GLOBIN GENE TRANSFER FOR THERAPY OF SICKLE CELL ANEMIA

球蛋白基因转移治疗镰状细胞性贫血

• 批准号: 6285917
• 负责人: MICHEL SADELAIN
• 金额: $ 36.82万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2005-05-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6285917
• 关键词: CD34 molecule; Lentivirus; NOD mouse; SCID mouse; bone marrow transplantation; cell cycle; cord blood; dihydrofolate reductase; disease /disorder model; erythroid stem cell; gene expression; gene therapy; globin; hematopoietic tissue transplantation; human genetic material tag; human tissue; mixed tissue /cell culture; nonhuman therapy evaluation; nucleic acid repetitive sequence; recombinant virus; sickle cell anemia; stem cell transplantation; transfection /expression vector; umbilical cord

DESCRIPTION (Applicant's Description Verbatim): Sickle cell anemia is one of the commonest inherited diseases in humans, characterized by a severe chronic hemolytic anemia with an unpredictable course. While current forms of chemotherapy do not represent a radical treatment, the use of bone marrow replacement is limited by complications of allogeneic transplantation and the need for aggressive conditioning regimens. Thus, the goal of this proposal is to develop a treatment for severe hemoglobinopathies that integrates a genetic correction in autologous hematopoietic stem cells (HSC) with a reasonable transplantation strategy. The approach we propose is based on efficient lentiviral-mediated transfer of a wild-type globin gene in cord blood or peripheral blood stem cells, together with a selection for genetically modified cells that is applied in vivo after transplantation. In vivo selection is useful for two purposes: (1) to increase the relative representation of genetically corrected blood cells and (2) to decrease the toxicity associated with the transplantation conditioning regimen. Our recent results establish that efficient gene transfer of a modified beta-globin gene and large elements of the beta-globin LCR can be achieved using recombinant lentiviruses. We have demonstrated that (1) a large LCR greatly increases mean globin expression compared to the core elements of the LCR that were previously investigated and (2) incorporation of an insulator element into a retroviral vector increases the probability of expression at random integration sites and decreases vector silencing. The major goals of this project are: (a) to improve erythroid-specific gene expression from a virally encoded beta-globin transcription unit; (b) to compare the betaAand gammaAglobin genes in terms of their level of expression in bone marrow chimeras and their therapeutic activity in mouse models of sickle cell disease; (c) to confer a competitive advantage to the transduced HSC for repopulation of the host marrow using resistance to methotrexate as a model. We propose a detailed analysis of the function of the LCR and of the chicken globin insulator in stringent in vitro and in vivo assays that are relevant to the critical evaluation of their therapeutic potential. These studies are based on investigations in murine models of sickle cell disease and in primary human CD34+ cells of normal subjects and patients. To analyze globin gene expression and the effectiveness of drug resistance in selecting out corrected cells that express therapeutic levels of the globin transgene, we will capitalize on our ability to efficiently derive erythroid progeny from long-term cultured CD34+ cells and our mouse/human xenochimeras based on NOD-scid/scidmice. We ultimately aim to establish by direct experimental evidence that expression of the lentivirus-encoded human globin gene is sustained over time in murine and human cells in vivo and that expression of the mutant dihydrofolate reductase permits efficient in vivo selection with methotrexate/trimetrexate.

描述（申请人的描述逐字描述）：镰状细胞贫血是一种 人类中最常见的遗传疾病，其特征是严重的慢性 溶血性贫血，病程不可预测。而当前形式的形式 化学疗法不代表自由基治疗，骨髓的使用 替代受到同种异体移植并发症的限制和 需要积极的条件方案。因此，该提议的目标是 开发对整合遗传的严重血红蛋白病的治疗 自体造血干细胞（HSC）校正合理 移植策略。我们提出的方法是基于有效的 慢病毒介导的野生型球蛋白基因在脐带血或 外周血干细胞，以及选择基因修饰的 移植后体内应用的细胞。体内选择是 有两个目的有用：（1）增加 遗传校正的血细胞和（2）降低毒性相关的毒性 具有移植条件方案。我们最近的结果建立了 改良的β-珠蛋白基因和大元素的有效基因转移 可以使用重组慢病毒来实现β-珠蛋白LCR的。我们有 证明（1）大LCR大大增加了平均球蛋白表达 与以前研究过的LCR的核心要素相比 （2）将绝缘子元件掺入逆转录病毒载体中增加 在随机积分位点表达的概率并减少矢量 沉默。该项目的主要目标是：（a）改进 来自病毒编码的β-珠蛋白的红细胞特异性基因表达 转录单元； （b）用 它们在骨髓嵌合体中的表达水平及其治疗性 镰状细胞疾病的小鼠模型中的活性； （c）授予竞争性 使用转导的HSC的优势用于使用 抗甲氨蝶呤作为模型的抗性。我们提出了对 LCR和Chicken Globin绝缘子的功能严格的体外功能 以及与对其的批判性评估相关的体内测定 治疗潜力。这些研究基于在鼠的调查 镰状细胞疾病的模型以及正常的原发性CD34+细胞 受试者和患者。分析球蛋白基因表达和有效性 在选择表达治疗的校正细胞中的耐药性 球蛋白转基因的水平，我们将利用我们的能力 有效地从长期培养的CD34+细胞和 我们的小鼠/人类Xenochimeras基于点数/scid/scidmice。我们最终旨在 通过直接的实验证据建立表达 慢病毒编码的人球蛋白基因在鼠和人类中持续存在 体内细胞和突变二氢叶酸还原酶允许的表达 用甲氨蝶呤/三甲酸酯甲氨蝶呤进行有效的体内选择。