HUMAN GC GENE TRANSDUCTION INTO HEMATOPOIETIC CELLS

高谢病是一种令人衰弱的鞘脂储存障碍 影响到美国大约30,000名患者。的目标是 这个项目是为了开发改进的基因转导方法，这将 允许通过表达功能水平的蛋白来治愈这种疾病 高雪病患者巨噬细胞葡萄糖脑苷酶活性的研究 用人类GC基因转导他们的造血细胞。 我们希望开发更有效的体外转导方法 造血干细胞，改善条件，体外和体内 在狗模型中的选择策略，并最终证实了我们在 少量灵长类动物在进行临床应用之前 人类。概述的实验将成为后续实验的基础 临床研究不仅是为了治愈高谢病，也是为了 为其他人类疾病的基因治疗提供了一个模型。 可以估计，至少10%的高雪病患 巨噬细胞必须表达GC基因才能获得GC活性 足以改善疾病的表现。出于这个原因，逆转录病毒 转导的造血细胞再生的转导和植入 必须提高电池的效率。为了实现这两个目标，我们首先提出 提高细胞的体外维持、扩增和转导能力 造血细胞再生。这将涉及以下战略： 确定逆转录病毒载体上清液的最佳暴露时间 建立干细胞增殖的最佳体外生长条件 并在不损失自我更新的情况下高效整合载体DNA 容量。第二，我们建议产生安全、高滴度的逆转录病毒GC 对犬和灵长类细胞具有高亲和力的载体。具体来说，我们 已经制作了单基因表达的GC载体(PG13/LGGC)，并将 生成共表达可选择基因的双基因表达GC载体。 第三，我们建议在狗身上进行研究，研究如何增加 移植的转导后的再生细胞克隆。为了实现这一目标， 我们将比较不同的调理方案，以减少 在输注转导细胞之前内源性再生细胞， 我们将开发细胞的体外和体内选择策略 表达可选择的标记基因，以减少非 转导细胞。最后，在为治疗病人做准备时，我们 建议在非人类灵长类动物模型中测试转导和 移植策略被发现是最有效的。概述 研究将解决应用基因疗法和干细胞的基本问题 生物学，并加快这些学科的应用，以 疾病的治疗。

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.