LIPOSOME BASED GLOBIN TRANSFER EX VIVO AND IN VIVO

基于脂质体的球蛋白体外和体内转移

• 批准号: 6242425
• 负责人: KARIN L GAENSLER
• 金额: $ 16.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6242425
• 关键词: Macaca mulatta; disease /disorder model; gene expression; gene therapy; genetic enhancer element; genetic promoter element; genetically modified animals; globin; hematopoietic stem cells; laboratory rat; liposomes; messenger RNA; plasmids; sickle cell anemia; tissue /cell culture; transfection

Sickle cell anemia, a genetic disorder caused by a specific mutation in codon 6 of the human beta globin gene, results in severe morbidity and early mortality. None of the existing therapies have proved as effective as initially hoped. Currently, the development of gene transfer for sickle cell anemia has emerged as an important therapeutic option. Recent advances in 3 separate areas: l) identifying elements which confer high level, erythroid-specific globin gene expression, 2) isolating and culturing human pluripotent hematopoietic stem cells, and 3) developing new gene transfer technologies, ultimately may permit correction of the underlying genetic defect causing sickle cell anemia. Our overall objective is to adapt our existing cationic liposome-based gene delivery technology in order to develop safe and effective gene therapy for sickle cell anemia. To accomplish this, we will pursue, in parallel, both ex vivo and in vivo, liposome-based, gene transfer approaches. These two strategies are both separate and complementary. We will take advantage of assays provided by the stem cell core facilities and the availability of a transgenic sickle cell anemia mouse model, both of which are provided within this program project, to test the efficacy of our in vitro and in vivo gene transfer procedures, respectively. Each approach is based on the availability of the cationic liposome formulations, expression plasmid constructions, and DNA:lipid combinations that we have already shown can efficiently transfer and express gene constructs both in cultured cells, and in animals. (1) Our first goal is to identify conditions necessary to achieve liposome-based, efficient, stable transfection of bone marrow- derived human CD34+ stem cells ex vivo. (2) Our second goal is to maximize the amount and duration of human beta-like globin gene expression in erythroid progenitor cells of mice after cationic liposome-mediated in vivo gene delivery. To achieve these aims, we will optimize the cationic liposome formulation, the promoter and enhancer element used, design of the expression plasmid topology, DNA:lipid ratio, and targeting-ligands attached to the liposome surface, specifically for ex vivo and in vivo transfer and expression of human beta globin genes. We will measure the amounts of human beta globin mRNA and protein produced in these cells and determine the proportion of target cells that express the gene product. We will target uptake and expression of the beta globin gene to erythroid progenitor cells in mice, following systemic delivery of DNA:liposome complexes. We will focus on targeting the expression of these genes by linking them to beta globin promoter and enhancer sequences from the beta globin locus control region (LCR). These sequences have been shown to confer high level, erythroid-specific expression of linked human beta- globin genes. In summary, we will use our combined experience with cationic liposome-mediated gene delivery and the regulation of globin gene expression in order to A) stably transfect human beta-globin genes in human CD34+ stem cells ex vivo and B) to deliver and express these genes in murine erythroid progenitor cells for significant periods in vivo.

镰状细胞贫血，这是一种遗传疾病，由特定突变引起 人β球蛋白基因的密码子6导致严重的发病率和 早期死亡率。现有的疗法均未被证明是有效的 正如最初希望的那样。目前，镰刀基因转移的发展 细胞贫血已成为重要的治疗选择。最近的 在三个单独的领域的进步：l）识别赋予高的元素 水平，红细胞特异性球蛋白基因表达，2）分离和 培养人多能造血干细胞，3） 新的基因转移技术最终可能允许校正 潜在的遗传缺陷导致镰状细胞贫血。我们的整体 目的是适应我们现有的基于阳离子脂质体的基因输送 为了开发镰刀安全有效的基因疗法的技术 细胞贫血。为了实现这一目标，我们将同时追求两者 和基于脂质体的基因转移方法。这两个 策略既是分开的又是互补的。我们将利用 干细胞核心设施提供的测定和可用性 转基因镰状细胞贫血小鼠模型，两者都提供 在此计划项目中，以测试我们的体外和 体内基因转移程序。每种方法都是基于 阳离子脂质体配方的可用性，表达质粒 构造和DNA：我们已经显示的脂质组合可以 在培养细胞中有效地转移和表达基因构建， 和动物。 （1）我们的第一个目标是确定必要的条件 实现基于脂质体的，有效的，稳定的骨髓转染 衍生的人CD34+干细胞离体。 （2）我们的第二个目标是最大化 人类β样球蛋白基因表达的数量和持续时间 阳离子脂质体介导的小鼠的红细胞祖细胞在 体内基因递送。为了实现这些目标，我们将优化阳离子 脂质体配方，使用的启动子和增强子元件，设计 表达质粒拓扑，DNA：脂质比和靶向配体 连接到脂质体表面，专门用于体内和体内 人β球蛋白基因的转移和表达。我们将衡量 在这些细胞中产生的人β球蛋白mRNA和蛋白质的量， 确定表达基因产物的靶细胞的比例。我们 将靶向β球蛋白基因对红细胞的摄取和表达 DNA的全身递送后，小鼠的祖细胞：脂质体 复合物。我们将专注于靶向这些基因的表达 将它们与Beta的Beta Globin启动子和增强子序列联系起来 球蛋白基因座控制区（LCR）。这些序列已显示为 授予连接的人β- 球蛋白基因。总而言之，我们将利用我们的综合经验 阳离子脂质体介导的基因递送和球蛋白基因的调节 表达为a）稳定转染人β-珠蛋白基因 人CD34+干细胞ex Vivo和b）传递并表达这些基因 在鼠类红细胞祖细胞中，体内长期存在。