GENE TRANSFER INTO HEMATOPOIETIC STEM CELLS

基因转移至造血干细胞

• 批准号: 6501106
• 负责人: ARTHUR W. NIENHUIS
• 金额: $ 12.63万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6501106
• 关键词: Lentivirus; Macaca mulatta; Retroviridae; SCID mouse; biotechnology; cell population study; clinical research; disease /disorder model; erythropoiesis; gene therapy; genetic regulatory element; globin; hematopoietic stem cells; hemoglobin F; human subject; human tissue; laboratory mouse; messenger RNA; polymerase chain reaction; recombinant virus; sickle cell anemia; sickling inhibitor; thalassemia; transfection /expression vector

This project is focused on the development of effective strategies for correcting the disease phenotype by therapeutic gene transfer into stem cells of mice and humans with sickle cell anemia. Our goals are to identify a vector system that is capable of achieving relatively efficient gene transfer into primitive human hematopoietic cells under conditions which preserve or expand their numbers and repopulating potential. A second goal is to develop a therapeutic expression cassette which can be used to directly increase the amount of gamma mRNA in transduced cells or to activate the endogenous gamma-globin genes through expression of a transcriptional factor, transdominant mutant therefor or antisense sequences targeted toward a relevant mRNA. A third goal is to model gene therapy protocols in mice with sickle cell disease using drug selection to amplify a genetically normal or gene corrected minority population of hematopoietic cells. The research proposed under the first specific aim is designed to test the hypothesis that the intrinsic biological advantages of lentiviral vectors, namely the relative stability of the pre-integration complex and the ability of this nucleoprotein complex do transverse the nuclear membrane, will allow a higher frequency of transduction of primitive human transfer into repopulating cells. Research is also proposed to close the gap in our knowledge regarding the recovery and transducibility of repopulating cells from patients with sickle cell anemia. The second specific aim is organized around the basic hypothesis that retroviral mediated gene transfer into hematopoietic stem cells can be used to achieve a therapeutic level of gamma-globin gene expression in maturing erythroblasts. Two strategies will be pursued concurrently. 1) development of an erythroid specific expression cassette that generates high levels of exogenous gamma-globin mRNA and 2) evaluation of various genetic elements with respect to their capacity to activate the exogenous gamma-globin genes. Research proposed specific aim 3 is designed to use murine models of human hemoglobin disorders, severe beta thalessemia or sickle cell disease, to test the hypothesis that a minority of cells that have been genetically modifier by retroviral mediated gene transfer can be amplified by drug selection using the dihydrofolate reductase selection system leading to cure of the hemoglobin disorder. Experiments are proposed to vigorously test the selection system in the context of competitive repopulation thereby providing useful information for developing gene therapy protocols for patients with sickle cell disease in the future.

该项目的重点是开发有效的策略，用于通过治疗基因转移到患有镰状细胞贫血的小鼠和人类干细胞中的疾病表型。我们的目标是确定能够在保留或扩大其数量并重新占用潜力的条件下将基因转移到原始人类造血细胞中的媒介系统。第二个目标是开发一种治疗性表达盒，可用于直接增加转导细胞中的伽马mRNA量或通过表达转录因子的表达，其转录突变剂或靶向相关mRNA的反义序列来激活内源性γ-球蛋白基因。第三个目标是使用药物选择在患有镰状细胞疾病的小鼠中对基因治疗方案进行建模，以扩大遗传正常或基因校正的造血细胞的少数群体。第一个特定目的提出的研究旨在检验以下假设：慢病毒载体的内在生物学优势，即，预融合复合物的相对稳定性以及该核蛋白复合物的能力确实会横向核膜，这将使原始人转移到循环细胞的转移频率较高。还提出了研究缩小我们对镰状细胞贫血患者再植入细胞恢复和可渗透的知识的差距。第二个特定目的是围绕基本假设组织的，即逆转录病毒介导的基因转移到造血干细胞中可以用来在成熟的红细胞成熟的成熟红细胞中实现γ--珠蛋白基因表达的治疗水平。将同时采用两种策略。 1）开发红色特异性表达盒，该盒产生高水平的外源性γ-球蛋白mRNA和2）评估各种遗传元素，以激活外源性γ-球蛋白基因的能力。研究提出的特定目标3旨在使用人类血红蛋白疾病的鼠模型，严重的β-甲ress症或镰状细胞疾病，以测试以下假设：逆转录病毒介导的基因转移曾经通过药物选择的逆转录病毒介导的基因转移可以通过药物选择来扩增二氢酸酯酸盐酶选择系统，从而扩大了导致dihydrovical seption的基因转移。提出了实验，以在竞争性重生的背景下大力测试选择系统，从而为未来的镰状细胞疾病患者开发基因治疗方案提供了有用的信息。