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（申请人的逐字描述）：镰状细胞性贫血是一种 人类最常见的遗传性疾病，其特征是严重的慢性 病程不可预测的溶血性贫血。虽然目前的形式 化疗并不代表根治性治疗，使用骨髓 置换受到同种异体移植并发症的限制 需要积极的调理方案。因此，本提案的目标是 开发一种治疗严重血红蛋白病的方法，该方法整合了遗传基因 自体造血干细胞（HSC）的校正具有合理的 移植策略。我们提出的方法基于高效 慢病毒介导的脐带血中野生型珠蛋白基因的转移或 外周血干细胞，以及经过选择的转基因 细胞移植后应用于体内。体内选择是 有两个目的：（1）增加相对代表性 基因校正血细胞和（2）减少相关毒性 与移植预处理方案。我们最近的结果表明 修饰的β-珠蛋白基因和大元件的有效基因转移 β-珠蛋白LCR的检测可以使用重组慢病毒来实现。我们有 证明 (1) 大的 LCR 大大增加了平均珠蛋白表达 与之前研究的 LCR 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 在随机整合位点表达的概率并减少向量 沉默。该项目的主要目标是： (a) 改进 病毒编码的β-珠蛋白的红系特异性基因表达 转录单位； (b) 比较 betaA 和 gammaAglobin 基因 它们在骨髓嵌合体中的表达水平及其治疗 镰状细胞病小鼠模型中的活性； (c) 赋予有竞争力的 使用转导的 HSC 来重新填充宿主骨髓的优势 以甲氨蝶呤耐药为模型。我们提出了详细的分析 LCR 和鸡珠蛋白绝缘体在严格体外的功能 以及与其严格评估相关的体内测定 治疗潜力。这些研究基于对小鼠的调查 镰状细胞病模型和正常人原代 CD34+ 细胞 受试者和患者。分析珠蛋白基因表达及其有效性 选择表达治疗药物的校正细胞时的耐药性 球蛋白转基因的水平，我们将利用我们的能力 从长期培养的 CD34+ 细胞中有效衍生红系后代 我们的小鼠/人异种嵌合体基于 NOD-scid/scidmice。我们的最终目标是 通过直接实验证据确定表达 慢病毒编码的人珠蛋白基因在小鼠和人类体内随时间的推移而持续存在 体内细胞和突变二氢叶酸还原酶的表达允许 使用甲氨蝶呤/曲美曲沙进行有效的体内选择。