TREATMENT OF BETA THALASSMIA BY VIRUS TRANSFER OF BETA G

通过病毒转移 β G 治疗 β 地中海贫血

• 批准号: 6668354
• 负责人: MICHEL SADELAIN
• 金额: $ 19.94万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6668354
• 关键词: Lentivirus; autologous transplantation; biotechnology; clinical research; cooperative study; cord blood; disease /disorder model; gene therapy; genetic regulatory element; globin; hematopoietic stem cells; human subject; laboratory mouse; nonhuman therapy evaluation; stem cell transplantation; thalassemia; tissue /cell culture

Beta-Thalassemia is one of the commonest inherited diseases in humans, characterized by a severe hemolytic anemia and ineffective erythropoiesis. While transfusion and chelation therapy do represent a radical treatment, the use of bone marrow replacement is limited 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 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 the wild-type beta-globin gene in cord blood or peripheral blood stem cells, together with a strategy for selection of genetically modified cells that are applied in vivo after transplantation. Our recent results establish that efficient gene transfer of a modified beta- globin gene and large elements of the beta-globin locus control region (LCR) can be achieved using recombinant lentiviruses. We have demonstrated that successful incorporation of large LCR elements increases mean beta-globin expression to therapeutically relevant levels. The major goals of this project are: (a) to improve erythroid-specific gene expression from a virally encoded beta-globin transcription unit; (b) to investigate whether thalassemia intermedia and thalassemia major can be cured in animal models of disease, expanding on our recent demonstration that we can achieve therapeutically relevant levels of human beta-globin in long-term murine bone marrow chimeras; (c) to confer a competitive advantage to the transduced HSC for repopulation of the host marrow using resistance to methotrexate as a model; and (d) to test our vectors in human hematopoietic cells. we propose a detailed analysis of the function of the LCR and of the chicken globin insulator (which we recently showed increases the probability of retroviral expression at random integration sites and decreases vector silencing) in stringent in vitro and in vivo assays that are relevant to the critical evaluation of their therapeutic potential. These studies include investigations in murine models of beta-thalassemia and in primary human CD34+ cells of normal subjects and patients. To analyze globin gene expression and the gene, we will capitalize on our ability to efficiently derive erythroid progeny from long-term cultured CD34+ cells and our experience with mouse/human xenochimeras using NOD-scid/scid mice. 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 adequate expression of the mutant dihydrofolate reductase permits efficient in vivo selection with methotrexate-trimetrexate.

β-甲性甲性疾病是人类中最常见的遗传疾病之一，其特征是严重的溶血性贫血和无效的红血病。虽然输血和螯合疗法确实代表了一种自由基治疗，但骨髓置换的使用受到限制受同种异体移植并发症的并发症的限制和需要进行侵袭性调节方案。因此，该提案的目的是开发一种将自体造血干细胞（HSC）与合理移植策略相结合的治疗方法。我们提出的方法是基于有效的慢病毒介导的野生型β-珠蛋白基因在脐带血或外周血干细胞中的转移，以及一种选择在移植后体内施用的基因修饰细胞的策略。我们最近的结果表明，可以使用重组慢病毒来实现改性β球蛋白基因的有效基因转移和β-珠蛋白基因座控制区域（LCR）的大元素。我们已经证明，大型LCR元件的成功合并将平均β-珠蛋白表达增加到治疗相关水平。该项目的主要目标是：（a）从病毒编码的β-珠蛋白转录单元中改善红细胞特异性基因表达； （b）调查丘脑中的核心中心和地中海贫血的主要疾病动物模型，以扩大我们最近的证明，即我们可以在长期鼠骨髓嵌合体中实现治疗相关的人β-珠蛋白水平； （c）赋予转导的HSC具有竞争优势，以使用对甲氨蝶呤的抗性作为模型重生宿主骨髓； （d）测试人类造血细胞中的载体。我们对LCR和鸡球蛋白绝缘子的功能进行了详细的分析（最近我们显示，在严格的体外和体内测定中，在随机整合位点增加了逆转录病毒表达的可能性）对其治疗潜力的批判性评估。这些研究包括对β-甲无血症的鼠模型以及正常受试者和患者的原发性人CD34+细胞的研究。为了分析球蛋白基因的表达和基因，我们将利用我们从长期培养的CD34+细胞中有效得出红细胞后代的能力，以及使用NOD-SCID/SCID小鼠在小鼠/人Xenochimeras方面的经验。我们最终的目的是通过直接的实验证据确定慢病毒编码的人球蛋白基因的表达在体内鼠和人类细胞中持续维持，并且对突变二氢叶酸还原酶的充分表达允许用甲氨蝶呤 - 三肽 - 三肽 - 三肽 - 三肽 - 二氢叶酸还原性的体内选择。