietic stem cells, improve conditioning and ex vivo and in vivo selection strategies in the dog model and finally confirm our results in a small number of primates before proceeding to a clinical application in humans. The experiments outlined will be the basis for an ensuing clinical study designed not only to cure Gaucher's disease, but also to serve as a model for gene therapy of other human diseases. It can be estimated that at least ten percent of a Gaucher patient's macrophages must express the GC gene in order to obtain a GC activity sufficient to improve disease manifestations. For that reason, retroviral transduction and engraftment of transduced hematopoietic repopulating cells have to be made more efficient. To achieve both, we first propose to improve the in vitro maintenance, expansion and transduction of hematopoietic repopulating cells. This will involve strategies to determine optimal exposure times to retrovirus vector supernatant and to develop optimal ex vivo growth conditions for stem cells to proliferate and integrate efficiently vector DNA without loss of self-renewal capacity. Second, we propose to generate safe, high-titer retrovirus GC vectors with high affinity for canine and primate cells. Specifically, we have produced a single-gene-expressing GC vector (PG13/LgGC) and will generate two-gene-expressing GC vectors that coexpress selectable genes. Third, we propose studies in dogs on how to increase the percentage of transduced repopulating cell clones that engraft. To accomplish this aim, we will compare different conditioning regimens that reduce the amount of endogenous repopulating cells prior to the infusion of transduced cells, and we will develop ex vivo and in vivo selection strategies for cells expressing a selectable marker gene in order to reduce the number of non- transduced cells. Finally, in preparation for treating patients, we propose to test in a nonhuman primate model the transduction and transplantation strategies found to be most effective. The outlined studies will address basic questions of applied gene therapy and stem cell biology and accelerate the application of these disciplines towards the treatment of disease.

戈谢病是一种使人衰弱的鞘脂储存障碍 影响了美国约3万名患者。的目标 该项目旨在开发改进的基因转导方法， 通过表达功能水平的 高雪氏病患者巨噬细胞中的葡萄糖脑苷脂酶（GC）， 用人GC基因转导其造血再生细胞。 我们希望开发更有效的体外转导方法， 造血干细胞、改善的调节以及离体和体内 选择策略，并最终证实了我们的结果， 在进行临床应用之前， 人类 概述的实验将是随后的实验的基础。 这项临床研究不仅旨在治愈戈谢病， 作为其他人类疾病基因治疗的模型。 据估计，至少有百分之十的戈谢病患者的 巨噬细胞必须表达GC基因以获得GC活性 足以改善疾病的表现。因此，逆转录病毒 转导的造血重建的转导和植入 必须使电池更有效率。为了实现这两个目标，我们首先建议 改善细胞的体外维持、扩增和转导， 造血再生细胞 这将涉及战略， 确定逆转录病毒载体上清液的最佳暴露时间， 开发干细胞增殖的最佳离体生长条件 并有效整合载体DNA而不丧失自我更新能力 容量第二，我们建议产生安全、高滴度的逆转录病毒GC 对犬和灵长类动物细胞具有高亲和力的载体。我们特别 已经产生了表达单基因的GC载体（PG 13/LgGC），并且将 产生共表达可选择基因的双基因表达GC载体。 第三，我们建议在狗身上研究如何增加 转导重新繁殖移植的细胞克隆。为了实现这一目标， 我们将比较不同的调节方案，减少量的 在输注转导细胞之前内源性再增殖细胞， 我们将开发体外和体内细胞选择策略， 表达选择性标记基因，以减少非选择性标记基因的数目。 转导的细胞。最后，在准备治疗患者时，我们 建议在非人灵长类动物模型中测试转导， 移植策略被认为是最有效的。概述的 研究将解决应用基因治疗和干细胞的基本问题 生物学，并加速这些学科的应用， 疾病的治疗。