NHLBI MINORITY INSTITUTION RESEARCH SCIENTIST DEVELOPMEN

描述 （改编自申请人摘要）镰状细胞病是一种严重的 一种以慢性溶血性贫血为特征的血红蛋白紊乱， 血管闭塞发作、继发性并发症、生活质量受损， 预期寿命缩短。 镰状细胞病是由 谷氨酸与缬氨酸在β- 珠蛋白链 结果红细胞合成镰状血红蛋白， 其在脱氧条件下聚合， 红细胞转化为镰状细胞 镰状细胞病没有治愈的方法 除了异基因干细胞移植。 但这 程序有其局限性，因为很难找到合适的 HLA相合的同胞供者，骨髓移植的高成本，以及 高发病率和死亡率。 相对较新的化疗， 羟基脲降低了疼痛发作的频率， 缓解了一些疾病症状，但不能治愈疾病。 那里 是开发创新治疗方法的基本需求， 镰状细胞病和减少其衰弱并发症的风险。 基因治疗似乎是一个很好的候选人，以提供一个治愈镰状细胞 疾病 然而，这是一项艰巨的任务，因为没有一个 珠蛋白基因递送理想载体，难以转导造血细胞 干细胞，需要红系特异性，高水平，平衡表达 珠蛋白基因。 基于逆转录病毒和腺相关基因转移 方法正在使用，但它们有其缺点，如DNA重排 整合后，转移基因的低表达和安全性问题 这是人类使用的病毒载体所固有的。 该提案试图探讨 开发产生γ- 红白血病细胞系中的珠蛋白基因， 替换造血干细胞中有缺陷的珠蛋白基因。 基于我们 自主复制序列（ARSH 1）的研究， 复制器，申请人将探索开发ARSH 1的可能性- 衍生的DNA分子作为Gent转移的替代载体。 这些 病媒将需要自主复制、稳定、可遗传， 转移基因的长期表达。 具体目标是 主要内容包括：1）新型载体的构建和初步鉴定 对于γ-珠蛋白基因递送到K-562细胞中，2）γ-珠蛋白基因的表征 K-562中转染载体的附加型复制活性和完整性 3）转染载体在K-562细胞中的表达。 一旦这个 载体的开发和表征，它可以在小鼠中进行研究 造血干细胞，以及后来，如果这些细胞维持复制， 转染后的干细胞可以进行移植 转化成镰状细胞转基因小鼠模型，并在体内表达胎儿 在动物模型中研究的血红蛋白。 如果成功，这些研究可以 为镰状细胞病的基因治疗提供了一种替代载体。

DESCRIPTION (Adapted from applicant's abstract) Sickle cell disease is a serious hemoglobin disorder characterized by a chronic hemolytic anemia with recurrent vasoocclusive episodes, secondary complications, impaired quality of life and a reduced life expectancy. Sickle cell disease is caused by the substitution of glutamic acid with valine at the position of amino acid six in the Beta- globin chain. As a result the red blood cells synthesize sickle hemoglobin, which polymerizes under deoxygenating conditions, changing the shape of the red blood cells into sickle cells. There is no cure for sickle cell disease with the exception of allogeneic stem cell transplantation. However, this procedure has its limitations because of the difficulty in finding a suitable HLA-identical sibling donor, the high cost of bone marrow transplantation, and the high rate of morbidity and mortality. Relatively new chemotherapies like hydroxyurea has decreased the frequency of the painful episodes, and alleviated some of the disease symptoms, but does not cure the disease. There is a basic need to develop innovative therapeutic approaches to cure and treat sickle cell disease and reduce the risks of its debilitating complications. Gene therapy seems to be a good candidate to provide a cure for sickle cell disease. However, it is a formidable task due to the unavailability of an ideal vector for globin gene delivery, difficulty in transducing hematopoietic stem cells, the need for erythroid specific, high level, balanced expression of the globin genes. Retroviral and adeno-associated gene transfer based methods are being used, but they have their drawbacks like DNA rearrangements upon integration, low expression of transferred genes, and safety concerns inherent to viral vectors for human use. This proposal attempts to explore the development of alternative vectors that yield high expression of gamma- globin genes in erythroleukemia cell lines, with the potential application to replace defective globin genes in hematopoietic stem cells. Based on our studies with an autonomously replicating sequence (ARSH1), a human putative replicator, the applicant will explore the possibility of developing ARSH1- derived DNA molecules as alternative vehicles for gent transfer. These vectors will need to replicate autonomously, be stable, heritable and have long term expression of the transferred gene. The specific aims of this proposal are: 1) Construction and primary characterization of novel vectors for gamma-globin gene delivery into K-562 cells, 2) Characterization of the episomal replication activity and integrity of the transfected vector in K-562 cells, 3) Expression of the transfected vector in K-562 cells. Once this vector is developed and characterized, it can be studied in mouse hematopoietic stem cells, and later on, if these cells sustain replication and expression of the vector, the transfected stem cells could be transplanted into a sickle cell transgenic mouse model, and in vivo expression of fetal hemoglobin studied in the animal model. If successful, these studies could produce an alternative vehicle for gene therapy of sickle cell disease.